WO1998000439A2 - Multivalent compounds for cross-linking receptors and uses thereof - Google Patents

Multivalent compounds for cross-linking receptors and uses thereof Download PDF

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WO1998000439A2
WO1998000439A2 PCT/US1997/011279 US9711279W WO9800439A2 WO 1998000439 A2 WO1998000439 A2 WO 1998000439A2 US 9711279 W US9711279 W US 9711279W WO 9800439 A2 WO9800439 A2 WO 9800439A2
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compound
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peptide
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William W. Bachovchin
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Trustees Of Tufts College
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Publication of WO1998000439A2 publication Critical patent/WO1998000439A2/en
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Definitions

  • the Field of the Invention relates to low molecular weight bivalent and multivalent crosslinking compounds capable of inducing association between naturally occurring receptors, and particularly to inducing association between surface receptors on T cells, hematopoietic cells or antigen presenting cells such as macrophages, dendritic cells or B cells, e.g., homobivalent induced CD26-CD26 association or heterobivalent induced CD26 association with the above-mentioned surface receptors.
  • Cell surface receptors transmit signals received on the outside of a cell to the inside through two basic mechanisms: (1) ligand-induced allosteric conformational change and (2) ligand-induced association.
  • the ligands for the ligand-induced, allosteric conformational change mechanism are typically small molecules, such as the catecholamines or the neuropeptide hormones.
  • Receptors activated by a ligand-induced dimerization include, for example, those for cell growth and differentiation factors. Factors which serve as ligands for these receptors are typically large polypeptide hormone and cytokines such as erythropoietin, granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating factor (GM-CSF), and human growth hormone (hGH). Many of the dimerization-activated receptors have cytoplasmic tails that contain protein kinase domains or docking sites.
  • cytoplasmic domains of dimerization-activated receptors do not have kinase domains themselves, but function the same as if they did because they associate with protein kinases via docking sites.
  • Receptors activated by oligomerization or aggregation are found most frequently in the immune system. They include, for example, the T cell surface receptors such as CD2, CD4, CD8, CD28, CD26, CD44, CD45, CD 10, and CD3/TCR (T cell antigen receptor) and the B cell surface receptors such as CD40, B7.1 and B7.2.
  • T cell surface receptors such as CD2, CD4, CD8, CD28, CD26, CD44, CD45, CD 10, and CD3/TCR (T cell antigen receptor)
  • B cell surface receptors such as CD40, B7.1 and B7.2.
  • the ligands for these cell receplors arc most often cell surface proteins themselves, and can be found on cognate cells. Aggregation-activated receptors frequently have short cytoplasmic domains which act to bind and thereby recruit other cell surface and/or cytosolic factors following the aggregation of their extracellular domains.
  • Antagonists block the binding of the natural ligand without inducing the conformational change in the receptor thereby blocking a signal transduction pathway.
  • Antists bind to the receptor in a manner which mimics the natural ligand closely enough to induce the same conformational change as natural ligand thereby initiating a signal transduction pathway. See Seed, et al. for a theoretical discussion on how to make an agonist from an antagonist (Seed, B., Making Agonists of Antagonists, Chemistry & Biology 1 : 125 (1994). See Austin, et al. for a discussion of the role of regulated protein dimerization in biology (Austin, et al. Chemistry & Biology 1 :131 (1994)).
  • Monomeric inhibitors block recall antigen-induced T cell activation and proliferation (G.R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP- IV) by Xa ⁇ -boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991)).
  • a number of anti- CD26 mAbs have the same inhibitory activity when used under non-crosslinking conditions (C. Morimoto, et al. 1F7, a novel cell surface molecule, involved in helper function of CD4 cells, Journal of Immunology 143, 3430-3439 (1989) and published erratum appears in J. Immunology.
  • T cell triggering molecule Tpl03 is associated with dipeptidyl aminopeptidase IV activity, J. Immunol. 144, 2980-2914 ( 1990)).
  • DP-IV is a poslproline cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any a ino acid) dipeptides from the amino terminus of polypeplides.
  • Xaa-boroPro Representative monomeric structures of these transition-stale-analog- based inhibitors, Xaa-boroPro, are e.g., Pro-boroPro and Ala-boroPro.
  • BoroPro refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group [B(OH) 2 ].
  • Pro-boroPro the most thoroughly characterized of these inhibitors has a Ki of 16 picomolar (pM) (W.G. Gutheil and W.W.
  • Vai-boroPro has even a higher affinity, with a Ki of 1.6 pM (W.G. Gutheil and W.W. Bachovchin. Supra; R.J. Snow, et al. Studies on Prolinc boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a B-N Bond, J. Am. Chem.
  • Cytotoxic drugs have untoward effects since they indiscriminately kill all proliferating cells. With the advent of monoclonal antibodies, it is possible to increase the specificity of these therapeutic tools. Monoclonal antibodies against the T cell receptors, e.g., T-cell receptor, CD4 and CD8 co-receptors, and to MFIC class II molecules, have all been evaluated for their respective benefit in experimental models for the treatment of autoimmune disease.
  • the major impediment to using monoclonal antibodies as a therapeutic tool in humans is that most monoclonal antibodies are made in mice, and humans rapidly develop an antibody response to mouse antibodies, which limits their potency because of neutralization and, worse, produces allergic reactions such as immune complex disease.
  • mice that are entirely human in origin can be obtained.
  • mice that lack endogenous immunoglobulin genes can be made transgenic for human heavy and light chain loci using yeast artificial chromosomes.
  • low molecular weight, bivalent or multivalent, synthetic crosslinking compounds are designed and developed. These synthetic crosslinking compounds may act either as agonists or antagonists and induce association between naturally occurring receptors, e.g., induce the association of one particular T cell surface receptor, such as CD26, with (a) itself or, (b) with another T cell or antigen presenting cell surface receptor (e.g., CD2, CD4, CDS, CD28, CD26, CD44, CD45, CD 10, CD3/TCR (or TCR/CD3)), CD40, B7.1 and B7.2.
  • T cell surface receptor such as CD26
  • another T cell or antigen presenting cell surface receptor e.g., CD2, CD4, CDS, CD28, CD26, CD44, CD45, CD 10, CD3/TCR (or TCR/CD3)
  • CD40 B7.1 and B7.2.
  • the low molecular weight, bivalent or multivalent, synthetic crosslinking compounds of this invention are small enough (less than about 30 amino acids, and more preferably about 20 amino acids) to obviate the immunogenicity associated with monoclonal antibodies.
  • the bivalent or multivalent, synthetic crosslinking compounds of this invention can be administered to a patient without being co-administered with an adjuvant.
  • most other peptides, proteins and carbohydrate antigens are usually poorly immunogenic, or not immunogenic at all, when administered without adjuvant.
  • the compounds of the invention are useful for crosslinking molecules on the same or different cells that are involved in immune system modulation.
  • Compounds of the invention fall within the following genus:
  • P 1 represents a first targeting moiety, preferably a peptide that can mimic the substrate binding site of a protease (preferably a serine protease or cysteine protease) that is expressed on the surface of the cell involved in immune system modulation (e.g., a T cell, a B cell, a stem cell, a bone marrow cell, including an antigen presenting cell);
  • a protease preferably a serine protease or cysteine protease
  • R 1 represents a reactive group that reacts with a functional group in a reactive center of the protease
  • P 2 represents a second targeting moiety, preferably a peptide, that may be the same or different from the first targeting moiety;
  • L represents a linker molecule (i) having a molecular weight ranging from about 100 daltons lo about 2000 daltons, (ii) having a length ranging from about 2 ⁇ to about 300 ⁇ ; and (iii) containing a chain of atoms selected from the group consisting of C, O, N, S, and phosphorus atoms, connected by single or by double bands.
  • P 1 can be D1 ⁇ A1 ⁇ A2 ⁇ A3 ⁇ A4 or D2 ⁇ A5 ⁇ A6 ⁇ A7 ⁇ A8 as described below in reference to certain embodiments of the invention.
  • P 1 is a peptide or a peptidomimetic.
  • R 2 can be absent, the same, or different from R 1 .
  • Cells which are involved in immune system modulation are blood cells including T cells, B cells, stem cells, bone marrow cells, dendritic cells, and other antigen presenting cells.
  • the P 1 targeting moiety can have a carboxyl terminal portion containing 1, 2, 3, or 4 amino acids which mimic the substrate binding site of the protease.
  • Exemplary proteases which are believed to be expressed on the surface of such cells and which are bound by the P 1 targeting moieties include post- prolyl cleaving enzymes, trypsin, chymotrypsin and elastase.
  • the particular amino acids in the naturally occurring substrates of these enzymes are well known in the art and are identified below.
  • the compounds of the invention contain from 1 to 30 amino acid residues (preferably the L isomers) and, more preferably, contain from 1 to 20 amino acid residues.
  • the P 1 targeting moieties contain from 1 to 10 amino acids, most preferably, from 1 to 2 amino acids.
  • the P 1 targeting moiety can contain L or D amino acids; however, it is preferred that at least the amino acids which mimic the substrate binding site be in the L configuration. In contrast, the amino acids which arc in a rctroinverso configuration (see the Examples) preferably are in the D configuration.
  • composition of the P 1 targeting moiety is not limited to amino acids but may include, in whole or in part, non-amino acid components, provided that such components do not interfere significantly (i.e., do not lower the Ki of the compound to less than about 10 "7 M with the site-specific recognition of the compound by the protease and provided that the non-amino acid components do not interfere with Ihe formation of a complex between the compound and the protease.
  • the portion of the P 1 targeting moiety that is involved in binding to the substrate binding site is formed of amino acids and the remaining portion of the P 1 targeting moiety is formed of non-amino acid components.
  • any portion of the P 1 targeting moiety can be modified, for example, to be coupled to a detectable reagent, or immobilized to a surface via a linker, provided that such modifications satisfy the foregoing inhibition constant and complex formation criteria.
  • Peptides which reportedly have utility for inhibiting post-prolyl cleaving enzymes and which, if coupled to a reactive group, form a covalent complex with a functional group in the reactive site of a post-prolyl cleaving enzyme are described in U.S. Patent No. 4,935,493, "Protease Inhibitors", issued to Bachovchin et al. ("Bachovchin '493"); U.S. 5,462,928, "Inhibitors of Dipeptidyl-aminopeptidase Type IV", issued to Bachovchin et al. ("Bachovchin '928"); U.S.
  • the P 1 targeting moiety mimics the substrate binding site of the post-prolyl cleaving enzyme DP IV (also referred to herein as "CD 26").
  • DP IV is a post-prolyl cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of a polypeptide substrate.
  • Representative structures of transition-state analog-based inhibitors Xaa-boroPro include Lys-BoroPro, Pro-BoroPro and Ala- BoroPro in which "boroPro” refers to the analog of proline in which the carboxylate group (COOI1) is replaced with a boronyl group [B(OH) 2 ].
  • Alternative crosslinking compounds of the invention have an analogous structure in which the boronyl group is replaced by a phosphonate or a fluoroalkylkctone (described below).
  • the invention also embraces compounds which mimic the substrate binding site of other post-prolyl cleaving enzymes.
  • IgA 1 proteases recognize the cleavage site Ser-Thr-Pro-Pro-X (where X is any amino acid).
  • Ser-Thr-Pro-Pro-R 1 is suitable for selectively binding to, and forming a complex with a functional group in the active site of an IgA 1 protease.
  • the Ser- Thr in this targeting moiety may be readily substituted with any of the 20 naturally occurring amino acids, most preferably those having non-bulky side groups, such as Ala and Gly.
  • a preferred P 1 R 1 binding moiety for binding to an IgA 2 protease has the formula Pro-Thr-Pro-R 1 .
  • Thr can be substituted by any of the naturally occurring amino acids, especially ones having non-bulky side groups, such as Ala, Gly or Ser.
  • Other examples of post-prolyl cleaving enzymes which can be targeted by the targeting moieties of the invention include other IgA enzymes, encephalon degrading enzymes, vasopressin degrading enzymes, and oxytocin degrading enzymes.
  • the P 1 targeting moieties of the invention can be designed to mimic the substrate binding sites of other, non-post-prolyi cleaving enzymes that may be expressed on the surface of cells involved in immune system modulation.
  • enzymes include, for example, cystcinc proteases and serine proteases such as trypsin, chymotrypsin and elastasc.
  • the substrate binding sites for these enzymes are well known and peptidomimetics for targeting binding to these sites have been reported.
  • a P 1 targeting moiety of the invention which mimics the substrate binding site of trypsin would include an arginine (Arg) or lysine (Lys) residue at its carboxyl-tcrminus with the carboxyl group of the Arg or Lys coupled to an appropriate reactive center, R 1 , to form a covalent bond with a functional group in the active site of trypsin.
  • arginine Arg
  • Lys lysine
  • Exemplary borolysine targeting moieties that can be used to form the compounds of the invention are described in U.S. Patent Nos. 5, 187,157 and 5,242,904, "Peptide Boronic Acid Inhibitors of Trypsin-like Proteases", issued to Kettner et al.
  • P 1 targeting moieties also can be designed to mimic the substrate binding site of chymolrypsin.
  • Such targeting moieties include a carboxyl terminal amino acid residue that is selected from the group consisting of phcnylalaninc (Phc), tryptophan (Tip), and tyrosine (Tyr).
  • the carboxyl groups of these amino acids are covalently coupled to an R 1 reactive group to form a binding moiety that selectively binds to, and forms a covalent complex with, a functional group in the active site of chymotrypsin.
  • Yet other P 1 targeting moieties of the invention can be designed which mimic the substrate binding site of an elastase.
  • a P 1 targeting moiety which mimics the substrate binding site of an elastase would include a carboxyl terminal amino acid residue that is alanine (Ala) or glycine (Gly) with the carboxyl group of these amino acids covalently coupled to the reactive group R 1 .
  • conventional chemical reactions can be used to form the foregoing P'R 1 binding moieties.
  • P'R 1 binding moieties of the invention can be designed and constructed to mimic the substrate binding site of virtually any protease for which the natural substrate is known or can be identified.
  • phagc display libraries and chemical combinatorial libraries from which synthetic compounds can be selected which mimic the substrate binding site of a protease permits the identification of further P 1 targeting moieties to which an R 1 reactive group can be covalently attached to form a binding moiety which mimics the substrate binding site of the protease and which forms a complex with a functional group in the protease reactive site.
  • Such libraries can be screened to identify non-naturally occurring putative targeting moieties by assaying protease cleavage activity in the presence and absence of the putative phage display library molecule or combinatorial library molecule and determining whether the molecule inhibits cleavage by the protease of its natural substrate or of a substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay).
  • a substrate analog e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay.
  • Those phage library and/or combinatorial library molecules which exhibit inhibition of the protease then can be covalently coupled to the reactive groups R 1 disclosed herein and again tested to determine whether these novel molecules selectively bind to the protease (e.g., by repeating the above-noted screening-assay).
  • a simple, high-through-put screening assay is provided for identifying non-naturally occurring targeting moieties of the invention.
  • the first targeting moieties of the invention are covalently coupled via a carboxyl group at their carboxyl terminal amino acid to a first reactive group, R 1 .
  • R 1 refers to a reactive group that is capable of reacting with a functional group in a reactive center of a protease expressed on the surface of a cell involved in immune system modulation. By reacting with a reactive center of the target protease, it is meant that the R 1 forms a covalent bond with a functional group that is located in the active site.
  • R 1 reactive groups that are embraced within the invention include the reactive groups referred to as group "T" in US 4,935,493, “Protease Inhibitors", issued to Bachovchin, et al. These include boronate groups, phosphonate groups, and fluoroalkylketone groups. The boronate groups are described in the detailed description of the invention and in the Examples.
  • the phosphonate and fluoroalkylketone groups are described below.
  • the linkage between the carboxyl terminus of the targeting moiety and the reactive group be in an L configuration.
  • the reactive group forms a covalent band with a functional group in the active site; however, there is no requirement for covalent bond formation in order to form a complex between the binding moiety and the active site.
  • the reactive groups of the invention that are phosphonate groups have the formula:
  • G is either H, F or an alkyl group containing 1 to about 20 carbon atoms and optional heteroatoms which can be N, S, or 0.
  • Additional exemplary proline phosphonate derivatives which contain a perfluoroalkyl group, a phenyl group or a substituted phenyl group and which can be used in accordance with the methods of the invention are those described in U. S. 5,543,396 (Powers '396).
  • the reactive groups of the invention that are fluoroalkylketone reactive groups have the formula:
  • Other kctoamides, ketoacids and kctocslers that are useful reactive groups for reacting with the reactive center of a protease are described in PCT/US91/09801 , "Peptides, Ketoamides, Ketoacids, and Ketocsters", Applicant: Georgia Tech Research Corp. ("GA Tech") which claims priority to U.S. 635,287, filed Dec. 28, 1990.
  • the reactive groups are selected from the groups having the formulas, 0 0
  • R is an alkyl, or aryl group and may be substituted or unsubstituted, an alphaketo ester
  • the second targeting moiety, P 2 binds to a molecule that is present on the surface of the same or different cell to which the first targeting moiety binds.
  • the second targeting moiety binds to a molecule (e.g., a receptor, a major histocompatibility complex (MHC) molecule) which is present on the surface of a T cell or on the surface of a B cell.
  • MHC major histocompatibility complex
  • the second targeting moiety has a structure which mimics the substrate binding site of a protease that is present on a cell that is involved in immune system modulation.
  • the second targeting moiety may be the same as the first targeting moiety, and the compounds of the invention are useful for crosslinking proteases that have the same or a similar substrate specificity on the same or different cells.
  • the compounds of the invention can be used to crosslink a first protease (e.g., a post-prolyl cleaving enzyme) on a first cell and a different protease (e.g., a trypsin, chymotrypsin, elastase or other serine protease or cysteine protease) that is expressed on the surface of the same or on a different second cell.
  • a first protease e.g., a post-prolyl cleaving enzyme
  • a different protease e.g., a trypsin, chymotrypsin, elastase or other serine protease or cysteine protease
  • Compounds which include identical P 1 and P 2 groups and identical R 1 and R 1 groups are referred to as "homodimers”.
  • the first and second targeting moieties are different and these compounds are referred to as "heterodimers”.
  • the second targeting moiety is an antigen that selectively binds to an MHC molecule on the surface of an antigen presenting cell.
  • Such embodiments of the invention are useful as vaccines for inducing an immune system response to the antigen.
  • such compounds are useful for inducing an immune system response to antigens that exhibit relatively low immunogenicity using conventional vaccine preparations.
  • the invention also provides for an improved vaccine and related methods for inducing an immune response to an antigen. Examples of antigens arc antigens characteristic of pathogens, cancer antigens, and allergens.
  • Antigens that are characteristic of autoimmune disease typically will be derived from the cell surface, cytoplasm, nucleus, mitochondria and the like of mammalian tissues. Examples include antigens characteristic of uvcilis (e.g. S antigen), diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, primary yxoedema, thyrotoxicosis, rheumatoid arthritis, pernicious anemia, Addison's disease, scleroderma, autoimmune atrophic gastritis, premature menopause (few cases), male infertility (few cases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anemia, idiopathic thrombocytopenic purpura, id
  • Antigens that are allergens are generally proteins or glycoproteins, although allergens may also be low molecular weight allergenic haptens that induce allergy after covalently combining with a protein carrier (Remington's
  • Allergens include antigens derived from pollens, dust, molds, spores, dander, insects and foods. Specific examples include the urushiols (pentadecylcatechol or heptadecylcatechol) of Toxicodendron species such as poison ivy, poison oak and poison sumac, and the sesquiterpenoid lactones of ragweed and related plants. Additional examples are provided below.
  • Antigens that arc characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples include antigens characteristic of tumor proteins, including proteins encoded by mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids.
  • Tumors include, but are not limited to, those from the following sites of cancer and types of cancer: lip, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon, rectum, liver, gall bladder, biliary tree, pancreas, larynx, lung and bronchus, melanoma of skin, breast, cervix, uteri, uterus, ovary, bladder, kidney, brain and other parts of the nervous system, thyroid, prostate, testes, Hodgkin 's disease, non-Hodgkin 's lymphoma, multiple myeloma and leukemia. Viral proteins associated with tumors would be those from the classes of viruses noted above.
  • Antigens characteristic of tumors may be proteins not usually expressed by a tumor precursor cell, or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor.
  • An antigen characteristic of a tumor may be a mutant variant of the normal protein having an altered activity or subcellular distribution. Mutations of genes giving rise to tumor antigens, in addition to those specified above, may be in the coding region, 5' or 3' noncoding regions, or introns of a gene, and may be the result of point mutations, frameshifts, deletions, additions, duplications, chromosomal rearrangements and the like.
  • One of ordinary skill in the art is familiar with the broad variety of alterations to normal gene structure and expression which gives rise to tumor antigens.
  • tumor antigens include: proteins such as Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel- 17 (gplOO) of melanoma, MART-1 (Melan-A) of melanoma, p i 5 protein of melanoma, tyrosinase of melanoma, MAGE 1 , 2 and 3 of melanoma, thyroid medullary, small cell lung cancer, colon and/or bronchial squamous.
  • proteins such as Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel- 17 (gplOO) of melanoma, MART-1 (Melan-A) of melanoma, p i 5 protein of melanoma, tyrosinase of melanoma, MAGE 1 , 2 and 3 of mela
  • proteinaceous tumor antigens may be presented by HLA molecules as specific peptides derived from the whole protein. Metabolic processing of proteins to yield antigenic peptides is well known in the art; for example see U.S. patent 5,342,774 (Boon et al.).
  • Preferred tumor antigens of the invention include the Melonoma tumor antigens (e.g., MAGE protein family (MAGE- 1 , MAGE-2, MAGE-3); MART- 1 (peptide 27-35); and gpl OO); and the Colon carcinoma antigens (e.g., peptides of the mutated APC gene product).
  • MAGE protein family MAGE- 1 , MAGE-2, MAGE-3
  • MART- 1 peptide 27-35
  • gpl OO Colon carcinoma antigens
  • Colon carcinoma antigens e.g., peptides of the mutated APC gene product.
  • Particularly preferred Melanoma tumor antigen sequences are those reported by Slingluff et al., in Curr. Opin. in Immunol. 6:733- 740 (1994):
  • the MAGE protein family also reportedly has been associated with more than one type of carcinoma: MAGE-1 (Melanoma, thyroid medullary, and small-cell lung carcinoma), MAGE-2 (Melanoma, small-cell lung, colon, bronchial squamous cell, and thyroid medullary carcinoma), and MAGE-3 (Melanoma, small- cell lung, colon, bronchial squamous cell, and thyroid medullary carcinoma).
  • tumor antigens that are peptides of the mutated APC gene product are those reported by Townsend et al., in Nature 371 :662 ( 1994)):
  • the second targeting moiety is a ligand that selectively binds to a receptor that is expressed on the surface of a cell (preferably a T cell or a B cell).
  • a cell preferably a T cell or a B cell.
  • exemplary receptors which have naturally occurring ligands that can be mimicked by the second targeting moieties of the invention include receptors selected from the following group: CD2, TCR/C3, CD4, CD8, CD 10, CD26, CD28, CD40, CD44, CD45, B7.1 and B7.2.
  • the second targeting moiety is an antibody or antibody fragment that selectively binds to an epitope expressed on the cell surface. The epitope can be a portion of any of the foregoing receptors.
  • phage display and other types of combinatorial libraries can be screened in a manner analogous to that described above to identify non-naturally occurring targeting moieties that are useful in forming the compounds of the invention.
  • the second targeting moiety be covalently attached to a second reactive group.
  • the second targeting moiety may have sufficient affinity for its binding partner (e.g., an MHC molecule) to permit cross linking between the same or different target molecules on the same or different cells without forming a covalent complex between the second targeting moiety and its targeted binding partner.
  • the second reactive group be the same as the first reactive group.
  • the compounds of the invention embrace molecules that include a first binding moiety which contains a first reactive group R 1 that is a boronate and a second binding moiety which contains a second reactive group that is a boronate, a phosphonate or a trifluoroalkylketone group.
  • a linker is covalently coupled to the first and second targeting moieties,
  • linkers including a description of linker composition, size, and procedures for coupling the linker to the targeting moieties are described in the detailed description of the invention and in the Examples. In general, such linkers are commercially available and are coupled to the targeting moieties using conventional coupling procedures which are well known to those of ordinary skill in the art.
  • Certain aspects and uses of the invention are based on the discovery that certain Koinodimers are capable of stimulating T cells and that this stimulatory ability is, at least in part, dependent upon the length of the linker.
  • Certain concentrations of the homodimers of the invention have been found to stimulate blood cells.
  • compounds of the invention are particularly useful for treating HIV+ patients, for example, by contacting the T cells obtained from an HIV+ patient with a therapeutically effective amount of one or more compounds of the invention under conditions that permit blood cell activation.
  • the stimulatory effect on T-cells of these compounds at stimulating concentrations is illustrated in the accompanying Examples.
  • the cells can be contacted with the crosslinking compounds in vivo or ex vivo.
  • This stimulator)' property of the homodimer compounds of the invention is unexpected and could not have been predicted based upon the reported inhibitory effect on the immune system of certain monomers (e.g., the compounds disclosed in Bachovchin '493) and certain homodimers (e.g., PCT/GB94/02615, "DP-IV-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring").
  • certain monomers e.g., the compounds disclosed in Bachovchin '493
  • certain homodimers e.g., PCT/GB94/02615, "DP-IV-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring").
  • the compounds of the invention can be used to inhibit the enzymatic activity of the proteases to which the targeting moieties selectively bind.
  • the compounds of the invention are useful for inhibiting post-prolyl cleaving enzymes, as well as for inhibiting other serine and cysteine proteases (e.g., chymotrypsin, trypsin, and elastase).
  • kits for modulating immune system function.
  • the compounds of the invention are administered to subjects in need of immune system modulation in amounts effective to modulate immune system function.
  • Modulation of immune system function includes, but is not limited to, increasing immune function such as by stimulating proliferation and specific immune function of blood cells nonspecifically or by specifically stimulating T and/or B cells and/or bone marrow cells, stem cells, early lineage progenitor cells to produce a prophylactic or therapeutic result relating to infectious disease, cancer, and the like.
  • the compounds of the invention and in particular, homodimers and/or heterodimers for the treatment of disorders characterized by reduced T cell levels in vivo, e.g., HIV and other disorders associated with a compromised immune system.
  • Modulation of immune system function also includes, but is not limited to, decreasing immune function such as by suppressing generally the immune system in transplant recipients or suppressing specifically the immune system to treat autoimmune disease, allergy and the like.
  • the homodimers and/or heterodimers of the invention are used to stimulate blood cell proliferation, as described in detail below. Specific conditions that may be treated according to the invention are deemed specific independent aspects of the invention and are described in detail in the tables and examples below.
  • a method for stimulating T cells is provided. A crosslinking compound of the invention is contacted with the T cells of a subject in need of such treatment in an amount effective to stimulate T cells.
  • the crosslinking compounds that are particularly useful f ⁇ r-stimulating T cells arc those compounds which crosslink DP IV molecules as described above, including the preferred crosslinking compounds.
  • the preferred crosslinking compounds include a linker, L which when positioned between binding moieties results in a minimum length of about 20 angstroms between these moieties.
  • the distance between binding moieties is from 20 to 60 angstroms, more preferably from 30 to 50 angstroms.
  • pharmaceutical preparations arc provided.
  • the pharmaceutical preparations contain a crosslinking compound as described above and, optionally, a pharmaceutically-acccptable carrier.
  • the pharmaceutical compositions are sterile.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • a hetero bivalent compound having the structure as shown in Fig. 1 A, wherein each component of the structure is as defined in Example 2 in reference to this figure, is provided.
  • Fig. 1U and Fig. I V represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
  • the linker molecule must be capable of linking atom Al of the binding moiety on the left of Fig. 1A to atom A5 of the binding moiety on the right of Fig. 1A.
  • this compound include: there are 4 atoms positioned between the group consisting of Dl and D2 and B of the binding moiety; the binding moiety is in an L-configuration; Yl, Y2, Y3, and Y4 are hydroxyl groups; the A4 bonded to the B is in the L-configuration and the A5 bonded to the B is in the L-configuration; the binding moiety is an L-amino acid residue conjugated to B, a boron molecule; the binding moiety is selected from the group consisting of L-Lys-L-boroPro and a derivative of L-Lys-L-boroPro.
  • linker molecule contains a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfhydryl group, an imidazole group, an alkene group (a carbon atom double bonded to another carbon atom), an acyl halogen group, e.g., an acylchloride, and CH 2 X, wherein X represents a halogen (e.g, the two binding moieties are linked when a nuclcophilic group displaces the halogen from the functional group of the CH 2 X linker molecule); wherein the linker molecule is further defined as having the structure as shown in Fig.
  • [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogcn and a sulfur atom;
  • [J] is selected from the group consisting of a CH 2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p, and q represent an integer from 1 to 50, inclusive;
  • [GJ preferably is an R group selected from the group consisting of L-amino acid residues selected from the group consisting of lysinc, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; wherein the linker molecule preferably is selected from the group consisting of hexanedioic acid (a
  • Another aspect of this invention is a compound having the structure as shown in Fig. IB.
  • the binding moieties are identical, e.g., having Al, A2, A3, and A4 in both binding moieties, and wherein each component of the structure is defined in Example 2 in reference to this Figure.
  • Another aspect of this invention is a compound having the structure as shown in fig. 2A, wherein each component of the structure is as defined in Example 4 in reference to this Figure.
  • An embodiment of this compound includes the structures shown in Figs. 1U and IV which represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
  • this compound include: there are 4 atoms positioned between D and B of Ihe binding moiety; the binding moiety is in an L- configuralion; Yl and Y2 are hydroxyl groups; the A4 bonded to the B is in the L- c on figuration; the binding moiety is an L-amino acid residue conjugated to B, a boron molecule; and the binding moiety is selected from the group consisting of L- Lys-L-boroPro and a derivative of L-Lys-L-boroPro.
  • linker molecule which includes: a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfhydryl group, an imidazole group, an alkene group, an acyl halogen group, and CH 2 X, wherein X represents a halogen; the linker molecule which is 'further defined as having the structure shown in Fig.
  • [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogen and a sulfur atom; [JJ is selected from the group consisting of a CH 2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p, and q represent an integer from 1 to 50, inclusive; wherein [G] preferably is an R group selected from the group consisting of L-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; wherein the linker molecule preferably is selected from the group consisting of adipic acid, between 2 and 1 consecutive amino acid residues,
  • Yet another embodiment of this compound is peptides ranging from about 7 to 25 amino acids; wherein exemplary peptide include: a) Myelin proteolipid protein peptide; b) Moth cytochrome C peptide; c) tetanus toxin peptide; d) HIV-1 GP 120 peptide; e) myelin basic protein peptide; f) tumor anti genie peptides, and g) anti genie peptides of infectious agents.
  • the Myelin proteolipid protein peptide includes human and mouse peptide selected from the group consisting of PLP peptide from the region of 85- 159 and in particular, 95-1 16, and PLP peptide 105-124, PLP peptide 139-151 and PLP peptide 190-209; the Moth cytochrome C peptide is peptide MCC 94-103; the myelin basic protein peptide is MBP peptide 1- 1 1 ; the tetanus toxin peptide is selected from the group consisting of tetanus toxoid peptide and P2 tetanus toxoid peptide; and the tumor antigenic peptides and antigenic peptides of infectious agents are described elsewhere in the application.
  • Another embodiment of this compound is where the naturally occurring receptor Is a B cell or T cell surface receptor; and the cell surface receptor is selected from the group consisting of TCR/C3, CD2, CD4, CD8, CD 10, CD26, CD28, CD44, CD45, CD40, B7.1 and B7.2.
  • Another aspect of this invention is a compound having the structure as shown in Fig. 1R, wherein each component of the structure is as defined in Example 3 in reference to this Figure.
  • An embodiment of this compound includes the structures as shown in
  • Fig. 1U and Fig. IV which represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
  • FIG. 1R Another embodiment of the compound shown in Fig. 1R is wherein (a) [G] overlap, is the side chain of a D- or L-isomcr of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (c) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (d) El and E3 arc distinct from each other.
  • FIG. 1R Yet another embodiment of the compound shown in Fig. 1R is wherein (a) [GJ m is the side chain of a D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is selected from the group consisting of 2-carboxy butyl, 2-carboxypropyl, 2-aminobutyl, 2- aminopropyl, and a hydrocarbon chain with an amino or carboxy side chain; (c) [J] p and [I] q represent, independently, hydrocarbon chains; (d) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (e) El and E3 are distinct from each other.
  • the compounds of the invention, in particular the homodimeric complexes can be used to stimulate activation or proliferation of human CD26- bearing lymphocytes, by contacting the lymphocytes with a proliferation or activ
  • the method preferably involves in vivo administration of the compound, admixed with a pharmaceutically acceptable carrier such as pharmaceutical, sterile saline.
  • a pharmaceutically acceptable carrier such as pharmaceutical, sterile saline.
  • the patient can be any patient who suffers from a disease state characterized by inadequate lymphocyte activation or concentration. Examples of such diseases are HIV infection, kidney failure, cancer (in particular, cancer accompanied by lymphocyte-depleting chemotherapy), and bone marrow disorders which result in depleted lymphocyte populations in the patient.
  • the compound is preferably administered to Ihe patient orally.
  • the compounds can also be used to stimulate proliferation or activation of lymphocytes in vitro, e.g., where a patient's autologous lymphocytes are removed, stimulated to increase activation and/or number of lymphocytes, and reinfused into the patient. This method can be used, for example, to increase the number of cytolytic T cells specific for a patient's tumor or T cells in HIV infected patients.
  • crosslinking compound means the compounds described above as well as salts thereof.
  • amino acid is meant to include imino acid.
  • boroPro is meant an alpha-amino boronic acid analog of proline bonded to an amino acid to form a dipeptide with boroPro as the C-terminal residue.
  • BooPro is used to designate such an analog having the carboxyl group of proline replaced with a B(OH) 2 group, where (OH) 2 represents two hydroxyl groups and B represents boron.
  • Xaa any amino acid residue, e.g., a lysine residue.
  • [Lysine-boroProline] 2 and "KbP-S-KbP, where S represents a linker spacer” are used interchangeably.
  • "Dimeric KbP with adipic acid as the spacer linker” are used interchangeably.
  • Dimeric and bivalent are used interchangeably.
  • Linker-spacer molecule cross-linker, cross-linker molecule, linker molecule and linker group are used interchangeably.
  • agonist is meant a molecule or compound which activates the signaling pathway in question.
  • antagonist is meant a molecule or compound which inhibits the signaling pathway in question.
  • CD26 ligand is any protein, glycoprotein, lipoprotein or polypeptide that binds to the T cell receptor CD26 and may provide a stimulatory or inhibitory signal.
  • CD26 Dipeptidyl Peptidase IV (DP IV) and dipeptidyl aminopeptidase IV are used interchangeably.
  • CD26 is a postprolinc cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of polypeptides.
  • CD26-specific binding species means a CD26-specific antibody, fragment or small molecular weight compound that binds to CD26.
  • Tethered or coupled ⁇ -amino acid is an ⁇ -amino acid with a carbon atom of its side chain tethered, linked or coupled to the N atom of the ⁇ -amino group.
  • alpha-carbon of an amino acid is the one to which the carboxylic acid group is attached.
  • alpha-amino acids is where the amino group is attached to the alpha- carbon. All naturally occurring amino acids arc alpha-amino acids or alpha-imino, which means that the amino and carboxylic acid groups are both attached to the same carbon atom.
  • Each amino acid can be thought of as a single carbon atom (the alpha carbon, C) to which there is attached one carboxyl group, one amino group, a side chain denoted R as shown below and a hydrogen.
  • R is a side chain
  • NH 2 is the alpha amino group
  • the first carbon (C) attached to the NH 2 group having a hydrogen (H) and an R group attached is the alpha carbon
  • the carbon double bonded to an oxygen and a hydroxyl group (OH) is the alpha carboxyl group.
  • the NH 2 and COOH groups are used to connect amino acids to one another.
  • the hydroxyl group (OH) of one amino acid on the carboxyl end and the hydrogen (H) on the N terminus are removed (H 2 0) when two amino acids are linked together.
  • H hydrogen
  • the amino group of one amino acid reacts with the carboxyl group of another by the elimination of water; the resulting chemical bond is called a peptide bond.
  • peptides is meant a small molecule, e.g., usually containing less than 50 amino acid residues, which do not generally possess a well-defined three- dimensional structure.
  • a or “Anstrom” is meant 10 "10 M.
  • the relative bond size used in different peptides is as follows: N-H: 1.0 A; C-FI: 1.1 A; C double bonded to 0: 1.2 A; N-CO: 1.3 A; C-O: 1 .4 A; C-C: 1.5 A; Alaninc: 6 A; Benzene: 6 A; water: 4 ⁇ ; and Phenylalanine: 7 A.
  • Pharmaceutical preparations and modes of administration are described herein.
  • Figs. 1A-1 V are diagrams showing the general structures of several preferred homobivalent and homomultivalent compounds or arc diagrams of components needed for these structures:
  • Fig. 1 A is a diagram of a general bivalent template with non-identical binding moieties.
  • Fig. IB is a diagram of a general homobivalent template.
  • Fig. 1C is a diagram of a general bivalent template with amino linkages using a dicarbonyl linker.
  • Fig. ID is a diagram of a bivalent example with amino linkages using an adipoyl linker.
  • Fig. IE is a diagram of a homobivalent example with amino linkages using an adipoyl linker [(Lysine-boroPro) 2 Adipatej.
  • Fig. IF is a diagram of another homobivalent example with amino linkages using an adipoyl linker.
  • Fig. 1G is a diagram of a general bivalent template with a carboxyl linkage using a diamino linker.
  • Fig. 1H is a diagram of a bivalent example with carboxyl linkages using a 1 ,4-Diaminobutane linker.
  • Fig. II is a diagram of a bivalent example with carboxyl linkages using a 1 ,4-Diaminobutane linker [(Aspartyl-boroProline) 2 l,4-Diaminobutane].
  • Fig. 1 J is a diagram of another bivalent example with carboxyl linkages using a f,4-Diaminobutane linker.
  • Fig. IK is a diagram of a general bivalent template with disulfide linkages using a dithiol linker.
  • Fig. 1L is a diagram of a bivalent example with disulfide linkages using a 1 ,4-Dithiobutane linker.
  • Fig. IM is a diagram of another bivalent example with disulfide linkages using a 1 ,4-Dithiobutane linker.
  • Fig. IN is a diagram of another bivalent example with disulfide linkages using a dithiothreitol linker [(Cysteine-boroProline) 2 dithiotheitol].
  • Fig. 10 is a diagram of a general bivalent template with imidazole linkages using a dicarbonyl linker.
  • Fig. IP is a diagram of another bivalent example with imidazole linkages using a dicarbonyl linker.
  • Fig. 1Q is a diagram of a bivalent example with imidazole linkages using an adipoyl linker: (FIistidine-boroProIine) 2 ⁇ dipate.
  • Fig. IR is a diagram of a general homofiinctional polymeric crosslinker template.
  • Fig. IS is a diagram of a homotrimeric example using adipoyl linkers.
  • Fig. IT is a diagram of a linker molecule template.
  • Fig. 1U is a diagram of a binding moiety containing Al , A2, A3, and
  • Fig. IV is a diagram of a binding moiety containing A5, A6, A7, and
  • Figs. 2A-2C are diagrams showing the general formula of several preferred heterobivalent compounds:
  • Fig. 2 A is a diagram of a general heterobivalent template.
  • Fig. 2B is a diagram of a heterobivalent example coupling a binding moiety to an MCC peptide (94-103) ⁇
  • FIG. 2C is a diagram of a heterobivalent example coupling a binding moiety to a PLP peptide (139-151).
  • Fig. 3 is a diagram showing the synthesis of adipoyl (Lys-boroPro) 2 , a homobivalent derivative of Lys-boroPro.
  • Fig. 4 is a graph showing a dose response curve observed with lower concentrations of KbP 2 -Adipate on anti-CD3 mAb stimulation of FI9 cells.
  • Fig. 5 is a graph showing a dose response curve observed with higher concentrations of KbP 2 -Adipate on anti-CD3 mAb stimulation of FI9 cells. Drug concentration is read as 10 X M.
  • Fig. 6 is a graph showing a dose response curve for anti-lF7.
  • Fig. 7 is a diagram showing intermolecular reactions that may occur al higher concentrations of KbP 2 -Adipate.
  • Fig. 8 is a diagram showing Lys-boroPro linked to Myelin Proteolipid Protein (PLP) Peptide 139-151.
  • Fig. 9 is a graph comparing the effects heterobivalent coupled KbP-S- MCC and uncoupled MCC 94-103 have on IL-2 production in 2B4 cells.
  • Fig. 10 is a diagram showing the structures of the open and cyclized forms of Xaa-boroProline inhibitors.
  • Figs. 1 1A-I ID are diagrams of different examples of bivalent compounds containing an olefin group.
  • Fig. 1 ID is a fluoroolefin isostere of Xaa- boroProline.
  • Fig. 12 is a graph illustrating the effect of (KbP) 2 -EGS at vaiying concentrations on IL-2 production by H9 cells.
  • Fig. 13 is the structure of EGS, the spacer-linker molecule used in the homodimer (KbP) 2 -EGS used in the experiments of Fig. 12.
  • Synthetic, low molecular weight, bivalent and multivalent crosslinking compounds with agonistic activity are designed and developed.
  • the molecule needs to be able to induce the association of receptors of a specific class in a manner similar to that induced by its natural ligand.
  • These agonistic molecules therefore need to be at least bidentate.
  • the individual binding units must be properly spaced for the desired association to occur.
  • homobivalent (homodimeric), homomultivalcnt and heterobivalent (heterodimeric) compounds of this invention represent a new class of biological modulators which can be used as therapeutic or diagnostic agents or both.
  • T cell surface receptors and their naturally occurring ligands arc used herein as examples to demonstrate how different homobivalent, homomultivalent, heteromultivalent, and heterobivalent synthetic crosslinking compounds function; these examples are therefore not intended to limit the invention.
  • the molecule, Xaa-boroPro which has been shown to have high affinity for the CD26 T cell surface receptor (See above discussion), can be a component molecule of the homobivalent, heteromultivalent, homomultivalent, or heterobivalent compounds outlined herein.
  • CD26 is a highly glycosylated type two transmembrane protein. It exists as a dimer with a subunit molecular weight of about 1 10 kDa.
  • the cDNAs encoding the human, mouse and rat proteins have been cloned and sequenced (D. Darmoul, et al., "Dipeptidyl peptidase IV (CD26) gene expression in enlcrocytc- like colon cancer cell lines FIT-29 and Caco-2: Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA levels during cell differentiation," J.Biol.Chem. 267, 4824-4833 (1992); D.
  • CD26 cDNA encodes a 766 amino acid polypeptide (having a molecular weight of 88,300) and the mouse cDNA encodes a 760 amino acid polypeptide (87,500 molecular weight).
  • the sequences of the mouse, rat and human CD26 share up to 98% homology.
  • Most of the CD26 molecule resides outside of the cell by being anchored to the cell plasma membrane through a 22 amino acid hydrophobic domain on the N terminus and only a small, six amino acid N terminal tail projects into the cytoplasm.
  • CD26 is found mainly on the surface of CD4+ T cells where it is believed to have important roles in T cell activation pathways (sec section on CD26 and T cell function below).
  • CD26 is also found on a small fraction of CD8+ cells. CD26 has been shown to be identical with an enzyme known as dipeptidyl peptidase amino peptidase Type IV (DP IV, sometimes also abbreviated DPP IV or DAP IV).
  • DP IV dipeptidyl peptidase amino peptidase Type IV
  • Enzymology of CD26 Active Site Structure and Inhibitor Design
  • the catalytic activity thus far identified for CD26 associated DP IV protease activity involves the cleaving of a dipeptide unit from the free amino terminus of polypeptides and proteins.
  • DP IV shows a strong preference for cleaving after a proline residue, i.e., a proline in the penultimate position from the amino terminus.
  • a free amino terminus appears to be a requirement but the enzyme displays little preference for any particular amino acid in this position (J.
  • DP IV can be regarded as a postproline cleaving aminopeptidase with a specificity for removing N-tcrminal Xaa-Pro dipeptides where Xaa can be any amino acid. Flowever, DP IV will also remove amino terminal Xaa-Ala dipeptides, although much less effectively.
  • the enzyme does, however, require that some amino acid be N terminal to proline because boroPro itself is not an effective inhibitor.
  • the P2 residue is probably required only for presenting a free amino group in the appropriate geometrical arrangement with respect to the proline bond to be cleaved because the addition of a group to the P2 N terminus, such as Ac, CBZ, or Fmoc, abolishes the inhibitory potency (G.R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991 )).
  • PI refers to the residue on Ihe N-tcrminal side of the sessile bond.
  • P2 refers to the residue on the N-terminal side of PI .
  • Abs are naturally bivalent, they are often able to mimic the natural ligand in inducing association of the receptor, e.g., an agonistic effect, which may be a dimerization or an aggregation. If an antibody fails to correctly mimic the natural-Iigand induced association, it will then often block the interaction with the natural ligand and therefore show an inhibitory or antagonistic activity.
  • CD26 is an association-activated co-stimulatory T cell receptor.
  • CD26 As an association-activated, co-stimulatory molecule has been recently confirmed in experiments in which the gene for CD26 was transfected into Jurkat T cell lines (T. Tanaka, et al., "Cloning and functional expression of the T cell activation antigen CD26," published erratum appears in J. Immunol., 150(5):2090 (Mar 1993); J.Immunol. 149, 481-486 (1992)).
  • CD26 has only a short six amino acid cytoplasmic tail. This argues against signal transduction via the cytosolic domain as is often the case for other cell surface receptors. CD26 may participate in T cell signal transduction through two hypothetical mechanisms: (1 ) through its association with other molecules in the membrane, and (2) through its DP IV protease activity.
  • bivalent or multivalent compounds or agents outlined herein involves essentially very similar chemistry. These bivalent and multivalent compounds are designed such that they induce associations between naturally occurring receptors, e.g., an association between a T cell surface CD26 receptor with itself (homobivalent) or an association between three T cell surface (CD26 receptors (homomullivalcnt, e.g., homotrivalent) or an association between CD26 receptor with the T cell receptor (TCR/CD3) or with the CD4 receptor (heterobivalent).
  • associations between naturally occurring receptors e.g., an association between a T cell surface CD26 receptor with itself (homobivalent) or an association between three T cell surface (CD26 receptors (homomullivalcnt, e.g., homotrivalent) or an association between CD26 receptor with the T cell receptor (TCR/CD3) or with the CD4 receptor (heterobivalent).
  • Flomomullivalcnt Compounds General Structure
  • the homobivalent and homomullivalcnt compounds taught herein can cither start with the general diagram for a bivalent template with non-identical binding moieties as shown in Fig. 1 A or with the general diagram for a homobivalent template as shown in Fig. IB.
  • both sides of the chemical structure as shown in Figs. 1A and IB are binding moieties.
  • Figs. 1C-1T are diagrams showing the structure of several preferred bivalent and multivalent compounds. Descriptions of the figures are provided above in the Brief Description of the Drawings.
  • Fig. 2A the general formula for a heterobivalent compound.
  • Figs. 2A-2C are diagrams showing the general formula of several preferred heterobivalent compounds:
  • Fig. 2 A is a diagram of a general heterobivalent template;
  • Fig. 2B is a diagram of a heterobivalent example coupling a binding moiety to an MCC peptide (94-103) using a compatible linker, e.g., an AAAAAA (SEQ ID NO: 21) linker group where A is L-alanine or D-alaninc; and
  • 2C is a diagram of a heterobivalent example coupling a binding moiety to a PLP peptide (139-151) using a compatible linker, e.g., an AAAAAA linker group where A is L-alanine or D-alanine.
  • a compatible linker e.g., an AAAAAA linker group where A is L-alanine or D-alanine.
  • peptide, polypeptide, or fragment thereof are used interchangeably and is defined to include a chain of amino acids ranging from about 3 to 50 residues, preferably from about 3 to 25 residues in length. More preferably, optimal size is on the order of about 10-18 amino acid residues in length.
  • Suitable antigenic peptides for any of the following autoimmune diseases, infectious diseases, allergic diseases, or cancers listed below and elsewhere in this document could be coupled to a bivalent template (see Fig. 2A), e.g., Xaa-boroPro, to form a heterobivalent compound (heterodimeric) molecule that could be used in this invention to treat that disease.
  • a bivalent template e.g., Xaa-boroPro
  • peptides with known specificity and high affinity for different T cell surface receptors.
  • These peptides (P) can be coupled to Xaa-boroPro or coupled to another binding moiety as shown in Fig. 2A to form different heterobivalent compounds exhibiting altered biological activity (increased or reduced as a result of the specific binding to the specific receptor.
  • TCR T cell Surface Receptor 3.
  • H-boroPro and Xaa/Lys-boroPro Homobivalent, homomultivalent, heterobivalent, and heteromultivalent compounds can begin with the synthesis of H-boroPro and LysboroPro as taught herein.
  • Use of H-boroPro and Lys-boroPro are for example purposes only, and is not intended to limit the scope of this invention.
  • Xaa/Lys-boroPro is an example of a molecule that can be used to form a binding moiety of a bivalent, homobivalent, homomultivalent, or heterobivalent compound as taught herein.
  • H-boroPro may be produced by a new procedure (Kelly, T.A., et al., "The efficient synthesis and simple resolution of a proline boronate ester suitable for enzyme inhibition studies," Tetrahedron 49, 1009-1016 (1993)). Both of these synthetic routes yield racemic H-boroPro pinanediol.
  • Stercochemically pure L, L and L, D diastereomcrs of Z-Lys-boroPro were prepared by first resolving racemic H-boroPro through crystallization with optically active blocking protecting groups ((IS, 2S, 3R, 5S)-+-pinanediol isomer) followed by coupling the isotopically pure L-boroPro and D-boroPro to the stercochemically pure L isomer of lysine (See United Stales Patent No. 5,462,928).
  • optically active blocking protecting groups (IS, 2S, 3R, 5S)-+-pinanediol isomer)
  • the L,L and L,D diastereomcrs of Lys-boroPro were prepared in high optical purity by coupling racemic H- boroPro by L-Lys and separating the resulting diastcrcomcric Z-Lys-boroPro-dicster into its component L,D and L,L diastereomers using reverse phase HPLC as previously described for diastereomeric Pro-boroPro (W.G. Gutheil and W.W. Bachovchin, "Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV.
  • these Xaa-boroPro compounds are coupled to other Xaa- boroPro compounds, e.g., itself, to form a homobivalent (homodimeric) or multivalent compound or coupled to a non-Xaa-boroPro peptide thereby forming a heterobivalent (heterodimeric) or multivalent compound.
  • Homobivalent, low molecular weight compounds capable of inducing an association, between two CD26 receptors arc taught in this Example.
  • Homobivalent synthetic crosslinking compounds have the properties normally associated with an antibody, i.e., high affinity, and specificity for CD26, and an ability to induce crosslinking.
  • any experiment with an anti-CD26 monoclonal antibody can be performed with one or more of the homobivalent compounds, e.g., immunoprecipitations.
  • the synthetic ligands have some properties that make them complimentary to anti-CD26 mAbs. These include: (i) their binding epitope is the DP IV active site; (ii) they exhibit cross-species specificity; and (iii) they offer flexibility in adjusting the spacing between the binding sites and the construction of chimeric or hclcrobidcntatc structure.
  • a series of different homobivalent derivatives of Lys-boroPro will be linked via their e-amino groups by a linker-spacer molecule containing two carboxylic acid groups, e.g., a six carbon iinker spacer or linker group, using conventional peptide coupling methods (see Fig. 3).
  • Fig. 3 is a diagram showing the synthesis of adipoyl (Lys-boroPro) 2 , a homobivalent derivative of Lys-boroPro.
  • This linkage method included coupling benzyloxycarbonyl- lysine-boroPro-diester (Z-lys-boroPro-diester) to a linker molecule, e.g., adipic acid or hexanedioic acid (HOOC(CH 2 ) 4 COOH).
  • the diester protecting group on the boronyl moiety can either be pinacol or pinanediol.
  • Dl is independently selected from the group consisting of NH and NH 2 , wherein N represents any isotope of nitrogen, wherein H represents an isotope of hydrogen; "-", independently, is selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from a group consisting of a C, a CS moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 are, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an 0, wherein X and Z, are, independently, selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope o oxygen; wherein each
  • Fig. 1 A The structure as shown above need not be identical, in that it can have the general structure as shown in Fig. 1 A:
  • the coupling reaction can be achieved by any of several standard peptide coupling methodologies.
  • the Lys-boroPro homobivalent (homodimeric) derivative was prepared by reacting protected Lys-boroPro in anhydrous THF with the acid chloride form of adipic acid, i.e., adipoyl chloride, which is commercially available (Aldrich Co.).
  • the N terminal Z protecting group was removed by catalytic hydrogenation.
  • Deprotection of the boronyl group was achieved by transesterification with phenyl boronic acid and extracted using a two phase, low pH water solution/organic solvent.
  • a of bivalent, dimeric Lys-boroPro derivatives with varying length spacer segments will be prepared (See discussion below on linker molecules).
  • linker molecules which have various internal heteroatoms and other functional groups, in addition to the terminal carboxylic groups, e.g., ethylcne glycolbissuccinate ("EGS", shown in Fig. 13).
  • EGS ethylcne glycolbissuccinate
  • using EGS in place of adipic acid provides a bivalent compound with a spacer of about twice the length of the adipoyl moiety.
  • the internal heteroatoms confer improved water solubility over a straight chain hydrocarbon of similar length.
  • Fig. 13 gives the structure of EGS, which was used as the linker molecule joining two KbP monomers, to form (KbP) 2 EGS.
  • the synthesis of this homodimer was carried out in a manner analogous to that described herein for KbP 2 adipate, using appropriate modifications.
  • EGS is commercially available from a variety of chemical supply companies.
  • H9 cells were preincubated with KbP 2 -Adipate for 0, 30, 60, or 150 min. After the preincubation, the cells were seeded into a 96 well flat bottom plate pre-coated with anti-CD3 monoclonal antibody OKT3 at 1 ,000, 5,000, 10,000, 20,000, 50,000, or 100,000 times dilution of stock. After 24 hr, the cells were lysed by freezing at 4°C.
  • IL-2 concentration in the H9 cells lysed was bioassayed using the IL-2 dependent cell line HT2, Watson, J.D. "Continuous proliferation of murine antigen specific helper T lymphocytes in culture," 1979, Journal of Experimental Medicine * . J 50: 1510. HT2 proliferation was measured by counting 3H-thymidine incorporation.
  • Figs. 4 and 5 are graphs showing a dose response curve observed with concentrations of KbP 2 -Adipate on anti-CD3 mAb stimulation of H9 cells, as manifested by 1L-2 production.
  • Fig. 12 shows the results of the experiments carried out with KbP 2 -EGS, in which the two KbP monomers are linked by the EGS spacer, which is on the order of twice the length of the adipate spacer used to make KbP 2 -Adipate homodimer.
  • the results of the experiments with KbP 2 -EGS exhibit a pattern analogous to the KbP 2 -Adipate results, as Fig. 12 illustrates.
  • D is, independently, selected from the group consisting of NH and NH 2 , wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "-" is, independently, selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from the group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 are, independently, selected from the group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Y
  • E 1 is attached to R' by a covalent bond which are together designated as El-R' or R'-El ; * .
  • E3 is attached to R by a covalent bond which are together designated as E3-R or R-E3;
  • R' represents the part of El-R' not undergoing a chemical reaction
  • R represents the part of R-E3 not undergoing a chemical reaction
  • El and E3 arc selected from the group consisting of a carboxylatc, amino, imidazole, sulfliydryl, aldehyde, ester, and any other reactive species; whcrcin [Jjp, E2, [Ijq and [G]m together arc a linker moiety, and wherein [G]m, [J]p, and [I]q represent, independently, linker molecules (i) having a molecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and where
  • X is any isotope of any atom capable of forming a single bond with carbon
  • H is any isotope of hydrogen
  • (d N is any isotope of nitrogen
  • Ph is any isotope of phosphorous
  • Y is any isotope of any atom capable of forming a single bond with phosphorous
  • (g) Z is any isotope of any atom capable of forming a single bond with phosphorous
  • U is any isotope of any atom capable of forming a double bond with phosphorous. Also, the figures shown below represent the binding moiety and R represents the remainder of the molecule in this polymeric compound:
  • the multivalent compound of this invention can range from a dimer, e.g., n is equal to one (1) or up to about 50-mcr, e.g., n is equal to forty-nine (49).
  • n is equal to one (1)
  • 50-mcr e.g., n is equal to forty-nine (49).
  • the binding moiety is repeated more than 2 times (a dimer) then the compound would necessarily be a "polymeric" compound with a finite number of repeating binding moieties.
  • heterobivalent also referred to as heterobidentate or heterodimeric
  • compounds or agents yields a class of agents capable of inducing an association between CD26 and distinct cell surface receptors.
  • heterobifunctional molecules have interesting biological activities and may be useful as drugs.
  • D is independently selected from the group consisting of NH and NH 2 , wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; " ⁇ ", independently, is selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from the group consisting of a C, a CX moiety, and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 arc, independently, selected from the group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl
  • P can be a peptide which selectively binds to CD26 but which does not include a reactive group for forming a covalent bond or complex vith the amino acids in the reactive center of the CD26.
  • a compound is considered a heterobivalent compound because only one reactive group is present.
  • Lys-boroPro is linked to the C terminal carboxylalc of enccphalitogcnic myelin proteolipid protein (PLP 139-151 ; Fig. 2C; sec below for discussion).
  • Lys- boroPro is linked to an antigenic moth cytochrome C peptide (MCC; Fig. 2B; see below for discussion). Both compounds were designed such that association between CD26 and another T cell receptor (TCR/CD3) would be induced.
  • TCR/CD3 T cell receptor
  • Peptide 139-151 to Induce Association Between CD26 Receptor and the T Cell Surface Receptor (TCR/CD3) Proteolipid protein (PLP) is the major protein of central nervous system myelin.
  • Kuchroo and co-workers have shown that mice immunized with a peptide corresponding to residues 139-151 of PLP, HSLGKWJLGHPJDKF (SEQ.ID.NO.22) (PLP 139- 151 ) develop acute experimental autoimmune encephalomyelitis (Kuchroo, V.K., et al., Induction of experimental allergic encephalomyelitis by myelin proteolipid-protein specific T cell clones and synthetic peptides, Pathobiology 59, 305-312 (1991); Kuchroo, V.K., et al., T-cell receptor alpha chain plays a critical role in antigen-specific suppressor eel!
  • T cell receptor (TCR) usage determines disease susceptibility in experimental autoimm ⁇ ue encephalomyelitis: studies with TCR V beta 8.2 transgenic mice, Journal of Experimental Medicine 179, 1659-1664 (1994); and Kuchroo, V.K., et al., A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediate by a diverse T cell repertoire, J. Immunol. 153, 3326-3336 (1994)).
  • PLP 139-151 also induces the proliferation of T cells in culture.
  • the mechanism involves the T cell receptor (TCR) recognition and binding of this peptide within the context of the major histocompatibility complex (MHC) class II.
  • MHC is a cluster of genes on human chromosome 6 or mouse chromosome 17 that encodes the MHC molecules.
  • the MHC class I molecules or proteins arc the present peptides generated in the cytosol to CD8 T cells.
  • the MHC class II molecules or proteins are the present peptides degraded in cellular vesicles to CD4 T cells.
  • the MHC is the most polymorphic gene cluster known to date in the human genome, having large numbers of allclcs al several different loci.
  • Ihe MHC proteins are often called "major histocompatibility antigens.” This allows easy manipulation of the antigenic peptide to convert it from an agonist to an antagonist (Jorgensen, J.L., et al., Molecular components of T-cell recognition, Anmi. Rev. Immunol. 10, 835-873 91992)).
  • Systematic amino acid replacement studies have demonstrated that Trp 144 and His 1 7, shown in bold in the sequence, are necessary for TCR binding while Leu 145 and Pro 148, shown underlined in the sequence above, are necessary for MHC binding.
  • Crystal structure data on the MHC class II receptor shows that the cleft on top of the molecule which binds the antigenic peptide is open on both sides, which allows longer pcplides to be presented by simply permitting them to extent away from the MHC receptor.
  • MHC class I receptors only accommodate short peptides from 9 to 12 amino acids and the antigenic peptide ends are not free.
  • TCR T cell surface receptor
  • CD26 CD26
  • Fig. 8 is a diagram showing Lys-boroPro linked to Myelin Proteolipid Protein (PLP) Peptide 139-151.
  • the heterodimer was constructed as HSLGKWLGHPDKF AAAAAA-eKbP (SEQ.ID.NO. 23 - eKbP) where HSLGKWLGHPDKF (SEQ.ID.NO. 22) was PLP 139-151 , AAAAAA (SEQ.ID.NO. 21) was a linker comprised of 6 alanincs and eKbP was Lysinc-boroProlinc in which the e- amino of Lysine is covalently attached to the -COOH terminus of
  • HSLGKWLGHPDKFAAAAAA (SEQ.ID.NO. 23).
  • the first synthetic step was to order a custom peptide from a synthetic peptide lab. Using long established protocols, the peptide was built from the C-terminus staring with alanine which was immobilized on a resin. Sequentially AAAAA FKPHGLWKGLSH (SEQ.ID.NO.25) were added using protected amino acids. The peptide was then removed from the resin to give a free -COOH terminus which could be reacted lo form a peptide bond. The other residues HSLGKWLGHPDKFAAAAA (SEQ.ID.NO. 24) were unreactive owing to protecting groups.
  • Lysine-boroProline in which the ⁇ -NH 2 of Lysine was protected, the B(OH) 2 of boro Prolinc was protected with pinanediol, and the e-NH 2 of Lysine was free was coupled to the peptide.
  • the spacer linker consisting of six consecutive Ala residues was chosen to provide a span sufficient to permit crosslinking ( ⁇ 3 ⁇ A).
  • the proliferative effect of PLP-S-KbP on several different T cell clones that recognize PLP 139-151 were tested. T cell clones that recognize an irrelevant epitope, were used as negative controls. The protocol used is described (Kuchroo, V.K., et al., B7- 1 and B7-2 Costimulatory Molecules Activate Differently the Thl Th2 Developmental Pathways: Application to Autoimmune Disease Therapy, Cell 80, 707-718 (1995)) and is incorporated herein by reference. Proliferation was measured by thymidine uptake.
  • Table 1 below compares the effect PLP-S-KbP, antigenic PLP 139-151 peptide and non- antigenic PLP 103-1 16 peptide has on proliferation in five different PLP 139-151 specific T cell clones.
  • the results show that PLP-S-kbP strongly enhances the proliferative response lo PLP 139-151 of all five 139-151 specific clones (see Table 1 below). Enhancement ranges from 100 to over 1000-fold, with respect to the concentration needed to produce a given response.
  • the Moth Cytochrome C (MCC) 94-103 peptide is another antigenic peptide employed in this invention.
  • MCC strongly induces IL-2 production and T cell proliferation when added to cultures of the murine 2B4 T cell hybridoma.
  • the critical residues for binding to the TCR receptor and to the class II MHC are known (Jorgensen, J.L., et al., Molecular components of T-cell recognition, Annu. Rev. Immunol. 10, 835-873 (1992)).
  • Lys- boroPro was coupled to the N terminus of this peptide using a spacer linker of about the same length as used to link Lys-boroPro to the PLP peptide. Standard coupling methodologies were employed. This molecule was designated KbP-S-MCC to signify coupling to the N terminus.
  • Fig. 9 compares the effect of the bivalent, dimeric KbP-S-MCC molecule and MCC 94-103 itself on IL-2 production in 2B4 cells.
  • the 2B4 T cell hybridoma proliferates in responses to moth cytochrome C peptide 94-103.
  • the peptide 2B4 system is ideal for these studies, since the contest points between MCC 94-103 and the TCR have been determined (Jorgensen, J.L., et al., "Molecular components of T-ccIl recognition," Annu. Rev. Immunol. 10, 835-873 (1992)).
  • 2B4 cells were cultured at 10 5 /well with H-2 k APC's and with varying concentrations of KbP-S-MCC and MCC itself. After 24 hours the supernatant was harvested and the IL-2 content determined in a bioassay using HT-2 indicator cells in the same manner as previously described for the PLP peptide assay.
  • Lys-boroPro a preferred binding moiety-reactive group
  • PLP peptides e.g., PLP peptides, MCC peptides
  • MCC peptides e.g., PLP peptides, MCC peptides
  • Lys-boroPro or an alternative
  • Other heterodimers comprising various derivatives of the PLP or the MCC peptide, which were previously shown to be antagonistic, will be prepared and studies will be performed to determine if linking these peptides to Lys-boroPro enhances their antagonistic activity or transforms them into an agonistic molecule.
  • tetanus toxoid peptide P2 is of interest because it provides a test system involving human peripheral blood mononuclear cells (PBMC) (Wyse-Coray, T, et al., "Use of antibody/peptides constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation," Cellular Immunology, 139(l):268-73, (1992)).
  • PBMC peripheral blood mononuclear cells
  • TCR/CD3 molecules capable of inducing the association between CD26 and the T cell surface receptor
  • TCR/CD3 can be prepared by coupling Lys-boroPro or other binding moiety to the C terminal or N terminal functional group of different peptides known to bind to the T cell receptor in the context of class II MHC receptors.
  • Lys-boroPro or other binding moiety to the C terminal or N terminal functional group of different peptides known to bind to the T cell receptor in the context of class II MHC receptors.
  • Similar protocols as those used to prepare [PLP 139-151]-KbP-S-MCC and KbP-S-MCC, both of which are described above and which involve straightforward standard peptide coupling methodology, can be employed.
  • Heterobivalent compounds containing Xaa-boroPro designed to induce association between the CD26 receptor and the CD4 receptor also can be prepared.
  • a molecule known to bind to CD4, e.g., peptides derived from the HIV-1 GP 120 protein, will be coupled to Xaa-boroPro (Ebcnbichler, C, et al., "Structure-function relationships of the HIV-1 envelope V3 loop tropism determinant: evidence for two distinct conformations," Aids 7, 639-46 (1993); Linsley, P.S., et al., "Effects of anti-gpl 20 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1," Journal of Virology 62, 3695-702 (1988)); Rini, J.M., et al., "Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen,"
  • An Xaa-boroPro molecule e.g., Lys-boroPro
  • Lys-boroPro will be coupled to one of the peptides from the HIV-1 GP 120 protein by using the coupling methodology as described above for coupling Lys-boroPro to the PLP peptide.
  • any of the other non-boronyl reactive groups described herein can be used in place of the boronyl group to obtain an alternative embodiment.
  • Mctcrobivalcnl compounds containing Xaa-boroPro designed to induce association between the CD26 receptor and other T cell surface receptors, e.g., granulocyte colony stimulating factor, can be prepared.
  • cytokine granulocyte colony stimulating factor (G-CSF or granulocyte macrophage colony stimulating factor, GM-CSF), is produced by T cells and macrophages and binds to its own receptor on the T cell surface (the granulocyte colony stimulating factor receptor).
  • G-CSF granulocyte macrophage colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • a bivalent, heterodimeric form of granulocyte colony stimulating factor could enhance the potency of granulocyte colony stimulating factor by stimulating growth or differentiation or both in cells of myelomonocytic lineage.
  • This compound can be prepared by using standard coupling methodology to couple granulocyte colony stimulating factor to Lys-boroPro.
  • different spacer linkers of varying sizes will be evaluated to determine the optimal length for inducing the association between CD26 receptor and the receptor for colony stimulating factor.
  • Stem cell factor (c-kit ligand) is essential in stem cell development and binds to the stem cell factor receptor on T cells.
  • CD44 binding probably has no direct signaling function, but instead promotes the binding of a receptor known as c-kit.
  • Lymphoid progenitor cells and early pro-B cells bind to hyaluronic acid on stromal cells via CD44, promoting the binding of their surface c-kit tyrosine kinase to stem cell factor (SCF) on the stromal cell surface, activating the kinase and inducing proliferation.
  • SCF stem cell factor
  • a bivalent, heterodimeric form of stem cell factor e.g., by coupling to Lys-boroPro, should enhance the potency of SCF.
  • Lys-boroPro-SCF heterodimeric compound
  • Different spacer linkers of varying sizes will be tested to determine the optimal length for inducing the association between CD26 receptor and the receptor for stem cell factor.
  • This example is designed to synthesize a bivalent compound linked to different peptides, whereby the peptides arc resistant to natural proteolysis, e.g., Xaa-boroPro coupled to "protected” PLP.
  • protected means the peptide has been synthesized in the reverse and has a change in chirality.
  • Retro-Inverso Isomers Evolution has ensured the almost exclusive occurrence of L-amino acids in naturally occurring proteins.
  • Virtually all proteases therefore cleave peptide bonds between adjacent L-amino acids; thus, artificial proteins or peptides composed of D-amino acids are largely resistant to proteolytic breakdown.
  • This resistance has been attractive to drug designers, but the exclusivity of biological systems for proteins made of L-amino acids means that such proteins cannot interact with the mirror image surface formed by enantiomeric proteins.
  • an all D-amino acid protein usually has no biological effect or activity.
  • Retroinverso peptides for incorporation in the compounds of the invention are prepared for peptides of known sequence in the following manner.
  • a peptide having a known sequence e.g., a tumor antigen peptide
  • the analog is synthesized using D-amino acids by attaching the amino acids in a peptide chain such that the sequence of amino acids in Ihe retroinverso peptide analog is exactly opposite of that in the selected peptide which serves as the model.
  • the peptide model is a tumor antigen such as the MAGE-1 peptide Al (formed of L-amino acids) having the sequence, ADPTGHSY
  • the retroinverso peptide analog of this tumor antigen formed of D-amino acids
  • the procedures for synthesizing a chain of D-amino acids to form the retroinverso peptides are known in the art and are illustrated in the above-noted references.
  • heterobivalent or heteromultivalent compounds of this invention will be prepared to include the "retro-inverso isomer" of the desired peptide. Protecting the peptide from natural proteolysis should therefore increase the effectiveness of the specific heterobivalent or heteromultivalent compound.
  • the biological activity of the retro-inverso heterodimer-Lys-boroPro linked to retroinverso PLP 139-151 can be compared to the control heterodimer-Lys-boroPro linked to conventional PLP 139-151. Activation of T cells can be monitored as described previously in Example 4, Section 1.
  • a higher biological activity is predicted for the retro-inverso containing heterodimer when compared to the non-retro-inverso containing analog owing to protection from degradation by native proteinases.
  • linker molecules capable of inducing the association of one cell surface CD26 receptor with another cell surface CD26 receptor or other rcccr3tor.
  • linker molecules A wide assortment of dicarboxylic spacer linker molecules are commercially available (see below for discussion). This includes linker molecules which have various internal heteroatoms and other functional groups, in addition to the terminal carboxylic groups, e.g., cthylene glycobissuccinatc.
  • linker molecules designed for constructing the homobivalent and heterobivalent compounds of this invention will fall within the general range of about 20 A to about 300 A; and more specifically, from about 30 A to about 100 A.
  • the spacer linker consisting of six consecutive Ala residues (as used above) was chosen because it was the smallest length spacer believed to be sufficient to crosslink (-30 A).
  • linker cthylene glycobissuccinatc (instead of adipic acid) provides a bivalent compound with a spacer of about twice the length of the adipoyl moiety. Also, the internal heteroatoms may confer improved water solubility over a straight chain hydrocarbon of similar length.
  • Chemical cross-linkers are valuable tools for scientists and are discussed in numerous books and catalogues, e.g., Pierce Catalog and Handbook, Rockford, HI. These reagents arc used to assist in the determination of near-neighbor relationships in proteins, three-dimensional structures of proteins, enzyme-substrate orientation, solid-phase immobilization, hapten-carrier protein conjugation and molecular associations in cell membranes. They are also useful for preparing antibody-enzyme conjugates, immunotoxins and other labeled protein reagents.
  • Conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits.
  • the number of components should be kept low or to a minimum.
  • cross-linking agents often employ functional groups that couple to amino acid side chains of peptides.
  • Bifunctional reagents are classified on the basis of the following: P. , Functional groups and chemical specificity
  • cross-linking groups are similar (homobifunctional) or different (hcterobifunctional)
  • the reagent can be radio-labeled or tagged with another label.
  • Reactive groups that can be targeted using a cross-linker include primary amines, suifhydryls, carbonyls, carbohydrates and carboxylic acids (to be discussed below).
  • any reactive group can be coupled nonselcctively using a cross-linker such as photorcactive phenyl azides.
  • Cross-linkers are available with varying lengths of spacer arms or bridges. These bridges connect the two reactive ends. The most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites for cross-linking, different lengths of bridges are required for the interaction. Normally, a cross-linker with a short spacer arm (4-8 A) is used and the degree of cross-linking is determined. If this is unsuccessful, a cross-linker with a longer spacer arm is used. Shorter spacer arms are often used in intramolecular cross-linking studies. Intermolecular cross-linking is favored with a crosslinker containing a longer space arm.
  • cross-linker-to-protcin molar ratios Many factors must be considered to determine optimum cross-linker-to-protcin molar ratios. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is normally desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal to ensure that the biological activity of the protein is retained. It is also important to consider the number of reactive groups on the surface of the protein. If there are numerous target groups, a lower cross-linkcr-to-protein ratio can be used. For a limited number of potential targets, a higher cross-linker-to-protein ratio may be required. This translates into more cross-linker per gram for a small molecular weight protein.
  • Conformational changes of proteins associated with a particular interaction may also be analyzed by performing cross-linking studies before and after the interaction. A comparison is made by using different arm-length cross-linkers and analyzing the success of conjugation.
  • the use of cross-linkers with different reactive groups and/or spacer arms may be desirable when the conformation of the protein changes such that hindered amino acids become available for cross-linking.
  • Conjugation reagents contain at least two reactive groups.
  • Homobifunctional cross- linkers can contain at least two identical reactive groups, and hetcrobifunctional reagents contain two or more different reactive groups. Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources.
  • Homobifunctional cross-linkers have at least two identical reactive groups and more often arc used in a one-step reaction procedure in which the compounds to be coupled are mixed, and the cross-linker is added to the solution. This cross-linking method may result in self-conjugation, intramolecular cross-linking and/or polymerization.
  • homobifunctional imidoesters There are two major types of homobifunctional imidoesters and homobifunctional N-hydroxysuccinimidyl (NHS) esters.
  • Commercially available homobifunctional imidoesters range in size from about 8 A to about 1 1.9 A.
  • Commercially available homobifunctional N-hydroxysuccinimidyl esters range in size from about 6.2 A to about 16.1 A.
  • Maleimides, alkyl and aryl halides, ⁇ -haloacyls and pyridyl disulfides are thiol reactive groups. These reagents react faster with sulfhydryls, making them thiol-selective.
  • Maleimides, alkyl and aryl halides, and ⁇ -haloacyls react with sulfhydryls to form thiol either bonds.
  • Pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable.
  • a nonselective homobifunctional is useful for conjugating functional groups, such as hydroxyls for which specific cross-linkers are not available.
  • nonselective homobifunctional cross-linker is BASED (Product #21564 Pierce Co.). This cross-linker has a long spacer arm and 2 aromatic rings which makes it very hydrophilic with a limited solubility in aqueous systems. This cross-linker also has a large diffusion capacity and should permeate membranes before conjugation initiates.
  • Heterobi functional Cross-linkers possess two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation.
  • Heterobifunctional reagents frequently are used when modification of amines is problematic. Amines may sometimes be found at the active sites of macromolecules, and the modification of these may lead to the loss of activity. Other moieties such as sulfhydryls, carboxyls, phenols and carbohydrates may be more appropriate targets.
  • a two-step strategy allows for the coupling of a protein that can tolerate the modification of its amines to a protein with other accessible groups. 5
  • a variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation are commercially available.
  • heterobifunctional cross-linkers contain an amine-reactive functional group.
  • Cross-linkers that are amine-reactive at one end and sulfhydryl-rcactivc at the other end arc quite common.
  • Commercially available heterofunctional cross-linkers range in size from about 0
  • heterobifunctional cross-linkers are sulfhydryl-reactive NHS-esters.
  • the sulfhydryl reactive groups are usually maleimides, pyridyl disulfides and ⁇ -haloacetyls.
  • Carbodifmidcs are carboxyl and aminc reactive.
  • Heterobifunctional reagents in which the reactivity can be controlled and that contain one group that is spontaneously non-reactive, have distinct advantages. This allows for specific attachment of the labile group first; the second reaction can then be initiated when appropriate.
  • a selection of heterobifunctional reagents that contain at least one 0 photoafilnity group are commercially available. This selection includes iodinatable and cleavable reagents that react nonspecifically at the azido group and with amines, sulfhydryls, carbohydrates and carbonyls. Often a bifunctional photoactivatable crosslinker has a better chance of forming a covalent cross-link than a bifunctional chemically reactive cross-linker.
  • Imidoester homobifunctional cross-linkers were among the first used to immobilize proteins onto solid-phase supports. They were used extensively for the study of protein structure and molecular associations in membranes. Although these cross-linkers are still used in protein subunit studies and solid-phase immobilization, they have been steadily replaced by the more stable, more efficient homobifunctional NHS-ester cross-linkers. Homobifunctional imidoesters maintain the net electronic charge on protein after crosslinking. Spacer arm lengths range from about 8.6 A to about 1 1.9 A. Imidoester cross- linkers react rapidly with amines at alkaline pH, but they have short half lives.
  • Imidoesters are also very useful for protein-protein cross-links. These cross-linkers can penetrate cell membranes and cross-link proteins within the membrane to study membrane composition, structure and protein-protein and protein-lipid interactions. Imidoesters are also useful for oligomer formation. For example, cross-linking proteins to form oligomers may reveal if a bivalent, dimeric or trimcric form of the protein is responsible for activity.
  • NHS-esters yield stable products upon reaction with primary or secondary amines. Coupling is efficient at physiological pH, NHS-ester cross-linkers arc also more stable in solution than their imidate counterparts. Homobifunctional NHS-ester conjugations are commonly used to cross-link amine-containing proteins in either one-step or two-step reactions.
  • Coupling through sulfhydryl groups is advantageous because it can be site-directed, yield cleavable products and allow for sequential coupling.
  • a protein in a complex mixture can be specifically labeled if it is the only one with a free sulfhydryl group on its surface.
  • the maleimide group is specific for sulfhydryl groups when the pH of the reaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reaction of the maleimides with sulfhydryls is 1000-fold faster than with amines. Maleimides do not react with tyrosines, histidines or methionines. b. Haloacetyls
  • the most common used ⁇ -Haloacetyls react with sulfhydryl groups at physiological pH.
  • the reaction of the iodoacetyl group with a sulfhydryl proceeds by nucleophilic substitution of iodine, with a thiol producing a stable thioether linkage.
  • Selectivity for sulfhydr l .groups is ensured by using only a slight excess of the iodoacetyl group over the number of sulfhydryl groups at pH 8.3.
  • iodoacetyl group can react with other amino acids.
  • Pyridyl Disulfides Pyridyl disulfides react with sulfhydryls groups to form a disulfide bond. Pyridine- 2-thionc is released as a by-product of this reaction. These reagents can be used as cross- linkers and to introduce sulfhydryl groups into proteins.
  • Carbodiimidcs Carbodiimides couple carboxyls to primary amines or hydrazides, resulting in formation of amide or hydrazone bonds. Carbodiimides are unlike other conjugation reactions in that no cross-bridge is formed between the molecules being coupled. Carboxy termini of proteins can be targeted, as well as glutamic and aspartic acid side chains. In the presence of excess cross-linker, polymerization is likely to occur because proteins contain carboxyls and amines. No cross-bridge is formed, and the amide bond is the same as a peptide bond, so reversal of the cross-linking is impossible without destruction of the protein. EDC (Pierce Co.) reacts with carbocyclic acid group and activates the carboxyl group, allowing it to be coupled to the amino group (R 4 NH 2 ) in the reaction mixture.
  • EDC Pulierce Co.
  • a photoaffinity reagent is a compound that is chemically inert but becomes reactive when exposed to ultraviolet or visible light.
  • Arylazides are photoaffinity reagents that are photolyzed at wavelengths between 250-460 nm, forming a reactive aryl nitrene. The aryl nitrcnc reacts nonsclcctivcly to form a covalent bond. Reducing agents must be used with caution because they can reduce the azido group.
  • Glyoxals are useful compounds for targeting the guanidinyl portion of arginine residues. Glyoxals will target arginines at mildly alkaline pH. There is some cross- reactivity (the greatest at higher pH) with lysines. b. Carbonyl Specific Cross-Linkers Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH 5-7.
  • Cross-linkers a. Cell Surface Cross-linking To ensure cell-surface specific cross-linking for identification of surface receptors or their ligands, it is best to use membrane-impermeable cross-linkers. In the past, researchers used water-insoluble cross-linkers and carefully controlled the amount of cross- linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and subsequent reaction with membrane proteins. Many references cite the use of membrane-permeable cross-linkers for cell surface cross-linking. b. Subunit Cross-linking and Protein Structural Studies Cross-linkers can be used to study the structure and composition of proteins in biological samples.
  • Some proteins are difficult to study because they exist in different conformations under varying pH or salt conditions.
  • One way to avoid conformational changes is to cross-link the subunits together.
  • Amine-, carboxyl- or sulfydryl-reactive reagents are employed for identification of particular amino acids or for the determination of the number, location and size of subunits in a protein.
  • Short-to-medium spacer arm cross-linkers are selected when intramolecular cross-linking is performed. If the spacer arm is too long, intermolecular cross-linking can occur.
  • Carbodiimides that result in no spacer arm, along with short length conjugating reagents are commercially available. c.
  • Cross-linkers are widely used for identification of near-neighbor protein relationships, ligand-receptor identification and interactions, and enzyme substrate orientations.
  • the cross-linkers chosen for these applications are usually longer than those used for subunit cross-linking.
  • Homobifunctional, amine-reactive NHS-esters or imidates and heterobifunctional, amine-reactive photoactivatable phenyl azides are the most commonly-used cross-linkers for these procedures.
  • a sulfhydryl- and amine- reactive cross-linker may be employed if one of the two proteins or molecules is known to contain sulfhydryls.
  • Cleavable or noncleavable cross-linkers typically are used.
  • the optimum length of the spacer arm of the cross-linker may be determined by the use of a panel of similar cross- linkers with different lengths.
  • NHS-ester, phenylazides are very useful for this type of cross-linking because they usually result in some successful, if not efficient, cross-linking.
  • Cross-linkers can be used to determine whether a particular protein is located on the surface or integral to the membrane. These studies are possible because water-soluble cross-linkers are membrane-impermeable, while water-insoluble cross-linkers are membrane permeable. d. Cell Membrane Structural Studies
  • Immunotoxins Specific antibodies can be covalently linked to toxic molecules and then used to target antigens on cells. Often these antibodies are specific for tumor associated antigens. Immunotoxins are brought into the cell by surface antigens and, once internalized, they proceed to kill the ceil by ribosome inactivation or other means. The type of cross-linker used to niaj c an immunotoxin can affect its ability to locate and kill the appropriate cells. For immunotoxins to be effective, the conjugate must be stable in vivo. In addition, once the immunotoxin reaches its target, it is important that the antibody be separable from the toxin to allow the toxin to kill the cell.
  • Thio-cleavable, disulfide-containing conjugates have been shown to be more cytotoxic to tumor cells than nonclcavable conjugates of ricin A immunotoxins. Cells are able to break the disulfide bond in the cross-linker, allowing the release of the toxin within the targeted cell.
  • heterobifunctional cross-linkers can also be used to make immunogen conjugates. Often peptides are synthesized with terminal cysteines to allow for their attachment to supports or to carrier proteins through a part of the molecule that is not important for activity or recognition. Suifhydryl-reactive, heterobifunctional cross-linkers can be coupled to carrier proteins through their other functional group and then can be linked to peptides through terminal cysteines. This method can be very efficient and yield an immunogen that is capable of eliciting a good response upon injection.
  • Proteins, peptides and other molecules can be immobilized on solid-phase matrices for use as affinity supports or for sample analysis.
  • the matrices may be agarose, beaded polymers*, polystyrene plates or balls, porous glass or glass slides, and nitrocellulose or other membrane materials.
  • Some supports can be activated for direct coupling to a ligand.
  • Other supports arc made with nuclcophilcs or other functional groups that can be linked to proteins or other ligands using cross-linkers.
  • Immobilization of the compounds of the invention to solid-supports can be accomplished using routine coupling chemistries.
  • the compounds of the invention arc immobilized by including in the compounds an accessible first functional group (e.g., an alcohol group) and contacting the compound with a solid-support containing a complementary second functional group (e.g., carboxyl groups) under conditions and for a period of time sufficient to permit the first and the second functional groups to react with one another to form a covalent bond (e.g., ester bond).
  • accessible in reference to a functional group, it is meant that the functional group is in a form which is reactive and is not stcrically precluded from reacting with the solid-support.
  • the functional groups for immobilizing the compounds of the invention to a solid- support can be introduced into the peptide binding moieties or the linker portions of these compounds.
  • amino acids that include functional groups in their side chains e.g., aspartale, glutamatc, cystcine residues
  • the compounds of the invention can be immobilized via a functional group in the linker molecule to a solid- support.
  • the linkers which arc used in this aspect of the invention can include, in addition to the first and the second linker reactive groups for binding to the first and the second peptide binding moieties, a further functional group for binding to the solid support.
  • Trifunctional molecules of this type are illustrated in Example 1 1.
  • the linker reactive groups that are used for coupling the linker molecule to the peptide binding moieties be different from functional groups that are used for coupling the linker to the solid-support.
  • Such functional groups can be introduced into the linker molecules at any time during or after the synthesis of these molecules.
  • Xaa-boroPro analogs containing an olefin group e.g., a fluoroolefin will be constructed to prevent cyclization and increase biological activity.
  • the biological activity of his olefin containing compound will be tested by the methods described previously in Example 2, section III, e.g., comparing the production of IL-2 using an olefin containing compound versus using a compound that does not contain an olefin.
  • the inhibitors exist as a slowly equilibrating mixture of two conformations: an open chain structure which is inhibitory (active species), and a cyclic structure which is non-inhibitory (inactive species).
  • Fig. 10 which is a diagram showing the structures of the open and cyclizcd forms of Xaa-boroPro inhibitors (conformational equilibrium of Xaa-boroProline inhibitors).
  • the open, active, inhibitory chain species is favored at low pH while the cyclized structure is favored at high pH.
  • the reaction is fully reversible: the open chain becomes predominate at low pH.
  • the open chain to cyclic species reaction involves a trans to cis iso erization of the proline and the formation of a new N-B bond.
  • the cyclized structure is the boron analog of a di et ⁇ pipcrazinc, a product often seen in peptide chemistry. Cyclization liberates one equivalent of H+ thereby explaining the requirement for base in the cyclization reaction and acid in the opening reaction.
  • the cyclic structure is quite stable in aqueous solutions of high pH.
  • fluoroolefin peptide isostercs have been used as tools for controlling peptide conformations (Boros, L.G., et al Fluoroolefin Peptide Isostcres - Tools for Controlling Peptide Conformations, Tetrahedron Letters, 35(33), 6033-6036 (1994)). Fluoroolefin containing dipeptide isosteres have also been shown to be effective inhibitors of dipeptidyl peptidase IV (CD26) (Welch, J.T. et al Fluoroolefin Containing Dipeptide Isostercs as Inhibitors of Dipeptidyl Peptidase IV (CD26), Tetrahedron, 52(1), 291-304 (1995)).
  • Multivalent Compound The inventors predict that biological bioavailability (biological function) for the compounds taught in this invention could be significantly increased (approximately 100- 1000 times) by preventing peptide conformational changes, e.g., intermolecular cyclization, by constructing a bivalent or multivalent compound containing an olefin group (a carbon atom double bonded to another carbon atom; see Fig. 1 ID), e.g., fluoroolefin.
  • Figs. 1 1A- 1 ID are diagrams of different examples of bivalent compounds containing an olefin group and are not meant to limit the scope of the invention.
  • Fig. 1 ID is a fluoroolefin isostere of Xaa-boroProline.
  • a fluoroolefin mimics a peptide bond but prevents cis trans isomerization and therefore prevents cyclization. Thus, if cyclization can be blocked, the inventors*prcdict that the bioavailability of the compounds taught herein can be increased by approximately 100 - 1000 fold.
  • any of the compounds of the invention can be designed and constructed to incorporate an olefin group using these procedures. Accordingly, it will be appreciated that the procedures can be used to prepare olcfin-containing boronyl compounds of the invention in which the olefin is unsubstituted or substituted with a halogen other than fluorine. Further, it will be appreciated that although a compound having a boronyl reactive group is illustrated, a reactive group other than a boronyl group can be prepared by using an analog of compound (a) in which an alternative reactive group is substituted for BC1 3 in the reaction.
  • Thc fluoroolefin analogs of Xaa-boroPros will be compared to Xaa-boroPros by measuring the potency of inhibition of CD26 proteinase activity.
  • the immunodulatory effects of these fluoroolefin containing analogs will be evaluated by in vivo experiments using animal models and in vitro experiments using cell culture.
  • the cell culture experiments will monitor cytokine production by cells of lymphoid origin, proliferation of cells of lymphoid origin, or both.
  • Fiuorescently labelled derivatives of monomeric LysboroPro can be prepared and used to determine if monomeric Lys-boroPro induces internalization of CD26 into cells. Fluorescence microscopy can be used to monitor intercellular trafficking of CD26.
  • One labelling approach is directly couple the side chain amino group of monomeric Z-Lys-bordPro-diester to a functional group on a fluorescent molecule such as the isothiocyanate of fluorescein isothiocynate (FITC). Lys-boroPro can be linked to FITC either directly or through a spacer linker molecule.
  • FITC fluorescein isothiocynate
  • Another labelling approach is to link the side chain amino group of Lys-boroPro to biotin, again either directly or through a spacer and then use the avidin, strcpavidinbiotin systems for detection.
  • Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. This protein contains four identical subunits having a combined molecular mass of 67,000-68,000 daltons. Each subunit binds one molecule of biotin, and studies have shown that tryptophan and lysine arc involved in the binding site for biotin. The sequencing of the subunit indicates it consists of 128 amino acids.
  • Avidin has an isoelectric point of 10- 10.5 and is very soluble in water and salt solutions. Avidin is stable over a wide range of pMs and temperatures. Extensive chemical modification has little effect on the activity of avidin, making it useful for detection and protein purification.
  • Streptavidin is another biotin-binding protein, and it is isolated from Sireptomyces avidinii. The molecular weight of streptavidin is about 60,000. Unlike avidin, streptavidin has no carbohydrate and has an acidic isoelectric point of 5. Streptavidin is much less soluble in water than avidin and can be crystallized from water or 505 isopropanol.
  • biotin-avidin system is well developed, well described and has been successfully utilized for the type of work proposed herein in many other systems; (2) improved flexibility because a large number of reagents are commercially available for use in this system: (3) improved sensitivity because this system provides for amplification of the signal from the biotinylated moiety (e.g., the biotinylated Lys-boroPro can be rendered fluorescent by reaction with either avidin (or streptavidin) conjugated either to a fluorochro e such as FITC or conjugated to an enzyme such as horseradish peroxidase).
  • avidin-FITC conjugate has many FITC groups per avidin molecule compared to only one of the first approach described above.
  • the avidin-enzyme conjugate approach provides a large amplification owing to the many molecules of substrate converted to detectable product per avidin molecule.
  • N jydroxysuccinmide biotin (NHS-biotin, available from Pierce Company) reacts readily with free primary amines to form a biotin conjugate linked with a peptide bond.
  • NHS-biotin, and NHS-LC-biotin (which has a 22.4 A spacer arm to separate biotin from the amino group of the molecule to which it is to be conjugated) is linked to the side chain amino group of Lysine of Lys-boroPro, and these molecules are characterized as to their ability to inhibit DP IV and their ability to serve as a detection tool for Lys-boroPro- protein complexes using the avidin detection systems.
  • Each of the compounds prepared as described above can be purified to homogeneity using HPLC and its identity can be confirmed by NMR spectroscopy, amino acid composition, or mass .spectroscopy as deemed necessary.
  • Fluorescently labelled dimeric and multimeric derivatives containing preferred P'R 1 monomers can be prepared and used to determine if bivalent and trivalent derivatives of, e.g., Lys-boroPro, can induce aggregation and internalization of cells surface CD26.
  • Producing such molecules can be accomplished in several ways.
  • One way is to use a trifunctional molecule of the type illustrated below.
  • the carboxy late groups can be coupled to Lys-boroPro molecules while the amino group can be coupled to an FITC molecule, or other fiuorophore, or more preferably, to biotin via a suitable length spacer.
  • Trifunctional molecules of the type shown below are available or can be synthesized relatively easily.
  • Trimeric forms of Lys-boroPro also are possible through straightforward extension of the approach described above for the bivalent forms.
  • Another approach takes advantage of the multjmeric nature of the biotin-avidin system.
  • Avidin has four subunits, each of which has a biotin binding site for biotin. If fluorescent trimeric inliibitors are needed, biotin can be coupled to Lys-boroPro through a suitable length spacer (the NHS-LC-biotin should have an adequate length spacer arm 22.4 A).
  • the effect of various amounts of avidin, both on T cell activation and on cell surface CD26 aggregation can be determined using FITC-conjugated avidin. This approach likely yields a mixture of divalent, trivalent, and tetravalent inliibitors.
  • the compounds prepared as described above can be purified by HPLC and their structure can be confirmed by NMR spectroscopy, amino acid composition, or mass spectroscopy as necessary.
  • Lys-BoroPro linked to insoluble supports may be useful for three reasons.
  • the first is for determining the effect of such solid phase immobilization of Lys-boroPro on T cell proliferation for comparison with analogous experiments with solid phase immobilized anti-CD26 mAbs.
  • Solid phase immobilized Lys-boroPro derivatives should induce aggregation of cell surface CD26, but differ from soluble, multimeric inhibitors in that they should prevent internal ization, or at least internal ization of inhibitor bound CD26.
  • the second use is for determining if Lys-boroPro binds to proteins other than CD26 to an appreciable extent.
  • the third use is to produce an affinity column for producing purified CD26 from various sources.
  • the bi and multivalent compounds of the invention can be immobilized on solid supports in many ways, each with certain advantages.
  • solid state immobilized avidin together with biotinylated Lys- boroPro can be tested because this approach offers substantial flexibility.
  • Solid state immobilized avidin, e.g., linked to agarosc is commercially available (Pierce Chemical Co.) with avidin and can be obtained in both multimeric and in monomeric forms.
  • the monomeric form is designed to allow for the removal and recovery of biotinylated proteins from the resin and therefore may be preferred for the second and third purposes described above.
  • biotinylated Lys-boroPro protein conjugates there are, however, other ways to provide for the removal and recovery of biotinylated Lys-boroPro protein conjugates.
  • high concentration of biotin may compete with and displace the biotinylated Lys-boroPro from the solid state immobilized avidin
  • free Lys-boroPro may displace the biotinylated inhibitor from the proteins
  • a biotin derivative with a cleavable group in the spacer arm can be used in preparing the biotinylated Lys-boroPro
  • lowering the pH to -4.0 will dramatically lower the affinity of Lys-boroPro for the active site of DP IV and thus should allow elution of the protein from the resin leaving behind biotinylated Lys-boroPro attached to avidin (G.R.
  • Diols such as pinancdiol or pinacol
  • elution buffer can be added to the elution buffer to bind to and thus tie up the hydroxyl groups on the boronyl group.
  • Each of the compounds prepared as described above can be purified to homogeneity using HPLC and its identity can be confirmed by NMR spectroscopy, amino acid composition, or mass spectroscopy as deemed necessary.
  • Assays to measure CD26 (DP IV) activity can be performed on both the homobivalent compounds, e.g., Lys-boroPro coupled to another, and the heterobivalent compounds, e.g., Lys-boroPro coupled to a peptide specific for a T cell surface receptor, e.g., moth cytochrome C peptide.
  • Methods for quantitatively measuring the interaction of small peptidomimctic inhibitors with CD26 or DP IV, as well as for the interaction of CD26 with larger ligands, e.g., the HIV Tat protein have been developed (W.G. Gutheil and W.W. Bachovchin.
  • bivalent Lys-boroPro molecules designed to induce association of cell surface CD26 can be measured in an antigen specific T cell response as previously described (G.R. Flentkc, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inliibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991); M. Subramanyam, et al., “Mechanism of IIIV-1 Tatjnduced inhibition of antigen-specific T cell responsiveness," J. Immunol. 150, 2544-2553 (1993)).
  • peripheral blood mononuclear cells are cultured with suboptimal doses of anti-CD3 mAbs, in the presence or absence of the CD26 associating agents.
  • a recall antigen response is measured to a suboptimal concentration of tetanus toxoid or Candida antigen, plus or minus the CD26 associating agents (M. Subramanyam, et al., "Mechanism of HIV- 1 Tat Induced inhibition of antigen- specific T cell responsiveness," J. Immunol. 150, 2544-2553 (1993)).
  • the cytochrome C system is used for measuring the response in the 2B4 T cell hybridoma (G.R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991)).
  • Cellular activation is measured by the production of IL-2 by the T cell hybridoma, and the level of IL-2 produced is quantified in a biological assay, using HT-2 indicator cells.
  • Receptor internalization can be determined by employing FITC labelled bivalent Lys-boroPro and performing flow cytometric analysis (N.H. Dang, Y. Torimoto, K. Sugita, J.F. Daley, P. Schow, C. Prado, S.F. Sehlossman, and C. Morimoto. Cell surface modulation of CD26 by anti-lF7 monoclonal antibody: Analysis of surface expression and human T cell activation, Journal of Immunology 145, 3963-3971 (1990)).
  • the cells are analyzed (i) with intact membranes, allowing only cell surface staining, and (ii) after permeabilization of the membrane with saponin, which allows the antibody to cross the membrane, although the membrane stays structurally intact (de Caestecker, M.P., Telfer, B.A., Hutchinson, I.V., and Ballardie, F.W., "The detection of intcrcytoplasmic interlcukin l et, interleukin l ⁇ and tumour necrosis factor ⁇ expression in human monocytes using two colour immunofluorescence flow cytometry," J. Immunol. Methods 4, 1 1 -20 ( 1992).
  • CD26 molecule becomes internalized after incubation with a bivalent or multivalent derivative of Lys-boroPro
  • a larger molecule is coupled to the bivalent molecule, preferentially an enzyme that can be easily detected, e.g., luciferase, alkaline phosphate or ⁇ -galactosidase. Expression of these proteins can be measured using a cellular extract. Kits are commercially available for this determination (e.g., Novacasatra Laboratories Ltd., Newcastle upon Tyne, UK).
  • mAbs are commercially available (e.g., Southern Biotech, Birmingham Alabama) for these enzymes, enabling detection of protein expression in single cells by flow cytometry.
  • saponin can be used to permeabilize the cells, enabling entry of the mAbs. This method is very sensitive and allows the simultaneous analysis of other cell surface or cytoplasmic proteins.
  • the invention provides a number of uses and advantages.
  • the low molecular weight, synthetic bivalent or multivalent compounds of the invention arc designed to induce the association between association-activated receptors and, therefore, offer considerable potential for regulating biological systems.
  • this new class of bioiogically active compounds, or compositions thereof are useful for inducing the association between association-activated receptors on human T cells and for the treatment of a wide variety of T-cell mediated diseases in animals; for example, autoimmune disease.
  • these bivalent or multivalent homo- or hetero-compounds or compositions thereof are useful as immune response modulating therapeutics (1) to treat disease conditions characterized by immunosuppression, e.g., AIDS or AIDS-related complex, other virally or environmentally induced conditions and certain congenital immune deficiencies; (2) to increase an immune function which has been impaired by the use of i munosuppressive drugs, (3) to treat systemic lupus erythematosis, rheumatoid arthritis, and multiple sclerosis.
  • the compounds of this invention can be used to deliver a member of the superantigen family to stimulate T cells.
  • Superantigens comprise a class of disease- associated, immunostimulatory molecules that bind class II MHC molecules and stimulate large numbers of T cells (Jardetzky, T.S., et al. "Threee Dimensional Structure of a Human Class II I- istocompatiblity Molecule Complexd with Superantigen", Nature 368, 711-718 (April, 1994)).
  • Members of the superantigen family include toxins from S. aureus and other bacteria, as well as viral superantigens from mouse mammary tumor virus.
  • the toxicity of the bacterial superantigens is thought to be mediated by their potent T-cell stimulating activities, leading to lymphokine release, respiratory distress and shock.
  • Superantigens have also been implicated in rabies, rheumatoid arthiritis, and mouse and human AIDS.
  • bivalent or multivalent homo- or hetero-compounds or compositions thereof can be used to stimulate the growth of hemapopoietic cells in culture.
  • Such cells can be transplanted into mammals, e.g., humans, to stcngthen or boost the hematopoietic, immune system, or both.
  • These compounds also can be used to treat patients suffering from disease or from deficiency of hematopoietic cells such as AIDS patients, patients undergoing chemotherapy and/or patients under radiotherapy for hcmatological or other cancers, and patients undergoing bone marrow transplants.
  • the compounds of the invention When administered to mammals, e.g., humans, the compounds of the invention may enhance the ability of the immune system to regenerate cells that arc immunosuppressed, e.g., CD4 and T cells.
  • the bivalent compounds of this invention can be administered to mammals, e.g., humans, in an effective amount alone or in combination with a pharmaceutically acceptable carrier, excipient, or diluent, in unit dosage form.
  • a pharmaceutically acceptable carrier, excipient, or diluent in unit dosage form.
  • Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions to administer these compounds to patients suffering from immunosuppression or an immune deficiency or presymptomatic of AIDS.
  • Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intrcapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene polyoxyproplyene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for these bivalent compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems or iiposomes.
  • Formulations of inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxethylene-9-lauryl ether, glycocholatc and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients for example, lactose
  • aqueous solutions containing, for example, polyoxethylene-9-lauryl ether, glycocholatc and deoxycholate
  • bivalent homo- or heterbivalent compounds or compositions thereof can be used to control the T cell activation process and thus be used to prevent unwanted immune responses.
  • the homobivalent compounds designed to induce the association between one CD26 T cell receptor and another CD26 T cell receptor can be useful in stimulating the proliferation of CD4+ cells. These compounds could, therefore, help restore CD4+ cells numbers in immunosuppressed patients, e.g., AIDS patients, and thereby reverse the decline in immune function.
  • the homobivalent compounds or compositions thereof (CD26 T cell receptor associated with another CD26 T cell receptor) of the invention can be used to enhance recall-antigen specific immune responses. Lymphocytes from most HIV-infcctcd individuals exhibit a qualitative defect in their ability to respond to recall antigens (A.S. Fauci. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 617-722 (1988)).
  • the stimulatory activities should improve lymphocyte function in HIV infected individuals by ameliorating the defective recall antigen responses which show up early after infection, and by improving CD4+T cell numbers.
  • the compounds of the invention and, in particular, the compounds of claim 1 have high affinity and specificity for CD26, they can be useful for the selective delivery of other therapeutic agents to, and into CD4+ T cells.
  • the compounds of the invention can be used to deliver pharmacological agents inside CD4+ cells that normally are unable to penetrate such cells.
  • many highly potent inhibitors of the HIV protease have been developed, which despite their high affinities for the HIV proteinase, are limited in blocking HIV in vivo owing to their inability to get inside the CD4+ cells.
  • These HIV proteinases hibitors inhibitors can be linked to the spacer arm of a bivalent CD26 ligand and delivered into CD26+ lymphocytes, which are the cells the virus primarily invades.
  • the compounds of the invention can be used to concentrate the drug in CD26 cells, thereby maximizing the desired pharmacological activity while minimizing unwanted toxic side effects on other cells.
  • This delivery vehicle therefore provides a mechanism to prevent or reduce AZT resistance by providing higher concentrations of AZT in CD4+ cells and lower concentrations elsewhere.
  • the CD26 internalization activity of the compounds disclosed herein can be used to provide a vehicle for delivering and concentrating other therapeutic agents into CD4+T cells.
  • heterobivalent compounds designed to induce the association between a CD26 T cell receptor and a different T cell receptor can be useful in stimulating a cell-mediated immune response against specific antigens.
  • Heterobivalent compounds or compositions thereof comprising a CD26 inhibitor and an antigenic peptide stimulate a cellular immune response against an antigenic peptide as compared to an antibody-mediated immune response.
  • This stimulation of cell-mediated response against specific antigens can be useful in patients with AIDS because these patients have high concentrations of anti-HIV antibodies.
  • the CD26-TCR association-inducing activity can be useful for stimulating a cell mediated immune response to specific antigens.
  • This biological activity should prove especially useful in vaccine development, particularly for AIDS because a cellular or T culinary response is the appropriate response for HIV-1 and is apparently lacking in AIDS patients.
  • heterobivalent compounds can be useful in the development and manufacture of peptide-based vaccines and therapeutic agents for the treatment of allergies and autoimmune diseases.
  • bifunctional agents can be useful in the development of vaccines for modulating a cell mediated immune response, especially for the development of a vaccine for treating AIDS.
  • the heterobivalent compounds designed to induce the association between a CD26 T cell receptor and a different T cell receptor, e.g., CD4, can be useful in activating T cell function.
  • This heterobivalent compound or compositions thereof can be used to selectively deliver agents to CD4+ CD26+ cells, or they can be useful in blocking HIV entry into CD4+ cells.
  • the heterobivalent compounds or compositions thereof can be used to deliver a toxin, e.g., ricin A immunotoxin or AZT, within a CD26+ T cell.
  • a toxin of choice can be coupled to a dimeric compound having high affinity for the CD26 T cell surface receptor.
  • a toxin can be coupled to a peptide having specificity for a specific T cell receptor. Either way, once the dimeric compound associates or binds to the CD26 T cell surface receptor, the toxin is internalized within the T cell, thereby delivering this toxin to a T cell.
  • the compounds or compositions thereof can be administered alone or in combination with one another, or in combination with other therapeutic agents.
  • treatment with one of the bivalent compounds can be combined with more traditional therapies for treating disorders of the immune.
  • the pharmaceutical preparations of the invention are applied in pharmaceutical ly-acceptable amounts and in pharmaceutically-acceptably compositions.
  • Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutical ly-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • the pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimcrosal.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the crosslinking compound, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
  • acceptable vehicles and solvents that may be employed arc water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • a variety of administration routes are available for treating a subject.
  • the particular mode of delivery selected will depend, of course, upon the particular crosslinking compound selected, the severity of the condition being treated and the dosage required for therapeutic efficacy.
  • the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes.
  • Such modes of administration also include obtaining T cells or bone marrow cells, stem cells or early lineage progenitor cells from a patient and contacting the isolated cells with the crosslinking compounds of the invention ex vivo, followed by reintrodUcing the treated cells to the patient.
  • the treated cells can be reintroduced to the patient in any manner known in the art for administering viable cells.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic and other treatment because of the convenience to the patient as well as the dosing schedule.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
  • the methods include the step of bringing the crosslinking compounds of the invention into association with a carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing the crosslinking compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the crosslinking compound of the invention.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the crosslinking compounds described above, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactoncs, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S.
  • Patent 5,075, 10 Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • peptide based systems such as mono- di- and tri-glycerides
  • wax coatings such as those described in U.S. Patent
  • a long-term sustained release implant may be particularly suitable for treatment of chronic conditions.
  • Long-term release as used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 10 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the crosslinking compounds described herein are administered in effective amounts.
  • An effective amount is a dosage of the crosslinking compound sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • an effective amount for stimulating T cell activation would be an amount sufficient to increase to a statistically significant extent T cell activation as for example, measured by proliferation or by increased T cell activity.
  • An effective amount for stimulating a desired immune response also can be measured, for example, by determining a change in the immune function in a subject (e.g., increased B cell response, increased cytotoxic T cell response, stimulation of bone marrow proliferation, or an ability to slow, halt, or prevent an infection or cancer).
  • An effective amount for treating an autoimmune disorder or allergic disorder would be that amount sufficient to lessen or inhibit altogether the immune or allergic response associated with the disorder so as to slow or halt the development of or the progression of the disorder.
  • the crosslinking compounds of the invention can be used to treat an autoimmune disorder (e.g., transplant rejection) prophylactically in subjects at risk of developing an immune response (e.g., recipients prior to transplant).
  • an effective amount for treating an immune system disorder is that amount which can slow or halt altogether the symptoms associated with the immune system disorder so as to prevent the disorder, slow its progression, or halt the progression of the immune system disorder. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • doses of active compounds will be from about 0.001 mg/kg per day to 1000 mg/kg per day. It is expected that doses range of 0.01 to 100 mg/kg will be suitable, preferably orally and in one or several administrations per day. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • ADDRESSEE Wolf, Greenfield & Sacks, P.C.

Abstract

Synthetic cross-linking homobivalent and heterobivalent compounds have been designed and developed. These compounds are low in molecular weight, have antagonistic or agonistic activity, and induce the association between two identical or similar natural receptors (homobivalent compounds) or induce the association between two different natural receptors (heterobivalent compounds).

Description

MULTIVALENT COMPOUNDS FOR CROSSLINKING RECEPTORS AND USES THEREOF
Cross Reference To Related Applications
This application is a continuation-in-part of Application Serial No. 08/837,305, filed April 1 1 , 1997, which is a continuation-in-part of Application Serial No. 08/671 ,756, filed June 28, 1996.
Statement as to Federally Sponsored Research
This invention was made, in part, with U.S. Government support under Grant No. 5-R01-AI31866 awarded by the National Institute of Allergy and Infectious Diseases.
The Field of the Invention This invention relates to low molecular weight bivalent and multivalent crosslinking compounds capable of inducing association between naturally occurring receptors, and particularly to inducing association between surface receptors on T cells, hematopoietic cells or antigen presenting cells such as macrophages, dendritic cells or B cells, e.g., homobivalent induced CD26-CD26 association or heterobivalent induced CD26 association with the above-mentioned surface receptors.
Background of the Invention Cell surface receptors transmit signals received on the outside of a cell to the inside through two basic mechanisms: (1) ligand-induced allosteric conformational change and (2) ligand-induced association.
The ligands for the ligand-induced, allosteric conformational change mechanism are typically small molecules, such as the catecholamines or the neuropeptide hormones.
The ligand-induced association mechanism involves an association of specific proteins on the cell surface and has only recently been discovered (relatively speaking), but already has been shown to be as widely used and as important as the first mechanism. Receptors activated by a ligand-induced dimerization include, for example, those for cell growth and differentiation factors. Factors which serve as ligands for these receptors are typically large polypeptide hormone and cytokines such as erythropoietin, granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating factor (GM-CSF), and human growth hormone (hGH). Many of the dimerization-activated receptors have cytoplasmic tails that contain protein kinase domains or docking sites. Ligand-induced dimerization of the extracellular domains of these receptors results in the juxtaposition of their cytoplasmic tails. They then presumably phosphorylate each other in trans and thereby initiate the cytosolic signaling pathway. In some cases the cytoplasmic domains of dimerization-activated receptors do not have kinase domains themselves, but function the same as if they did because they associate with protein kinases via docking sites.
* * Receptors activated by oligomerization or aggregation are found most frequently in the immune system. They include, for example, the T cell surface receptors such as CD2, CD4, CD8, CD28, CD26, CD44, CD45, CD 10, and CD3/TCR (T cell antigen receptor) and the B cell surface receptors such as CD40, B7.1 and B7.2. The ligands for these cell receplors arc most often cell surface proteins themselves, and can be found on cognate cells. Aggregation-activated receptors frequently have short cytoplasmic domains which act to bind and thereby recruit other cell surface and/or cytosolic factors following the aggregation of their extracellular domains.
The allosterically activated receptor class has been the primary focus of drug discovery, design, and development efforts for decades. These efforts have yielded many pharmacologically agents. In principle, two distinct types of agents are possible: antagonists and agonists. Antagonists block the binding of the natural ligand without inducing the conformational change in the receptor thereby blocking a signal transduction pathway. Agonists bind to the receptor in a manner which mimics the natural ligand closely enough to induce the same conformational change as natural ligand thereby initiating a signal transduction pathway. See Seed, et al. for a theoretical discussion on how to make an agonist from an antagonist (Seed, B., Making Agonists of Antagonists, Chemistry & Biology 1 : 125 (1994). See Austin, et al. for a discussion of the role of regulated protein dimerization in biology (Austin, et al. Chemistry & Biology 1 :131 (1994)).
Several association-activation receptors have recently become the targets of drug discovery efforts, owing to the important roles they play in various cellular signaling. Low-molecular weight synthetic molecules, that block the interaction of receptors and their ligands and interfere with signal transduction (i.e., antagonists), have been identified using the methods employed with the allosterically activated class. These low-molecular weight synthetic molecules are potential drugs.
Monomeric inhibitors block recall antigen-induced T cell activation and proliferation (G.R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP- IV) by Xaα-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991)). A number of anti- CD26 mAbs have the same inhibitory activity when used under non-crosslinking conditions (C. Morimoto, et al. 1F7, a novel cell surface molecule, involved in helper function of CD4 cells, Journal of Immunology 143, 3430-3439 (1989) and published erratum appears in J. Immunology. 144(5):2027 (Mar 1990)). Most anti- CD26 mAbs are stimulatory, rather than inhibitory when used under crosslinking conditions (R.W. Barton, et al. Binding of the T cell activation monoclonal antibody Tal to dipeptidyl peptidase IV, Journal of Leukocyte Biology 48, 291-296 (1990); L.A. Bristol, et al. Thymocyte costimulating antigen is CD26 (dipeptidyl- peptidasc IV). Co-stimulation of granulocyte, macrophage, and T lineage cell proliferation via CD26, Journal of Immunology 149, 367-372. (1992); L.A. Bristol, et al. Characterization of a novel rat thymocyte costimulating antigen by the monoclonal antibody 1.3, Journal of Immunology 148, 332-338 (1992); B. Fleischer, et al. Triggering of cytotoxic T lymphocytes and N cells via the Tpl03 pathway is dependent on the expression of the T cell receptor/CD3 complex, Joumal of Immunology 141, 1 103-11077 (1988); M. Hegen, et al. The T cell triggering molecule Tpl03 is associated with dipeptidyl aminopeptidase IV activity, J. Immunol. 144, 2980-2914 ( 1990)).
A class of low molecular weight synthetic monomeric molecules with high affinity for CD26 have previously been developed and characterized (G.R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991); W.G. Gutheil and W.W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diaslereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). These molecules have been shown to be potent and specific synthetic inhibitors for CD26's associated DP IV proteinase activity. DP-IV is a poslproline cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any a ino acid) dipeptides from the amino terminus of polypeplides.
Representative monomeric structures of these transition-stale-analog- based inhibitors, Xaa-boroPro, are e.g., Pro-boroPro and Ala-boroPro. BoroPro refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group [B(OH)2]. Pro-boroPro, the most thoroughly characterized of these inhibitors has a Ki of 16 picomolar (pM) (W.G. Gutheil and W.W.
Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723- 8731 (1993)). Vai-boroPro has even a higher affinity, with a Ki of 1.6 pM (W.G. Gutheil and W.W. Bachovchin. Supra; R.J. Snow, et al. Studies on Prolinc boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a B-N Bond, J. Am. Chem. Soc. 116, 10860-10869 (1994)). Thus, these Xaa-boroPro inhibitors are about 10+6 fold more potent than the next best known inhibitors. In comparison, antibodies usually have affinities for their targets between 10'8 and 10"9 M.
United States Patent Nos. 4,935,493 (the *493 patent) and 5,462,928 (the '928 patent), both of which are incorporated herein by reference, disclose protease inhibitors and transition state analogs (the '493 patent) and methods for treating transplant rejection in a patient, arthritis, or systemic lupus crythematosis (SLE) by administering a potent inhibitor of the catalytic activity of soluble amino peptidase activity of dipeptidyl peptidase type IV (DP-IV; (G.R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556- 1559 ( 1991)).
Until now, most drug discovery and development efforts have been directed at the allosteric conformational change-activated class of receptors. Also, the efforts directed at the association-activated class have focused on monomeric agents capable of blocking binding of a natural ligand, and therefore blocking signal transduction mediated by these receptors.
Cytotoxic drugs have untoward effects since they indiscriminately kill all proliferating cells. With the advent of monoclonal antibodies, it is possible to increase the specificity of these therapeutic tools. Monoclonal antibodies against the T cell receptors, e.g., T-cell receptor, CD4 and CD8 co-receptors, and to MFIC class II molecules, have all been evaluated for their respective benefit in experimental models for the treatment of autoimmune disease. The major impediment to using monoclonal antibodies as a therapeutic tool in humans, is that most monoclonal antibodies are made in mice, and humans rapidly develop an antibody response to mouse antibodies, which limits their potency because of neutralization and, worse, produces allergic reactions such as immune complex disease. Once this has occurred, all mouse monoclonal antibodies become useless in thai patient. To avoid this problem, antibodies which are not recognized as foreign by the human immune system are currently being made via different ways. One approach is to clone human V regions into a phage display library and select for binding to human cells. Using this method, monoclonal antibodies that are entirely human in origin can be obtained. Second, mice that lack endogenous immunoglobulin genes can be made transgenic for human heavy and light chain loci using yeast artificial chromosomes. Third, one may graft the antigen-binding loops of a mouse monoclonal antibody onto the framework of a human immunoglobulin molecule (a process known as humanization).
Each of these methods produce monoclonal antibodies which are far less immunogenic in humans than the parent mouse monoclonal antibodies, but with each methods comes a host of additional problems or road-blocks. For example, antiidiolypic neutralizing antibodies are often produced in patients receiving monoclonal antibody therapy.
Summary of the Invention * . In general, low molecular weight, bivalent or multivalent, synthetic crosslinking compounds are designed and developed. These synthetic crosslinking compounds may act either as agonists or antagonists and induce association between naturally occurring receptors, e.g., induce the association of one particular T cell surface receptor, such as CD26, with (a) itself or, (b) with another T cell or antigen presenting cell surface receptor (e.g., CD2, CD4, CDS, CD28, CD26, CD44, CD45, CD 10, CD3/TCR (or TCR/CD3)), CD40, B7.1 and B7.2. The low molecular weight, bivalent or multivalent, synthetic crosslinking compounds of this invention are small enough (less than about 30 amino acids, and more preferably about 20 amino acids) to obviate the immunogenicity associated with monoclonal antibodies. The bivalent or multivalent, synthetic crosslinking compounds of this invention can be administered to a patient without being co-administered with an adjuvant. In contrast, most other peptides, proteins and carbohydrate antigens are usually poorly immunogenic, or not immunogenic at all, when administered without adjuvant.
The compounds of the invention are useful for crosslinking molecules on the same or different cells that are involved in immune system modulation. Compounds of the invention fall within the following genus:
(I) [P2(R2)m]n-L-P'R']
wherein P1 represents a first targeting moiety, preferably a peptide that can mimic the substrate binding site of a protease (preferably a serine protease or cysteine protease) that is expressed on the surface of the cell involved in immune system modulation (e.g., a T cell, a B cell, a stem cell, a bone marrow cell, including an antigen presenting cell);
R1 represents a reactive group that reacts with a functional group in a reactive center of the protease;
P2 represents a second targeting moiety, preferably a peptide, that may be the same or different from the first targeting moiety;
R2 represents a second reactive group that may be the same or different from the first reactive group; m = 0 or 1 and n is a whole number from 1 to 10, and
L represents a linker molecule (i) having a molecular weight ranging from about 100 daltons lo about 2000 daltons, (ii) having a length ranging from about 2θΛ to about 300 Λ; and (iii) containing a chain of atoms selected from the group consisting of C, O, N, S, and phosphorus atoms, connected by single or by double bands. Thus, P1 can be D1 ~A1 ~A2~A3 ~A4 or D2~A5~A6~A7~A8 as described below in reference to certain embodiments of the invention. In important embodiments of the invention, P1 is a peptide or a peptidomimetic.
In certain embodiments of the invention, if P:=P', then R2 can be absent, the same, or different from R1. In general, n is 1 and the compounds of the invention are referred to as homodimers (i.e., P2=P') or heterodimers (i.e., P2≠P'). Cells which are involved in immune system modulation are blood cells including T cells, B cells, stem cells, bone marrow cells, dendritic cells, and other antigen presenting cells. The P1 targeting moiety can have a carboxyl terminal portion containing 1, 2, 3, or 4 amino acids which mimic the substrate binding site of the protease. Exemplary proteases which are believed to be expressed on the surface of such cells and which are bound by the P1 targeting moieties include post- prolyl cleaving enzymes, trypsin, chymotrypsin and elastase. The particular amino acids in the naturally occurring substrates of these enzymes are well known in the art and are identified below.
In general, the compounds of the invention contain from 1 to 30 amino acid residues (preferably the L isomers) and, more preferably, contain from 1 to 20 amino acid residues. In the most preferred embodiments, the P1 targeting moieties contain from 1 to 10 amino acids, most preferably, from 1 to 2 amino acids. The P1 targeting moiety can contain L or D amino acids; however, it is preferred that at least the amino acids which mimic the substrate binding site be in the L configuration. In contrast, the amino acids which arc in a rctroinverso configuration (see the Examples) preferably are in the D configuration.
The composition of the P1 targeting moiety is not limited to amino acids but may include, in whole or in part, non-amino acid components, provided that such components do not interfere significantly (i.e., do not lower the Ki of the compound to less than about 10"7M with the site-specific recognition of the compound by the protease and provided that the non-amino acid components do not interfere with Ihe formation of a complex between the compound and the protease. In certain embodiments, the portion of the P1 targeting moiety that is involved in binding to the substrate binding site is formed of amino acids and the remaining portion of the P1 targeting moiety is formed of non-amino acid components. In general, any portion of the P1 targeting moiety can be modified, for example, to be coupled to a detectable reagent, or immobilized to a surface via a linker, provided that such modifications satisfy the foregoing inhibition constant and complex formation criteria.
Peptides which reportedly have utility for inhibiting post-prolyl cleaving enzymes and which, if coupled to a reactive group, form a covalent complex with a functional group in the reactive site of a post-prolyl cleaving enzyme are described in U.S. Patent No. 4,935,493, "Protease Inhibitors", issued to Bachovchin et al. ("Bachovchin '493"); U.S. 5,462,928, "Inhibitors of Dipeptidyl-aminopeptidase Type IV", issued to Bachovchin et al. ("Bachovchin '928"); U.S. 5,543,396, "Prolinc Phosphonate Derivatives", issued to Powers et al., ("Powers '396"); U.S. 5,296,604, "Proline Derivatives and Compositions for Their Use as Inhibitors of HIV Protease", issued to Hanko et al., ("Hanko '604"); PCT/US92/09845, "Method for Making a Prolineboronate Ester", and its U.S. priority applications (USSN 07/796, 1*4£ and 07/936, 198), Applicant Boehringer Ingelheim Pharmaceuticals, Inc. ("Boehringer"); and PCT/GB94/02615, "DP-I V-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring").
In important embodiments, the P1 targeting moiety mimics the substrate binding site of the post-prolyl cleaving enzyme DP IV (also referred to herein as "CD 26"). DP IV is a post-prolyl cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of a polypeptide substrate. Representative structures of transition-state analog-based inhibitors Xaa-boroPro, include Lys-BoroPro, Pro-BoroPro and Ala- BoroPro in which "boroPro" refers to the analog of proline in which the carboxylate group (COOI1) is replaced with a boronyl group [B(OH)2]. Alternative crosslinking compounds of the invention have an analogous structure in which the boronyl group is replaced by a phosphonate or a fluoroalkylkctone (described below).
The invention also embraces compounds which mimic the substrate binding site of other post-prolyl cleaving enzymes. For example, IgA 1 proteases recognize the cleavage site Ser-Thr-Pro-Pro-X (where X is any amino acid). Accordingly, Ser-Thr-Pro-Pro-R1 is suitable for selectively binding to, and forming a complex with a functional group in the active site of an IgA 1 protease. The Ser- Thr in this targeting moiety may be readily substituted with any of the 20 naturally occurring amino acids, most preferably those having non-bulky side groups, such as Ala and Gly. It also is possible to substitute non-naturally occurring amino acids, such as 2-azetidinecarboxylic acid or pipecolic acid (which have 6-mcmbered, and 4-membered ring structures respectively) for either of the Pro residues. Those skilled in the art will recognize that there are other such changes which can be made without significantly affecting the binding and complex forming character of these compounds.
In the case of IgA 2 protease, the cleavage site in the natural substrate is Pro-Thr-Pro-X with hydrolysis occurring between Pro and X. Thus, a preferred P1 R1 binding moiety for binding to an IgA 2 protease has the formula Pro-Thr-Pro-R1. Thr can be substituted by any of the naturally occurring amino acids, especially ones having non-bulky side groups, such as Ala, Gly or Ser. Other examples of post-prolyl cleaving enzymes which can be targeted by the targeting moieties of the invention include other IgA enzymes, encephalon degrading enzymes, vasopressin degrading enzymes, and oxytocin degrading enzymes. The P1 targeting moieties of the invention can be designed to mimic the substrate binding sites of other, non-post-prolyi cleaving enzymes that may be expressed on the surface of cells involved in immune system modulation. These enzymes include, for example, cystcinc proteases and serine proteases such as trypsin, chymotrypsin and elastasc. The substrate binding sites for these enzymes are well known and peptidomimetics for targeting binding to these sites have been reported. For example, a P1 targeting moiety of the invention which mimics the substrate binding site of trypsin would include an arginine (Arg) or lysine (Lys) residue at its carboxyl-tcrminus with the carboxyl group of the Arg or Lys coupled to an appropriate reactive center, R1, to form a covalent bond with a functional group in the active site of trypsin. Exemplary borolysine targeting moieties that can be used to form the compounds of the invention are described in U.S. Patent Nos. 5, 187,157 and 5,242,904, "Peptide Boronic Acid Inhibitors of Trypsin-like Proteases", issued to Kettner et al. ("Kettner ' 157" and Kettner '5,242,904) and in U.S. Patent No. 5,288,707, "Borolysine Peptidomimetics", issued to Metternich ("Metternich"). Intermediates that are useful for preparing these inhibitors and related procedures are described in U.S. 5,250,720, "Intermediates for Preparing Peptide Boronic Acid Inhibitors of Trypsin-like Proteases" ("Kettner '720") and U.S. 5,384,410, "Removal of Boronic Acid Protecting Groups by Transesterification" ("Kettner '410").
P1 targeting moieties also can be designed to mimic the substrate binding site of chymolrypsin. Such targeting moieties include a carboxyl terminal amino acid residue that is selected from the group consisting of phcnylalaninc (Phc), tryptophan (Tip), and tyrosine (Tyr). Preferably, the carboxyl groups of these amino acids are covalently coupled to an R1 reactive group to form a binding moiety that selectively binds to, and forms a covalent complex with, a functional group in the active site of chymotrypsin. Yet other P1 targeting moieties of the invention can be designed which mimic the substrate binding site of an elastase. For example, a P1 targeting moiety which mimics the substrate binding site of an elastase would include a carboxyl terminal amino acid residue that is alanine (Ala) or glycine (Gly) with the carboxyl group of these amino acids covalently coupled to the reactive group R1 . In general, conventional chemical reactions can be used to form the foregoing P'R1 binding moieties. Thus, P'R1 binding moieties of the invention can be designed and constructed to mimic the substrate binding site of virtually any protease for which the natural substrate is known or can be identified. The development of phagc display libraries and chemical combinatorial libraries from which synthetic compounds can be selected which mimic the substrate binding site of a protease permits the identification of further P1 targeting moieties to which an R1 reactive group can be covalently attached to form a binding moiety which mimics the substrate binding site of the protease and which forms a complex with a functional group in the protease reactive site. Such libraries can be screened to identify non-naturally occurring putative targeting moieties by assaying protease cleavage activity in the presence and absence of the putative phage display library molecule or combinatorial library molecule and determining whether the molecule inhibits cleavage by the protease of its natural substrate or of a substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay). Those phage library and/or combinatorial library molecules which exhibit inhibition of the protease then can be covalently coupled to the reactive groups R1 disclosed herein and again tested to determine whether these novel molecules selectively bind to the protease (e.g., by repeating the above-noted screening-assay). In this manner, a simple, high-through-put screening assay is provided for identifying non-naturally occurring targeting moieties of the invention. In general, the first targeting moieties of the invention are covalently coupled via a carboxyl group at their carboxyl terminal amino acid to a first reactive group, R1. As used herein, R1 refers to a reactive group that is capable of reacting with a functional group in a reactive center of a protease expressed on the surface of a cell involved in immune system modulation. By reacting with a reactive center of the target protease, it is meant that the R1 forms a covalent bond with a functional group that is located in the active site. R1 reactive groups that are embraced within the invention include the reactive groups referred to as group "T" in US 4,935,493, "Protease Inhibitors", issued to Bachovchin, et al. These include boronate groups, phosphonate groups, and fluoroalkylketone groups. The boronate groups are described in the detailed description of the invention and in the Examples. The phosphonate and fluoroalkylketone groups are described below. In general, it is preferred that the linkage between the carboxyl terminus of the targeting moiety and the reactive group be in an L configuration. It is preferred that the reactive group forms a covalent band with a functional group in the active site; however, there is no requirement for covalent bond formation in order to form a complex between the binding moiety and the active site. The reactive groups of the invention that are phosphonate groups have the formula:
O
II -C-CF2-G
where G is either H, F or an alkyl group containing 1 to about 20 carbon atoms and optional heteroatoms which can be N, S, or 0. Additional exemplary proline phosphonate derivatives which contain a perfluoroalkyl group, a phenyl group or a substituted phenyl group and which can be used in accordance with the methods of the invention are those described in U. S. 5,543,396 (Powers '396). As used herein, the reactive groups of the invention that are fluoroalkylketone reactive groups have the formula:
O
-P-J
J-0
where each J, independently, is O-alkyl, N-alkyI, or alkyl (each containing about 1- 20 carbon atoms) and, optionally, heteroatoms which can be n=N, S, or O. Other kctoamides, ketoacids and kctocslers that are useful reactive groups for reacting with the reactive center of a protease (e.g., a serine protease or a cystcine protease) are described in PCT/US91/09801 , "Peptides, Ketoamides, Ketoacids, and Ketocsters", Applicant: Georgia Tech Research Corp. ("GA Tech") which claims priority to U.S. 635,287, filed Dec. 28, 1990.
In certain embodiments, the reactive groups are selected from the groups having the formulas, 0 0
C - C - NH2, an alphaketo amide;
0 0
- C - C - OR, where R is an alkyl, or aryl group and may be substituted or unsubstituted, an alphaketo ester; and
0 0
- C - C - OH, an alphaketo acid.
The reactive groups of the invention also include the reactive groups described in PCT/GB94/02615, "DP-IV-Serine Protease Inhibitors" (Ferring). These include the above-noted boronyl groups [B(OH)2], as well as pyrrolidides and the following reactive groups, any of which may be substituted or unsubstituted provided that the substitution does not adversely affect the functional activity of the reactive group or the binding moiety to which it is attached: CN, C≡C, CHO and CH=NPh, wherein Ph refers to phenyl. These examples are illustrative only and are not intended to limit the scope of the invention. As noted in Ferring, compounds containing these representative reactive groups can be prepared by an adaptation of the general route described by E. Schon el al., Biol, Chem. Hoppe-Seyler:372:305- 31 1 ( 1991) and by W.W. Bachovchin et al., J. Biol. Chem. 265:3738-3743 (1990). (See, also, the above-referenced Bachovchin United States patents.
The second targeting moiety, P2, binds to a molecule that is present on the surface of the same or different cell to which the first targeting moiety binds. Preferably, the second targeting moiety binds to a molecule (e.g., a receptor, a major histocompatibility complex (MHC) molecule) which is present on the surface of a T cell or on the surface of a B cell. In certain embodiments, the second targeting moiety has a structure which mimics the substrate binding site of a protease that is present on a cell that is involved in immune system modulation. Thus, the second targeting moiety may be the same as the first targeting moiety, and the compounds of the invention are useful for crosslinking proteases that have the same or a similar substrate specificity on the same or different cells. For example, the compounds of the invention can be used to crosslink a first protease (e.g., a post-prolyl cleaving enzyme) on a first cell and a different protease (e.g., a trypsin, chymotrypsin, elastase or other serine protease or cysteine protease) that is expressed on the surface of the same or on a different second cell. In certain preferred embodiments, the first and second targeting moieties are identical (i.e., 2__pi) antj e second reactive group R2 may be absent (i.e., m=0), the same or different from the first reactive group R1 (i.e., R' ≠ R2). Compounds which include identical P1 and P2 groups and identical R1 and R1 groups are referred to as "homodimers". In yet other embodiments, the first and second targeting moieties are different and these compounds are referred to as "heterodimers".
* * In yet other embodiments, the second targeting moiety is an antigen that selectively binds to an MHC molecule on the surface of an antigen presenting cell. Such embodiments of the invention are useful as vaccines for inducing an immune system response to the antigen. In particular, such compounds are useful for inducing an immune system response to antigens that exhibit relatively low immunogenicity using conventional vaccine preparations. Accordingly, the invention also provides for an improved vaccine and related methods for inducing an immune response to an antigen. Examples of antigens arc antigens characteristic of pathogens, cancer antigens, and allergens.
Antigens that are characteristic of autoimmune disease typically will be derived from the cell surface, cytoplasm, nucleus, mitochondria and the like of mammalian tissues. Examples include antigens characteristic of uvcilis (e.g. S antigen), diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, primary yxoedema, thyrotoxicosis, rheumatoid arthritis, pernicious anemia, Addison's disease, scleroderma, autoimmune atrophic gastritis, premature menopause (few cases), male infertility (few cases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis (few cases), ulcerative colitis, Sjogren's syndrome, Wegener's granulomatosis, poly/dcrmatomyositis, and discoid lupus erythromatosus. Additional examples are provided below.
Antigens that are allergens are generally proteins or glycoproteins, although allergens may also be low molecular weight allergenic haptens that induce allergy after covalently combining with a protein carrier (Remington's
Pharmaceutical Sciences). Allergens include antigens derived from pollens, dust, molds, spores, dander, insects and foods. Specific examples include the urushiols (pentadecylcatechol or heptadecylcatechol) of Toxicodendron species such as poison ivy, poison oak and poison sumac, and the sesquiterpenoid lactones of ragweed and related plants. Additional examples are provided below.
Antigens that arc characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples include antigens characteristic of tumor proteins, including proteins encoded by mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids. Tumors include, but are not limited to, those from the following sites of cancer and types of cancer: lip, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon, rectum, liver, gall bladder, biliary tree, pancreas, larynx, lung and bronchus, melanoma of skin, breast, cervix, uteri, uterus, ovary, bladder, kidney, brain and other parts of the nervous system, thyroid, prostate, testes, Hodgkin 's disease, non-Hodgkin 's lymphoma, multiple myeloma and leukemia. Viral proteins associated with tumors would be those from the classes of viruses noted above. Antigens characteristic of tumors may be proteins not usually expressed by a tumor precursor cell, or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor. An antigen characteristic of a tumor may be a mutant variant of the normal protein having an altered activity or subcellular distribution. Mutations of genes giving rise to tumor antigens, in addition to those specified above, may be in the coding region, 5' or 3' noncoding regions, or introns of a gene, and may be the result of point mutations, frameshifts, deletions, additions, duplications, chromosomal rearrangements and the like. One of ordinary skill in the art is familiar with the broad variety of alterations to normal gene structure and expression which gives rise to tumor antigens. Specific examples of tumor antigens include: proteins such as Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel- 17 (gplOO) of melanoma, MART-1 (Melan-A) of melanoma, p i 5 protein of melanoma, tyrosinase of melanoma, MAGE 1 , 2 and 3 of melanoma, thyroid medullary, small cell lung cancer, colon and/or bronchial squamous. cell cancer, BAGE of bladder, melanoma, breast, and squamous cell carcinoma, gp75 of melanoma, oncofetal antigen of melanoma; carbohydrate/lipids such as mucl mucin of breast, pancreas, and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes such as mutant p53 of carcinoma, mutant ras of colon cancer and HER-2/ eu proto-oncogcnc of brcasl carcinoma; viral products such as human papilloma virus proteins of squamous cell cancers of cervix and esophagus. It is also contemplated that proteinaceous tumor antigens may be presented by HLA molecules as specific peptides derived from the whole protein. Metabolic processing of proteins to yield antigenic peptides is well known in the art; for example see U.S. patent 5,342,774 (Boon et al.).
Preferred tumor antigens of the invention include the Melonoma tumor antigens (e.g., MAGE protein family (MAGE- 1 , MAGE-2, MAGE-3); MART- 1 (peptide 27-35); and gpl OO); and the Colon carcinoma antigens (e.g., peptides of the mutated APC gene product). Particularly preferred Melanoma tumor antigen sequences are those reported by Slingluff et al., in Curr. Opin. in Immunol. 6:733- 740 (1994):
Gene/protein MHC
MAGE-1 Al
CwlόOl MAGE-3 Al Tyrosinase A2
A24 gplOO/pMel-17 A2
MART-1/Melan-A A2
Figure imgf000020_0001
The MAGE protein family also reportedly has been associated with more than one type of carcinoma: MAGE-1 (Melanoma, thyroid medullary, and small-cell lung carcinoma), MAGE-2 (Melanoma, small-cell lung, colon, bronchial squamous cell, and thyroid medullary carcinoma), and MAGE-3 (Melanoma, small- cell lung, colon, bronchial squamous cell, and thyroid medullary carcinoma). See, also, Morioka, et al., "A Decapeptide (Gln-Asp-Leu-Thr-Met-Lys-Tyr-Gln-Ile-Phe) from Human Melanoma Is Recognized by CTL in Melanoma Patients", J. Immunol. 153:5650 (1994), for additional tumor antigens (e.g., PI A, Connexin 37, MAGE-1, MAGE-3, MART 1/Aa, gpl OO, Tyrosinase) and/or information relating to the tissue distribution of selected tumor antigens.
Particularly preferred tumor antigens that are peptides of the mutated APC gene product are those reported by Townsend et al., in Nature 371 :662 ( 1994)):
New Sequence SF.Q.ID No. SSST/LCTSKADKSSGNQGGNGVF1VVNAWYS 9
SEDL/TΛGYCKCFEEFVLASRCK 10
Figure imgf000020_0002
EQRQ/GIKVQL1LF1LRAL INTSSSNHIL 1 1
DSRNVFLI ITGHGEPMVQKQIEWVL1MELIKM
1353 8bp del HKAV/FRSEISLQKWCSDTQ ST 12 1398 I bp del DSFE/SVRLPAPFRVNHAVEW 13
1420 I bp del IISP/VIFQ1ALDKPCHQAEVKHLHHLLK
QLKPSEKYLKIKHLLLKRERVDLSKLQ 14
1439 I bp del RSKT/LHHLLKQLKPSEKYLK1KHLL 15
LKRERVDLS LQ
PPQT/GEKYLKIKHLLLKRERVDLSKLQ 16
DADT/YY1LPRKVLQMDFLVHPA 17
DTLL/L PR VLOMDFLVHPA 18
Figure imgf000021_0001
LHFA/SRWIFLFIQPECSEPR 19 In alternative embodiments, the second targeting moiety is a ligand that selectively binds to a receptor that is expressed on the surface of a cell (preferably a T cell or a B cell). Exemplary receptors which have naturally occurring ligands that can be mimicked by the second targeting moieties of the invention include receptors selected from the following group: CD2, TCR/C3, CD4, CD8, CD 10, CD26, CD28, CD40, CD44, CD45, B7.1 and B7.2. According to yet other embodiments, the second targeting moiety is an antibody or antibody fragment that selectively binds to an epitope expressed on the cell surface. The epitope can be a portion of any of the foregoing receptors.
Regardless of the nature of the second targeting moiety target (e.g., protease, receptor, MHC complex, epitope), phage display and other types of combinatorial libraries can be screened in a manner analogous to that described above to identify non-naturally occurring targeting moieties that are useful in forming the compounds of the invention.
There is no requirement that the second targeting moiety be covalently attached to a second reactive group. For example, the second targeting moiety may have sufficient affinity for its binding partner (e.g., an MHC molecule) to permit cross linking between the same or different target molecules on the same or different cells without forming a covalent complex between the second targeting moiety and its targeted binding partner. There also is no requirement that the second reactive group be the same as the first reactive group. Thus, for example, the compounds of the invention embrace molecules that include a first binding moiety which contains a first reactive group R1 that is a boronate and a second binding moiety which contains a second reactive group that is a boronate, a phosphonate or a trifluoroalkylketone group. A linker is covalently coupled to the first and second targeting moieties,
P1 and P2, in a manner that does not adversely affect the ability of these moieties to bind to their respective targeted binding partners. Exemplary linkers, including a description of linker composition, size, and procedures for coupling the linker to the targeting moieties are described in the detailed description of the invention and in the Examples. In general, such linkers are commercially available and are coupled to the targeting moieties using conventional coupling procedures which are well known to those of ordinary skill in the art.
Certain aspects and uses of the invention are based on the discovery that certain Koinodimers are capable of stimulating T cells and that this stimulatory ability is, at least in part, dependent upon the length of the linker. In addition,
Applicant has discovered that there exists a length between binding moieties (about 20 angstroms) below which this homodimer is no longer capable of stimulating T cells. This ability of any type of homodimer for crosslinking DP IV proteases to stimulate T cells is unexpected in view of published reports that such DP IV homodimers exiiibit a T cell inhibitory activity. (See, e.g., PCT/GB94/02615, "DP- IV-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring") and the U.S. patent application which claims priority to Ferring). Ferring describes certain symmetric homodimers containing two active-site directed inhibitors of DP-IV linked via the side chains of their amino acid residues. Thus, the invention provides a new use for the homodimer DP-IV inhibitors disclosed in Ferring, namely, the use of the Ferring homodimers for stimulating T cells in a patient in need of such treatment.
Certain concentrations of the homodimers of the invention have been found to stimulate blood cells. Thus, compounds of the invention are particularly useful for treating HIV+ patients, for example, by contacting the T cells obtained from an HIV+ patient with a therapeutically effective amount of one or more compounds of the invention under conditions that permit blood cell activation. The stimulatory effect on T-cells of these compounds at stimulating concentrations is illustrated in the accompanying Examples. The cells can be contacted with the crosslinking compounds in vivo or ex vivo. This stimulator)' property of the homodimer compounds of the invention is unexpected and could not have been predicted based upon the reported inhibitory effect on the immune system of certain monomers (e.g., the compounds disclosed in Bachovchin '493) and certain homodimers (e.g., PCT/GB94/02615, "DP-IV-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring").
The compounds of the invention can be used to inhibit the enzymatic activity of the proteases to which the targeting moieties selectively bind. Thus, the compounds of the invention are useful for inhibiting post-prolyl cleaving enzymes, as well as for inhibiting other serine and cysteine proteases (e.g., chymotrypsin, trypsin, and elastase).
According to another aspect of the invention, methods are provided for modulating immune system function. The compounds of the invention are administered to subjects in need of immune system modulation in amounts effective to modulate immune system function. Modulation of immune system function includes, but is not limited to, increasing immune function such as by stimulating proliferation and specific immune function of blood cells nonspecifically or by specifically stimulating T and/or B cells and/or bone marrow cells, stem cells, early lineage progenitor cells to produce a prophylactic or therapeutic result relating to infectious disease, cancer, and the like. Specifically included is the use of the compounds of the invention, and in particular, homodimers and/or heterodimers for the treatment of disorders characterized by reduced T cell levels in vivo, e.g., HIV and other disorders associated with a compromised immune system. Modulation of immune system function also includes, but is not limited to, decreasing immune function such as by suppressing generally the immune system in transplant recipients or suppressing specifically the immune system to treat autoimmune disease, allergy and the like. In one important embodiment the homodimers and/or heterodimers of the invention are used to stimulate blood cell proliferation, as described in detail below. Specific conditions that may be treated according to the invention are deemed specific independent aspects of the invention and are described in detail in the tables and examples below. According to yet another aspect of the invention, a method for stimulating T cells is provided. A crosslinking compound of the invention is contacted with the T cells of a subject in need of such treatment in an amount effective to stimulate T cells. The crosslinking compounds that are particularly useful fόr-stimulating T cells arc those compounds which crosslink DP IV molecules as described above, including the preferred crosslinking compounds. As noted above, the preferred crosslinking compounds include a linker, L which when positioned between binding moieties results in a minimum length of about 20 angstroms between these moieties. Preferably, the distance between binding moieties is from 20 to 60 angstroms, more preferably from 30 to 50 angstroms. According to still another aspect of the invention, pharmaceutical preparations arc provided. The pharmaceutical preparations contain a crosslinking compound as described above and, optionally, a pharmaceutically-acccptable carrier. Preferably, the pharmaceutical compositions are sterile. The term pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
According to yet another aspect of the invention, a hetero bivalent compound, having the structure as shown in Fig. 1 A, wherein each component of the structure is as defined in Example 2 in reference to this figure, is provided.
An embodiment of this compound is wherein the structures shown in Fig. 1U and Fig. I V represent, independently, a binding moiety, wherein R represents the remainder of the molecule. By definition, the linker molecule must be capable of linking atom Al of the binding moiety on the left of Fig. 1A to atom A5 of the binding moiety on the right of Fig. 1A.
Other embodiments of this compound include: there are 4 atoms positioned between the group consisting of Dl and D2 and B of the binding moiety; the binding moiety is in an L-configuration; Yl, Y2, Y3, and Y4 are hydroxyl groups; the A4 bonded to the B is in the L-configuration and the A5 bonded to the B is in the L-configuration; the binding moiety is an L-amino acid residue conjugated to B, a boron molecule; the binding moiety is selected from the group consisting of L-Lys-L-boroPro and a derivative of L-Lys-L-boroPro. Another embodiment of this compound is wherein the linker molecule contains a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfhydryl group, an imidazole group, an alkene group (a carbon atom double bonded to another carbon atom), an acyl halogen group, e.g., an acylchloride, and CH2X, wherein X represents a halogen (e.g, the two binding moieties are linked when a nuclcophilic group displaces the halogen from the functional group of the CH2X linker molecule); wherein the linker molecule is further defined as having the structure as shown in Fig. IT and wherein [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogcn and a sulfur atom; [J] is selected from the group consisting of a CH2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p, and q represent an integer from 1 to 50, inclusive; wherein [GJ preferably is an R group selected from the group consisting of L-amino acid residues selected from the group consisting of lysinc, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; wherein the linker molecule preferably is selected from the group consisting of hexanedioic acid (adipic acid), EGS, 1 ,4-diaminobutane, 1,4-dithiobutane, dithiothreitol, lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; wherein the linker molecule preferably contains at least two amino groups when the binding moieties contain glutamic acid residues; wherein the linker molecule preferably contains at least two amino groups when the binding moieties contain aspartic acid residues; wherein the linker molecule preferably contains at least two sulfhydryl groups when the binding moieties contain cysteine residues; and wherein the linker molecule length ranges from about 30 A to about 100 A.
Another aspect of this invention is a compound having the structure as shown in Fig. IB. In this compound, the binding moieties are identical, e.g., having Al, A2, A3, and A4 in both binding moieties, and wherein each component of the structure is defined in Example 2 in reference to this Figure.
An embodiment of the compound where only Al, A2, A3, and A4 appear in both binding moieties, is the structures shown in Fig. 1U and Fig. IV which represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
Another aspect of this invention is a compound having the structure as shown in fig. 2A, wherein each component of the structure is as defined in Example 4 in reference to this Figure. An embodiment of this compound includes the structures shown in Figs. 1U and IV which represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
Other embodiments of this compound include: there are 4 atoms positioned between D and B of Ihe binding moiety; the binding moiety is in an L- configuralion; Yl and Y2 are hydroxyl groups; the A4 bonded to the B is in the L- c on figuration; the binding moiety is an L-amino acid residue conjugated to B, a boron molecule; and the binding moiety is selected from the group consisting of L- Lys-L-boroPro and a derivative of L-Lys-L-boroPro. Additional embodiments of this compound is the linker molecule which includes: a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfhydryl group, an imidazole group, an alkene group, an acyl halogen group, and CH2X, wherein X represents a halogen; the linker molecule which is 'further defined as having the structure shown in Fig. IT, wherein [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogen and a sulfur atom; [JJ is selected from the group consisting of a CH2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p, and q represent an integer from 1 to 50, inclusive; wherein [G] preferably is an R group selected from the group consisting of L-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; wherein the linker molecule preferably is selected from the group consisting of adipic acid, between 2 and 1 consecutive amino acid residues, 1 ,4-diaminobutanc, 1 ,4-dithiobulanc, and dithiothreitol; and wherein the linker molecule span ranges from about 30 A to about 100 A.
Yet another embodiment of this compound is peptides ranging from about 7 to 25 amino acids; wherein exemplary peptide include: a) Myelin proteolipid protein peptide; b) Moth cytochrome C peptide; c) tetanus toxin peptide; d) HIV-1 GP 120 peptide; e) myelin basic protein peptide; f) tumor anti genie peptides, and g) anti genie peptides of infectious agents. Preferably, the Myelin proteolipid protein peptide includes human and mouse peptide selected from the group consisting of PLP peptide from the region of 85- 159 and in particular, 95-1 16, and PLP peptide 105-124, PLP peptide 139-151 and PLP peptide 190-209; the Moth cytochrome C peptide is peptide MCC 94-103; the myelin basic protein peptide is MBP peptide 1- 1 1 ; the tetanus toxin peptide is selected from the group consisting of tetanus toxoid peptide and P2 tetanus toxoid peptide; and the tumor antigenic peptides and antigenic peptides of infectious agents are described elsewhere in the application.
Another embodiment of this compound is where the naturally occurring receptor Is a B cell or T cell surface receptor; and the cell surface receptor is selected from the group consisting of TCR/C3, CD2, CD4, CD8, CD 10, CD26, CD28, CD44, CD45, CD40, B7.1 and B7.2.
Another aspect of this invention is a compound having the structure as shown in Fig. 1R, wherein each component of the structure is as defined in Example 3 in reference to this Figure. An embodiment of this compound includes the structures as shown in
Fig. 1U and Fig. IV which represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
Another embodiment of the compound shown in Fig. 1R is wherein (a) [G]„, is the side chain of a D- or L-isomcr of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (c) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (d) El and E3 arc distinct from each other.
Yet another embodiment of the compound shown in Fig. 1R is wherein (a) [GJm is the side chain of a D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is selected from the group consisting of 2-carboxy butyl, 2-carboxypropyl, 2-aminobutyl, 2- aminopropyl, and a hydrocarbon chain with an amino or carboxy side chain; (c) [J]p and [I]q represent, independently, hydrocarbon chains; (d) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (e) El and E3 are distinct from each other. The compounds of the invention, in particular the homodimeric complexes, can be used to stimulate activation or proliferation of human CD26- bearing lymphocytes, by contacting the lymphocytes with a proliferation or activation-inducing concentration of the compound.
' ' The method preferably involves in vivo administration of the compound, admixed with a pharmaceutically acceptable carrier such as pharmaceutical, sterile saline. The patient can be any patient who suffers from a disease state characterized by inadequate lymphocyte activation or concentration. Examples of such diseases are HIV infection, kidney failure, cancer (in particular, cancer accompanied by lymphocyte-depleting chemotherapy), and bone marrow disorders which result in depleted lymphocyte populations in the patient. The compound is preferably administered to Ihe patient orally. The compounds can also be used to stimulate proliferation or activation of lymphocytes in vitro, e.g., where a patient's autologous lymphocytes are removed, stimulated to increase activation and/or number of lymphocytes, and reinfused into the patient. This method can be used, for example, to increase the number of cytolytic T cells specific for a patient's tumor or T cells in HIV infected patients.
As used herein, crosslinking compound means the compounds described above as well as salts thereof. These and other aspects of the invention will be described in greater detail below.
All patents, patent applications, references and other documents that are identified in this patent application are incorporated in their entirety herein by reference.
Definitions By "amino acid" is meant to include imino acid. By "boroPro" is meant an alpha-amino boronic acid analog of proline bonded to an amino acid to form a dipeptide with boroPro as the C-terminal residue. "BoroPro" is used to designate such an analog having the carboxyl group of proline replaced with a B(OH)2 group, where (OH)2 represents two hydroxyl groups and B represents boron.
By Xaa is meant any amino acid residue, e.g., a lysine residue. * • Also, for this invention, "[Lysine-boroProline]2" and "KbP-S-KbP, where S represents a linker spacer" are used interchangeably. "Dimeric KbP with adipic acid as the spacer linker," "[di(L-Lysine-L-boroProline)adipate," and "KbP2- Adipate" are used interchangeably. Dimeric and bivalent are used interchangeably.
Linker-spacer molecule, cross-linker, cross-linker molecule, linker molecule and linker group are used interchangeably. By "agonist" is meant a molecule or compound which activates the signaling pathway in question.
By "antagonist" is meant a molecule or compound which inhibits the signaling pathway in question.
"CD26 ligand" is any protein, glycoprotein, lipoprotein or polypeptide that binds to the T cell receptor CD26 and may provide a stimulatory or inhibitory signal.
CD26, Dipeptidyl Peptidase IV (DP IV) and dipeptidyl aminopeptidase IV are used interchangeably. CD26 is a postprolinc cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of polypeptides.
"CD26-specific binding species" means a CD26-specific antibody, fragment or small molecular weight compound that binds to CD26. "Tethered or coupled α-amino acid" is an α-amino acid with a carbon atom of its side chain tethered, linked or coupled to the N atom of the α-amino group.
By alpha-carbon of an amino acid is the one to which the carboxylic acid group is attached. By alpha-amino acids is where the amino group is attached to the alpha- carbon. All naturally occurring amino acids arc alpha-amino acids or alpha-imino, which means that the amino and carboxylic acid groups are both attached to the same carbon atom. Each amino acid can be thought of as a single carbon atom (the alpha carbon, C) to which there is attached one carboxyl group, one amino group, a side chain denoted R as shown below and a hydrogen.
H OH
H,N - C - C = 0
R
Wherein: "R" is a side chain; "NH2" is the alpha amino group; the first carbon (C) attached to the NH2 group having a hydrogen (H) and an R group attached is the alpha carbon; and the carbon double bonded to an oxygen and a hydroxyl group (OH) is the alpha carboxyl group.
The NH2 and COOH groups are used to connect amino acids to one another. The hydroxyl group (OH) of one amino acid on the carboxyl end and the hydrogen (H) on the N terminus are removed (H20) when two amino acids are linked together. To form a protein, the amino group of one amino acid reacts with the carboxyl group of another by the elimination of water; the resulting chemical bond is called a peptide bond.
By "peptides" is meant a small molecule, e.g., usually containing less than 50 amino acid residues, which do not generally possess a well-defined three- dimensional structure.
By A or "Anstrom" is meant 10"10 M. The relative bond size used in different peptides is as follows: N-H: 1.0 A; C-FI: 1.1 A; C double bonded to 0: 1.2 A; N-CO: 1.3 A; C-O: 1 .4 A; C-C: 1.5 A; Alaninc: 6 A; Benzene: 6 A; water: 4 Α; and Phenylalanine: 7 A. Pharmaceutical preparations and modes of administration are described herein.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Brief Description of the Drawings Figs. 1A-1 V are diagrams showing the general structures of several preferred homobivalent and homomultivalent compounds or arc diagrams of components needed for these structures:
Fig. 1 A is a diagram of a general bivalent template with non-identical binding moieties. Fig. IB is a diagram of a general homobivalent template.
Fig. 1C is a diagram of a general bivalent template with amino linkages using a dicarbonyl linker. Fig. ID is a diagram of a bivalent example with amino linkages using an adipoyl linker.
Fig. IE is a diagram of a homobivalent example with amino linkages using an adipoyl linker [(Lysine-boroPro)2Adipatej. Fig. IF is a diagram of another homobivalent example with amino linkages using an adipoyl linker.
Fig. 1G is a diagram of a general bivalent template with a carboxyl linkage using a diamino linker.
Fig. 1H is a diagram of a bivalent example with carboxyl linkages using a 1 ,4-Diaminobutane linker.
Fig. II is a diagram of a bivalent example with carboxyl linkages using a 1 ,4-Diaminobutane linker [(Aspartyl-boroProline)2l,4-Diaminobutane].
Fig. 1 J is a diagram of another bivalent example with carboxyl linkages using a f,4-Diaminobutane linker. Fig. IK is a diagram of a general bivalent template with disulfide linkages using a dithiol linker.
Fig. 1L is a diagram of a bivalent example with disulfide linkages using a 1 ,4-Dithiobutane linker.
Fig. IM is a diagram of another bivalent example with disulfide linkages using a 1 ,4-Dithiobutane linker.
Fig. IN is a diagram of another bivalent example with disulfide linkages using a dithiothreitol linker [(Cysteine-boroProline)2dithiotheitol].
Fig. 10 is a diagram of a general bivalent template with imidazole linkages using a dicarbonyl linker. Fig. IP is a diagram of another bivalent example with imidazole linkages using a dicarbonyl linker.
Fig. 1Q is a diagram of a bivalent example with imidazole linkages using an adipoyl linker: (FIistidine-boroProIine)2Λdipate. Fig. IR is a diagram of a general homofiinctional polymeric crosslinker template.
Fig. IS is a diagram of a homotrimeric example using adipoyl linkers.
Fig. IT is a diagram of a linker molecule template.
Fig. 1U is a diagram of a binding moiety containing Al , A2, A3, and
A4.
Fig. IV is a diagram of a binding moiety containing A5, A6, A7, and
A8.
Figs. 2A-2C are diagrams showing the general formula of several preferred heterobivalent compounds:
Fig. 2 A is a diagram of a general heterobivalent template. Fig. 2B is a diagram of a heterobivalent example coupling a binding moiety to an MCC peptide (94-103)^
* » Fig. 2C is a diagram of a heterobivalent example coupling a binding moiety to a PLP peptide (139-151).
Fig. 3 is a diagram showing the synthesis of adipoyl (Lys-boroPro)2, a homobivalent derivative of Lys-boroPro.
Fig. 4 is a graph showing a dose response curve observed with lower concentrations of KbP2-Adipate on anti-CD3 mAb stimulation of FI9 cells. Fig. 5 is a graph showing a dose response curve observed with higher concentrations of KbP2-Adipate on anti-CD3 mAb stimulation of FI9 cells. Drug concentration is read as 10XM.
Fig. 6 is a graph showing a dose response curve for anti-lF7. Fig. 7 is a diagram showing intermolecular reactions that may occur al higher concentrations of KbP2-Adipate.
Fig. 8 is a diagram showing Lys-boroPro linked to Myelin Proteolipid Protein (PLP) Peptide 139-151.
Fig. 9 is a graph comparing the effects heterobivalent coupled KbP-S- MCC and uncoupled MCC 94-103 have on IL-2 production in 2B4 cells.
Fig. 10 is a diagram showing the structures of the open and cyclized forms of Xaa-boroProline inhibitors.
Figs. 1 1A-I ID are diagrams of different examples of bivalent compounds containing an olefin group. Fig. 1 ID is a fluoroolefin isostere of Xaa- boroProline.
Fig. 12 is a graph illustrating the effect of (KbP)2-EGS at vaiying concentrations on IL-2 production by H9 cells.
Fig. 13 is the structure of EGS, the spacer-linker molecule used in the homodimer (KbP)2-EGS used in the experiments of Fig. 12.
Detailed Description
Synthetic, low molecular weight, bivalent and multivalent crosslinking compounds with agonistic activity are designed and developed. To have agonist activity, the molecule needs to be able to induce the association of receptors of a specific class in a manner similar to that induced by its natural ligand. These agonistic molecules therefore need to be at least bidentate. Additionally, the individual binding units must be properly spaced for the desired association to occur.
The homobivalent (homodimeric), homomultivalcnt and heterobivalent (heterodimeric) compounds of this invention represent a new class of biological modulators which can be used as therapeutic or diagnostic agents or both.
T Cell Surface Receptors
T cell surface receptors and their naturally occurring ligands arc used herein as examples to demonstrate how different homobivalent, homomultivalent, heteromultivalent, and heterobivalent synthetic crosslinking compounds function; these examples are therefore not intended to limit the invention. The molecule, Xaa-boroPro, which has been shown to have high affinity for the CD26 T cell surface receptor (See above discussion), can be a component molecule of the homobivalent, heteromultivalent, homomultivalent, or heterobivalent compounds outlined herein.
Biochemistry of CD26 T Cell Surface Receptor
CD26 is a highly glycosylated type two transmembrane protein. It exists as a dimer with a subunit molecular weight of about 1 10 kDa. The cDNAs encoding the human, mouse and rat proteins have been cloned and sequenced (D. Darmoul, et al., "Dipeptidyl peptidase IV (CD26) gene expression in enlcrocytc- like colon cancer cell lines FIT-29 and Caco-2: Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA levels during cell differentiation," J.Biol.Chem. 267, 4824-4833 (1992); D. Marquct, et al., "cDNA cloning for mouse thymocyte-activaling molecule: A multifunctional ecto- dipeptidyl peptidase IV (CD26) included in a subgroup of serine protease." J.Biol.Chem. 267, 2200-2208 (1992); T. Tanaka, et al., "Cloning and functional expression of the T cell activation antigen CD26," J. Immunol. 149, 481 -486 ( 1992); published erratum appears in J.Immunol. 50(5):2090 (Mar 1993)). Fluman CD26 cDNA encodes a 766 amino acid polypeptide (having a molecular weight of 88,300) and the mouse cDNA encodes a 760 amino acid polypeptide (87,500 molecular weight). The sequences of the mouse, rat and human CD26 share up to 98% homology. Most of the CD26 molecule resides outside of the cell by being anchored to the cell plasma membrane through a 22 amino acid hydrophobic domain on the N terminus and only a small, six amino acid N terminal tail projects into the cytoplasm. Among lymphoid cells, CD26 is found mainly on the surface of CD4+ T cells where it is believed to have important roles in T cell activation pathways (sec section on CD26 and T cell function below). CD26 is also found on a small fraction of CD8+ cells. CD26 has been shown to be identical with an enzyme known as dipeptidyl peptidase amino peptidase Type IV (DP IV, sometimes also abbreviated DPP IV or DAP IV).
Enzymology of CD26: Active Site Structure and Inhibitor Design The catalytic activity thus far identified for CD26 associated DP IV protease activity involves the cleaving of a dipeptide unit from the free amino terminus of polypeptides and proteins. DP IV shows a strong preference for cleaving after a proline residue, i.e., a proline in the penultimate position from the amino terminus. A free amino terminus appears to be a requirement but the enzyme displays little preference for any particular amino acid in this position (J. Heins, et al., "Mechanism of proiine-specific protcinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proiine-specific endopeptidase from Flavobacterium meningosepticum," Biochimica Et Biophysica Acta 954, 161-169 (1988)) DP IV can be regarded as a postproline cleaving aminopeptidase with a specificity for removing N-tcrminal Xaa-Pro dipeptides where Xaa can be any amino acid. Flowever, DP IV will also remove amino terminal Xaa-Ala dipeptides, although much less effectively. The enzyme does, however, require that some amino acid be N terminal to proline because boroPro itself is not an effective inhibitor. The P2 residue is probably required only for presenting a free amino group in the appropriate geometrical arrangement with respect to the proline bond to be cleaved because the addition of a group to the P2 N terminus, such as Ac, CBZ, or Fmoc, abolishes the inhibitory potency (G.R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991 )). This indicates that the active site is constructed to recognize a prolinc residue at P I and a free amino group at P2. The P2 amino acid side chain is probably directed away from the enzyme and free in solution. PI refers to the residue on Ihe N-tcrminal side of the sessile bond. P2 refers to the residue on the N-terminal side of PI .
CD26 and T Cell Function
The work of Schon et al. was among the first to implicate DP IV as important to T cell function (E. Schon, et al., "Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes," Biological Chemistry Hoppe Seyler 372, 305-31 1 (1991 ); E. Schon, et al., "The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes," Biomedica Biochimica Ada 44, (1985); E. Schon, et al., "Dipeptidyl peptidase IV in human T lymphocytes. An approach to the role of a membrane peptidase in the immune system," Biomedica Biochimica Acta 45, 1523- 1528 (1986); E. Schon, et al., "The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro," European Journal of Immunology 17, 1821- 1826 (1987); E. Schon et al., "Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma intcrferon due to specific inhibition of dipeptidyl peptidase IV," Scandinavian Journal of Immunology 29, 127-132 ( 1989)). This work reported that DP IV inhibitors and anti-DP IV poiyclonal antibodies suppressed T cell activation in culture. It has been demonstrated that Pro-boroPro and Ala-boroPro inhibited an immune response in vivo in mice such that antibody production in response to an antigen challenge was reduced; this was direct evidence for a role for DP IV/CD26 in immune function in vivo.
Also, most of the evidence implicating CD26/DP IV as important to proper T cell function and immune system regulation comes from studies of the effects of various anli-CD26 mAbs on T cell functions. Because Abs are naturally bivalent, they are often able to mimic the natural ligand in inducing association of the receptor, e.g., an agonistic effect, which may be a dimerization or an aggregation. If an antibody fails to correctly mimic the natural-Iigand induced association, it will then often block the interaction with the natural ligand and therefore show an inhibitory or antagonistic activity.
The anti-CD26 mAbs thus far identified have either activating, inhibitory, or both effects on various T cell responses. These results show that CD26 is an association-activated co-stimulatory T cell receptor.
The role for CD26 as an association-activated, co-stimulatory molecule has been recently confirmed in experiments in which the gene for CD26 was transfected into Jurkat T cell lines (T. Tanaka, et al., "Cloning and functional expression of the T cell activation antigen CD26," published erratum appears in J. Immunol., 150(5):2090 (Mar 1993); J.Immunol. 149, 481-486 (1992)).
The results indicated that mAb-mediated cross linking of CD26 on CD26+ Jurkat cell resulted in enhanced Ca2+ mobilization and IL-2 production in response to anti-CD3 suboptimal stimulation in the presence of phorbol esters. Untransfected Jurkat cells do not express CD26 and do not produce IL-2 in significant amounts in response to anti-CD26 costimulation with suboptimal anti- CD3 treatment in the presence of phorbol esters. Mechanisms of CD26 Mediated Signal Transduction
CD26 has only a short six amino acid cytoplasmic tail. This argues against signal transduction via the cytosolic domain as is often the case for other cell surface receptors. CD26 may participate in T cell signal transduction through two hypothetical mechanisms: (1 ) through its association with other molecules in the membrane, and (2) through its DP IV protease activity.
EXAMPLES Introduction
Throughout this application and in particular, in each of the Examples and drawings, particular embodiments are described and illustrated. It is to be undcrstood that any of the reactive groups disclosed herein can be substituted for the particular reactive groups (e.g., boronyl group) shown in the drawings or described in the particular Examples.
EXAMPLE 1 GENERAL SYNTHESIS OF HOMOBIVALENT, HOMOMULTIVALENT AND MULTIVALENT CROSSLINKING COMPOUNDS
The synthesis of bivalent or multivalent compounds or agents outlined herein involves essentially very similar chemistry. These bivalent and multivalent compounds are designed such that they induce associations between naturally occurring receptors, e.g., an association between a T cell surface CD26 receptor with itself (homobivalent) or an association between three T cell surface (CD26 receptors (homomullivalcnt, e.g., homotrivalent) or an association between CD26 receptor with the T cell receptor (TCR/CD3) or with the CD4 receptor (heterobivalent).
For the most part, straightforward peptide coupling chemistry is employed. The standard peptide coupling chemistry methods and procedures used in this invention are readily available. Examples of books using these methods include, but arc not limited to, the following citations incorporated herein by reference: P.D. Bailey, An Introduction to Peptide Chemistry. Ed.: John Wiley & Sons, 1990; Miklos Bodansky, Peptide Chemistry. A Practical Textbook. Ed.: Springer- Verlag, 1988; Miklos Bodansky, Principles of Peptide Synthesis. "Reactivity and Structure Concepts in Organic Chemistry," Volume 16, Ed.: Springer- Verlag, 1984; and Miklos Bodansky, Principles of Peptide Synthesis. "Reactivity and Structure Concepts in Organic Chemistry," Volume 21 , Ed.: Springer- Verlag, 1984.
1. Flomomullivalcnt Compounds: General Structure The homobivalent and homomullivalcnt compounds taught herein can cither start with the general diagram for a bivalent template with non-identical binding moieties as shown in Fig. 1 A or with the general diagram for a homobivalent template as shown in Fig. IB. Thus, both sides of the chemical structure as shown in Figs. 1A and IB are binding moieties. Figs. 1C-1T are diagrams showing the structure of several preferred bivalent and multivalent compounds. Descriptions of the figures are provided above in the Brief Description of the Drawings.
2. Heterobivalent Compounds: General Structure The heterobivalent and heteromultivalent compounds and agents taught herein may begin with the following general diagram as shown in Fig. 2A, the general formula for a heterobivalent compound. Figs. 2A-2C are diagrams showing the general formula of several preferred heterobivalent compounds: Fig. 2 A is a diagram of a general heterobivalent template; Fig. 2B is a diagram of a heterobivalent example coupling a binding moiety to an MCC peptide (94-103) using a compatible linker, e.g., an AAAAAA (SEQ ID NO: 21) linker group where A is L-alanine or D-alaninc; and Fig. 2C is a diagram of a heterobivalent example coupling a binding moiety to a PLP peptide (139-151) using a compatible linker, e.g., an AAAAAA linker group where A is L-alanine or D-alanine.
For this invention, peptide, polypeptide, or fragment thereof, are used interchangeably and is defined to include a chain of amino acids ranging from about 3 to 50 residues, preferably from about 3 to 25 residues in length. More preferably, optimal size is on the order of about 10-18 amino acid residues in length.
Known antigenic peptides for any of the following autoimmune diseases, infectious diseases, allergic diseases, or cancers listed below and elsewhere in this document (without limitation) could be coupled to a bivalent template (see Fig. 2A), e.g., Xaa-boroPro, to form a heterobivalent compound (heterodimeric) molecule that could be used in this invention to treat that disease. Thus, once coupled or linked to the bivalent or multivalent template (see Fig. 2Λ), these peptides form different heterobivalent compounds which can alter biological -40-
activity (increased or reduced) as a result of bivalent interaction inducing the association of two receptors.
AUTOIMMUNE DISEASES AND KNOWN ANTIGENIC PEPTIDES
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
ALLERGIC DISEASES Ayp R?n.rN ANTTG NS
Figure imgf000045_0002
Listed below are examples of different known peptides with known specificity and high affinity for different T cell surface receptors. These peptides (P) can be coupled to Xaa-boroPro or coupled to another binding moiety as shown in Fig. 2A to form different heterobivalent compounds exhibiting altered biological activity (increased or reduced as a result of the specific binding to the specific receptor.
HETEROVIBALENT PEPTIDES
Figure imgf000046_0001
TCR: T cell Surface Receptor 3. Synthesis of H-boroPro and Xaa/Lys-boroPro Homobivalent, homomultivalent, heterobivalent, and heteromultivalent compounds can begin with the synthesis of H-boroPro and LysboroPro as taught herein. Use of H-boroPro and Lys-boroPro are for example purposes only, and is not intended to limit the scope of this invention. Thus, Xaa/Lys-boroPro is an example of a molecule that can be used to form a binding moiety of a bivalent, homobivalent, homomultivalent, or heterobivalent compound as taught herein.
Standard peptide coupling chemistry methods and procedures used in this invention are taught in the books identified at the beginning of this Example. In particular, H-boroPro was prepared by the synthetic route previously developed and described (G.R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function," PNAS (U.S.A.) 88, 1556-1559 (1991); also described in United States Patent No. 5,462,928) Alternatively, H-boroPro may be produced by a new procedure (Kelly, T.A., et al., "The efficient synthesis and simple resolution of a proline boronate ester suitable for enzyme inhibition studies," Tetrahedron 49, 1009-1016 (1993)). Both of these synthetic routes yield racemic H-boroPro pinanediol.
Stercochemically pure L, L and L, D diastereomcrs of Z-Lys-boroPro were prepared by first resolving racemic H-boroPro through crystallization with optically active blocking protecting groups ((IS, 2S, 3R, 5S)-+-pinanediol isomer) followed by coupling the isotopically pure L-boroPro and D-boroPro to the stercochemically pure L isomer of lysine (See United Stales Patent No. 5,462,928). Alternatively, the L,L and L,D diastereomcrs of Lys-boroPro were prepared in high optical purity by coupling racemic H- boroPro by L-Lys and separating the resulting diastcrcomcric Z-Lys-boroPro-dicster into its component L,D and L,L diastereomers using reverse phase HPLC as previously described for diastereomeric Pro-boroPro (W.G. Gutheil and W.W. Bachovchin, "Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition," Biochemistry 32, 8723-8731 (1993)). Thus, there are several routes through which to obtain any of the four possible stereoisomers of Lys-boroPro. However, only the DP IV inhibitory L,L isomer and the DP IV non inhibitory L,D isomer were normally prepared for use as a control in the immunological experiments. In general, the derivatives prepared herein use optically pure diastereomers and therefore contain only either the L,L or the L,D isomer of Lys-boroPro.
Once prepared, these Xaa-boroPro compounds are coupled to other Xaa- boroPro compounds, e.g., itself, to form a homobivalent (homodimeric) or multivalent compound or coupled to a non-Xaa-boroPro peptide thereby forming a heterobivalent (heterodimeric) or multivalent compound.
EXAMPLE 2
SYNTHESIS OF HOMOBIVALENT AND HETEROBIVALENT COMPOUNDS AND ASSESSMENT OF FUNCTIONAL ACTIVITY
Homobivalent, low molecular weight compounds capable of inducing an association, between two CD26 receptors arc taught in this Example. Homobivalent synthetic crosslinking compounds have the properties normally associated with an antibody, i.e., high affinity, and specificity for CD26, and an ability to induce crosslinking. Thus, any experiment with an anti-CD26 monoclonal antibody can be performed with one or more of the homobivalent compounds, e.g., immunoprecipitations.
The synthetic ligands, however, have some properties that make them complimentary to anti-CD26 mAbs. These include: (i) their binding epitope is the DP IV active site; (ii) they exhibit cross-species specificity; and (iii) they offer flexibility in adjusting the spacing between the binding sites and the construction of chimeric or hclcrobidcntatc structure.
I. Synthesis of Homobivalent Xaa-boroPro Derivatives and Heterobivalent Compounds
To produce molecules which induce the association between one cell surface CD26 with another cell surface CD26 and also retain DP IV inhibitory activity, a series of different homobivalent derivatives of Lys-boroPro will be linked via their e-amino groups by a linker-spacer molecule containing two carboxylic acid groups, e.g., a six carbon iinker spacer or linker group, using conventional peptide coupling methods (see Fig. 3). Fig. 3 is a diagram showing the synthesis of adipoyl (Lys-boroPro)2, a homobivalent derivative of Lys-boroPro. This linkage method included coupling benzyloxycarbonyl- lysine-boroPro-diester (Z-lys-boroPro-diester) to a linker molecule, e.g., adipic acid or hexanedioic acid (HOOC(CH2)4COOH). The diester protecting group on the boronyl moiety can either be pinacol or pinanediol.
The general structure for this homobivalent (homodimeric) compound is shown in Fig. IB:
Y1
/ Y3
D1 ~ A1 - A2 - A3 - A4 - B \ D1 - A1 - A2 - A3 - A4 - B /
Y2
Y4
wherein Dl, is independently selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents an isotope of hydrogen; "-", independently, is selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from a group consisting of a C, a CS moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 are, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an 0, wherein X and Z, are, independently, selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope o oxygen; wherein each Yl, Y2, Y3, and Y4 are, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiologic conditions; and L represents a linker molecule (i) having a olecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous.
The structure as shown above need not be identical, in that it can have the general structure as shown in Fig. 1 A:
Figure imgf000050_0001
wherein Dl and D2, independently, are selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "~", independently, is selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al and A5 are, independently, selected from a group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, A4, A6, A7, and A8 are, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O wherein X and Z, are, independently, selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl, Y2, Y3, and Y4 arc, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiologic conditions; and L represents a linker molecule (i) having a molecular weight ranging between about 100 daltons and about 200 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous.
The coupling reaction can be achieved by any of several standard peptide coupling methodologies. For example, the Lys-boroPro homobivalent (homodimeric) derivative was prepared by reacting protected Lys-boroPro in anhydrous THF with the acid chloride form of adipic acid, i.e., adipoyl chloride, which is commercially available (Aldrich Co.). Following coupling, the N terminal Z protecting group was removed by catalytic hydrogenation. Deprotection of the boronyl group was achieved by transesterification with phenyl boronic acid and extracted using a two phase, low pH water solution/organic solvent.
II. Determination of Optimal Chemical Spacer Linkers
To determine the optimal spacer linker segment or linker molecule for inducing the association of one cell surface CD26 with another cell surface CD26 receptor, a scries of bivalent, dimeric Lys-boroPro derivatives with varying length spacer segments will be prepared (See discussion below on linker molecules). A wide assortment of dicarboxylic spacer linker molecules are commercially available. This includes linker molecules which have various internal heteroatoms and other functional groups, in addition to the terminal carboxylic groups, e.g., ethylcne glycolbissuccinate ("EGS", shown in Fig. 13). For example, using EGS in place of adipic acid provides a bivalent compound with a spacer of about twice the length of the adipoyl moiety. Also, the internal heteroatoms confer improved water solubility over a straight chain hydrocarbon of similar length.
Fig. 13 gives the structure of EGS, which was used as the linker molecule joining two KbP monomers, to form (KbP)2 EGS. The synthesis of this homodimer was carried out in a manner analogous to that described herein for KbP2 adipate, using appropriate modifications. EGS is commercially available from a variety of chemical supply companies.
Both (KbP)2 adipate and (KbP)2 EGS were used in experiments with the T cell line H9 to determine their effects on activation (as measured by II-2 production) and/or proliferation. Some of these experiments employed the homodimeric molecules as co- stimulatory factors with other, known T cell stimulatory factors such as the monoclonal antibody OKT3. The results of these experiments are shown in Figs. 5 and 12.
III. Experimental Methods To determine the effect KbP2-Adipate had on anli-CD3 mAb stimulation of H9 cells, e.g., stimulation of suppressing IL-2 production, the following protocol was used. In quadruplicate wells, H9 cells were preincubated with KbP2-Adipate for 0, 30, 60, or 150 min. After the preincubation, the cells were seeded into a 96 well flat bottom plate pre-coated with anti-CD3 monoclonal antibody OKT3 at 1 ,000, 5,000, 10,000, 20,000, 50,000, or 100,000 times dilution of stock. After 24 hr, the cells were lysed by freezing at 4°C. IL-2 concentration in the H9 cells lysed was bioassayed using the IL-2 dependent cell line HT2, Watson, J.D. "Continuous proliferation of murine antigen specific helper T lymphocytes in culture," 1979, Journal of Experimental Medicine*. J 50: 1510. HT2 proliferation was measured by counting 3H-thymidine incorporation.
Similar experiments were carried out with (KbP)2-EGS and the co-stimulatory antibody OKT3; the results of these experiments are shown in Fig. 12.
IV. Results
Figs. 4 and 5 are graphs showing a dose response curve observed with concentrations of KbP2-Adipate on anti-CD3 mAb stimulation of H9 cells, as manifested by 1L-2 production.
The inventors predict that the observed stimulatory effect with higher KbP2- Adipatc concentrations was due to intermolccular reactions between two different bivalent, dimeric molecules. It is suggested that intermolecular reactions between two different bivalent, dimeric molecules could form via a B-N bond, with the amino group of one divalent molecule binding to a boron atom of a second divalent molecule. This process can Continue to form polymers of various lengths. At equilibrium after polymerization, new bivalent compounds with linker spans greater than two times the size of the linker span of one dimeric compound may be formed. For example, the resultant new bivalent compound, as diagramed in Fig. 7, has lost a binding moiety from each dimer but now has a linker span of greater than twice the original linker span size. Fig. 7 is a diagram showing intermolecular reactions that may occur at higher concentrations of KbP2-
Adipate. Fig. 12 shows the results of the experiments carried out with KbP2-EGS, in which the two KbP monomers are linked by the EGS spacer, which is on the order of twice the length of the adipate spacer used to make KbP2-Adipate homodimer. The results of the experiments with KbP2-EGS exhibit a pattern analogous to the KbP2-Adipate results, as Fig. 12 illustrates. For example, there is a co-stimulatory effect with the T cell activating antibody OKT3. Further, the greatest stimulatory effect was observed at low concentrations (between 10*6 M and 10"12 M) of KbP2-EGS, and after the peak stimulatory effect (alone or with OKT3), increasing concentrations (10"5 M) decreased the activating effect observed.
EXAMPLE S HOMOMULTIVALENT POLYMERIC COMPOUNDS DESIGNED TO INDUCE ASSOCIATION BETWEEN T CELL SURFACE RECEPTORS
A variety of different examples of homomultivalent compounds are taught in this example. The standard peptide synthesis methods described above can be used to prepare these compounds. The general multivalent template has the following structure as shown in Fig.
IR:
Figure imgf000053_0001
wherein D is, independently, selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "-" is, independently, selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from the group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 are, independently, selected from the group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl and Y2 are, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiologic conditions; n represents an integer between 1 and 200, inclusive; wherein El and E3 are distinct reactive species in which:
(a) R and R' arc the remainder of the molecules not relevant to this reaction;
(b) E 1 is attached to R' by a covalent bond which are together designated as El-R' or R'-El ; * . (c) E3 is attached to R by a covalent bond which are together designated as E3-R or R-E3;
(d) R' represents the part of El-R' not undergoing a chemical reaction;
(e) R represents the part of R-E3 not undergoing a chemical reaction;
(f) El undergoes a chemical reaction with E3 to form the product El'-E3' and a byproduct F, wherein F is selected from the group consisting of
2H* and 2e" , H2O, and any other byproduct;
(g) where IT is the cation of any isotope of hydrogen and e" is an electron; (h) where II represents any isotope of hydrogen and O represents any isotope of oxygen; (i) where El' and E3' are covalently bonded;
(j) El docs not undergo a chemical reaction with another El ; (k) E3 does not undergo a chemical reaction with another E3; and (I) El and E3 arc selected from the group consisting of a carboxylatc, amino, imidazole, sulfliydryl, aldehyde, ester, and any other reactive species; whcrcin [Jjp, E2, [Ijq and [G]m together arc a linker moiety, and wherein [G]m, [J]p, and [I]q represent, independently, linker molecules (i) having a molecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous; and wherein m, p, and q represent, independently, an integer from 1 to 50, inclusive and wherein E2 is selected from the group consisting of CX, CH, N, PhYZ, PhU, and any other moiety capable of forming covalent bonds with [J]p, [G]π„ and [I]q and wherein:
(a) C is any isotope of carbon;
(b) X is any isotope of any atom capable of forming a single bond with carbon;
(c) H is any isotope of hydrogen; (d N is any isotope of nitrogen;
(e) Ph is any isotope of phosphorous;
(f) Y is any isotope of any atom capable of forming a single bond with phosphorous;
(g) Z is any isotope of any atom capable of forming a single bond with phosphorous; and
(h) U is any isotope of any atom capable of forming a double bond with phosphorous. Also, the figures shown below represent the binding moiety and R represents the remainder of the molecule in this polymeric compound:
Y1
D1 - A1 - A2 ~ A3 ~ A4 B
Y2
and
Figure imgf000056_0001
The multivalent compound of this invention can range from a dimer, e.g., n is equal to one (1) or up to about 50-mcr, e.g., n is equal to forty-nine (49). When the binding moiety is repeated more than 2 times (a dimer) then the compound would necessarily be a "polymeric" compound with a finite number of repeating binding moieties. EXAMPLE 4
SYNTHESIS OF HETEROBIVALENT COMPOUNDS DESIGNED
TO INDUCE ASSOCIATION BETWEEN CD26 RECEPTOR
AND THE T CELL SURFACE RECEPTOR (TCR/CD3)
Constructing heterobivalent (also referred to as heterobidentate or heterodimeric), compounds or agents yields a class of agents capable of inducing an association between CD26 and distinct cell surface receptors. Such heterobifunctional molecules have interesting biological activities and may be useful as drugs.
The general structure for a heterobivalent compound of this invention as shown in Fig. 2A:
Figure imgf000056_0002
wherein D is independently selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "~", independently, is selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from the group consisting of a C, a CX moiety, and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3, and A4 arc, independently, selected from the group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl and Y2 are, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiologic conditions; L represents a linker molecule (i) having a molecular weight ranging between about 100 daltons and about 200 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous; and P represents a peptide ranging from 3 to 30 amino acids having sufficient sequence homology to bind to a naturally occurring receptor. P can be a peptide which selectively binds to CD26 but which does not include a reactive group for forming a covalent bond or complex vith the amino acids in the reactive center of the CD26. Such a compound is considered a heterobivalent compound because only one reactive group is present.
Two different heterobivalent compounds (heterodimers) of Lys-boroPro have been constructed and tested. These two compounds arc presented as examples only and are not intended to limit the invention. In one heterobivalent compound, Lys-boroPro is linked to the C terminal carboxylalc of enccphalitogcnic myelin proteolipid protein (PLP 139-151 ; Fig. 2C; sec below for discussion). In the second hctcro-compound, Lys- boroPro is linked to an antigenic moth cytochrome C peptide (MCC; Fig. 2B; see below for discussion). Both compounds were designed such that association between CD26 and another T cell receptor (TCR/CD3) would be induced. The data presented below demonstrate that both heterobidentate molecules were much more stimulatory than using the peptides alone.
1. Synthesis of Lys-boroPro Linked to Myelin Proteolipid Protein (PLP)
Peptide 139-151 to Induce Association Between CD26 Receptor and the T Cell Surface Receptor (TCR/CD3) Proteolipid protein (PLP) is the major protein of central nervous system myelin. Kuchroo and co-workers have shown that mice immunized with a peptide corresponding to residues 139-151 of PLP, HSLGKWJLGHPJDKF (SEQ.ID.NO.22) (PLP 139- 151 ) develop acute experimental autoimmune encephalomyelitis (Kuchroo, V.K., et al., Induction of experimental allergic encephalomyelitis by myelin proteolipid-protein specific T cell clones and synthetic peptides, Pathobiology 59, 305-312 (1991); Kuchroo, V.K., et al., T-cell receptor alpha chain plays a critical role in antigen-specific suppressor eel! function, PNAS (U.S.A.) 88, 8700-8704 (1991 ); Kuchroo, V.K., et al, Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage, J. Immunol. 148, 3776-3782 (1992); Kuchroo, V.K., et al., Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T cell clones to mediate experimental allergic encephalomyelitis, J. Immunol. 151 , 4371 -4382 (1993); Kuchroo, V.K., ct al., T cell receptor (TCR) usage determines disease susceptibility in experimental autoimmαue encephalomyelitis: studies with TCR V beta 8.2 transgenic mice, Journal of Experimental Medicine 179, 1659-1664 (1994); and Kuchroo, V.K., et al., A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediate by a diverse T cell repertoire, J. Immunol. 153, 3326-3336 (1994)). PLP 139-151 also induces the proliferation of T cells in culture. The mechanism involves the T cell receptor (TCR) recognition and binding of this peptide within the context of the major histocompatibility complex (MHC) class II. The MHC is a cluster of genes on human chromosome 6 or mouse chromosome 17 that encodes the MHC molecules. The MHC class I molecules or proteins arc the present peptides generated in the cytosol to CD8 T cells. The MHC class II molecules or proteins are the present peptides degraded in cellular vesicles to CD4 T cells. The MHC is the most polymorphic gene cluster known to date in the human genome, having large numbers of allclcs al several different loci. Because this polymorphism is usually detected using antibodies or specific T cells, Ihe MHC proteins are often called "major histocompatibility antigens." This allows easy manipulation of the antigenic peptide to convert it from an agonist to an antagonist (Jorgensen, J.L., et al., Molecular components of T-cell recognition, Anmi. Rev. Immunol. 10, 835-873 91992)). Systematic amino acid replacement studies have demonstrated that Trp 144 and His 1 7, shown in bold in the sequence, are necessary for TCR binding while Leu 145 and Pro 148, shown underlined in the sequence above, are necessary for MHC binding.
Crystal structure data on the MHC class II receptor shows that the cleft on top of the molecule which binds the antigenic peptide is open on both sides, which allows longer pcplides to be presented by simply permitting them to extent away from the MHC receptor. In contrast, MHC class I receptors only accommodate short peptides from 9 to 12 amino acids and the antigenic peptide ends are not free. The above facts therefore suggest that a bivalent, heterodimer of Lys-boroPro linked to PLP 139-151 could be constructed that would simultaneously bind to the T cell surface receptor (TCR) and CD26 (see Fig. 8) and the MHC II on the antigen presenting cell. Fig. 8 is a diagram showing Lys-boroPro linked to Myelin Proteolipid Protein (PLP) Peptide 139-151.
In the case of PLP, the heterodimer was constructed as HSLGKWLGHPDKF AAAAAA-eKbP (SEQ.ID.NO. 23 - eKbP) where HSLGKWLGHPDKF (SEQ.ID.NO. 22) was PLP 139-151 , AAAAAA (SEQ.ID.NO. 21) was a linker comprised of 6 alanincs and eKbP was Lysinc-boroProlinc in which the e- amino of Lysine is covalently attached to the -COOH terminus of
HSLGKWLGHPDKFAAAAAA (SEQ.ID.NO. 23). The first synthetic step was to order a custom peptide from a synthetic peptide lab. Using long established protocols, the peptide was built from the C-terminus staring with alanine which was immobilized on a resin. Sequentially AAAAA FKPHGLWKGLSH (SEQ.ID.NO.25) were added using protected amino acids. The peptide was then removed from the resin to give a free -COOH terminus which could be reacted lo form a peptide bond. The other residues HSLGKWLGHPDKFAAAAA (SEQ.ID.NO. 24) were unreactive owing to protecting groups. Lysine-boroProline in which the α-NH2 of Lysine was protected, the B(OH)2 of boro Prolinc was protected with pinanediol, and the e-NH2 of Lysine was free was coupled to the peptide. The coupling was a peptide bond (-(C=0)-NH-) formed by standard peptide chemistry techniques. The result was then deprotectcd to yield the final product.
The spacer linker consisting of six consecutive Ala residues was chosen to provide a span sufficient to permit crosslinking (~3θA). The proliferative effect of PLP-S-KbP on several different T cell clones that recognize PLP 139-151 were tested. T cell clones that recognize an irrelevant epitope, were used as negative controls. The protocol used is described (Kuchroo, V.K., et al., B7- 1 and B7-2 Costimulatory Molecules Activate Differently the Thl Th2 Developmental Pathways: Application to Autoimmune Disease Therapy, Cell 80, 707-718 (1995)) and is incorporated herein by reference. Proliferation was measured by thymidine uptake. Table 1 below compares the effect PLP-S-KbP, antigenic PLP 139-151 peptide and non- antigenic PLP 103-1 16 peptide has on proliferation in five different PLP 139-151 specific T cell clones. The results show that PLP-S-kbP strongly enhances the proliferative response lo PLP 139-151 of all five 139-151 specific clones (see Table 1 below). Enhancement ranges from 100 to over 1000-fold, with respect to the concentration needed to produce a given response. For example, against the first T cell clone listed, 5B8.G8.E6.H12, 0.1 μM of PLP-S-KbP induces almost double the response that a 100-fold higher concentration of PLP 139-K51 itself induces, for an enhancement of almost 200-fold. Similarly, nearly a 2000-fold enhancement is induced in the 4E3.B1 1.D9.H10.H6 cells since 0.1 μM of PLP- S-KbP produces almost double the response a 1000-fold higher concentration of PLP 139- 151 itself induces.
These results indicate that a low molecular weight synthetic molecule designed to crosslink CD26 and the TCR, e.g., PLP-S-KbP, strongly enhanced the T cell response to the T cell receptor recognized antigen.
TABLE 1
PROLIFERATIVE EFFECT OF PLP-S-KBP ON SEVERAL
T CELL CLONES SPECIFIC FOR PLP 139-151
Dose (μM)
Figure imgf000061_0001
*3H counts II. Synthesis of Lys-boroPro Linked to Moth Cytochrome C Peptide (MCC) 94-103 to Induce Association Between CD26 Receptor and the T Cell Surface Receptor (TCR/CD3)
The Moth Cytochrome C (MCC) 94-103 peptide is another antigenic peptide employed in this invention. MCC strongly induces IL-2 production and T cell proliferation when added to cultures of the murine 2B4 T cell hybridoma. The critical residues for binding to the TCR receptor and to the class II MHC are known (Jorgensen, J.L., et al., Molecular components of T-cell recognition, Annu. Rev. Immunol. 10, 835-873 (1992)). Because residues critical for MHC and TCR binding are located near the C terminus, Lys- boroPro was coupled to the N terminus of this peptide using a spacer linker of about the same length as used to link Lys-boroPro to the PLP peptide. Standard coupling methodologies were employed. This molecule was designated KbP-S-MCC to signify coupling to the N terminus.
Fig. 9 compares the effect of the bivalent, dimeric KbP-S-MCC molecule and MCC 94-103 itself on IL-2 production in 2B4 cells. The 2B4 T cell hybridoma proliferates in responses to moth cytochrome C peptide 94-103. The peptide 2B4 system is ideal for these studies, since the contest points between MCC 94-103 and the TCR have been determined (Jorgensen, J.L., et al., "Molecular components of T-ccIl recognition," Annu. Rev. Immunol. 10, 835-873 (1992)). 2B4 cells were cultured at 10 5/well with H-2k APC's and with varying concentrations of KbP-S-MCC and MCC itself. After 24 hours the supernatant was harvested and the IL-2 content determined in a bioassay using HT-2 indicator cells in the same manner as previously described for the PLP peptide assay.
The results demonstrate that coupling Lys-boroPro to MCC strongly enhances the response to the antigenic MCC peptide. Even at the lowest concentration of KbP-S-MCC tested, e.g., 0.4 μM KbP-S-MCC, KbP-S-MCC induced a response at least double that of the maximum response obtained with the MCC peptide alone, which required ~ 10-fold higher concentration (see Fig. 9). These results indicate that a low molecular weight synthetic molecule designed to crosslink CD26 and the TCR, e.g., KbP-S-MCC, strongly enhanced the T cell response to the T cell receptor recognized antigen.
III. Synthesis of Lys-boroPro Linked to Other Peptides to Induce Association Between CD26 Receptor and the T cell Surface Receptor (TCR/CD3)
This example describes the synthesis of a preferred binding moiety-reactive group (Lys-boroPro) to certain peptides (e.g., PLP peptides, MCC peptides). It is contemplated that the procedures also can be used for synthesizing compounds in which Lys-boroPro (or an alternative) is coupled to a tumor antigenic peptide or an antigenic peptide of infectious disease. Other heterodimers comprising various derivatives of the PLP or the MCC peptide, which were previously shown to be antagonistic, will be prepared and studies will be performed to determine if linking these peptides to Lys-boroPro enhances their antagonistic activity or transforms them into an agonistic molecule.
Also, tetanus toxoid peptide P2 is of interest because it provides a test system involving human peripheral blood mononuclear cells (PBMC) (Wyse-Coray, T, et al., "Use of antibody/peptides constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation," Cellular Immunology, 139(l):268-73, (1992)). It has been shown that tetanus toxoid peptide P2 peptide induces a response in all HLA haplotypes tested so far (Panina-Bordignon, P., et al., "Universally immunogenic T cell epitopes: Promiscuous binding to human MHC class II and promiscuous recognition by T cells," Eur. J. Immunol. 19, 2237-2242 (1989)). Thus, bivalent, heterodimers of Lys-boroPro coupled (linked) to the P2 peptide of tetanus toxoid will be prepared. Different spacer linkers of varying sizes will be tested to determine the optimal length to be used with this peptide. The coupling chemistry needed to produce the bivalent, heterodimer, Lys-boroPro linked lo tetanus toxoid peptide P2 peptide, is the same as described above.
Additionally, other molecules capable of inducing the association between CD26 and the T cell surface receptor, e.g., TCR/CD3, can be prepared by coupling Lys-boroPro or other binding moiety to the C terminal or N terminal functional group of different peptides known to bind to the T cell receptor in the context of class II MHC receptors. Similar protocols as those used to prepare [PLP 139-151]-KbP-S-MCC and KbP-S-MCC, both of which are described above and which involve straightforward standard peptide coupling methodology, can be employed.
EXA PLE 5 HETEROBIVALENT COMPOUNDS DESIGNED TO
INDUCE ASSOCIATION BETWEEN CD26 RECEPTOR AND THE CD4 T CELL
SURFACE RECEPTOR
Heterobivalent compounds containing Xaa-boroPro designed to induce association between the CD26 receptor and the CD4 receptor also can be prepared. A molecule known to bind to CD4, e.g., peptides derived from the HIV-1 GP 120 protein, will be coupled to Xaa-boroPro (Ebcnbichler, C, et al., "Structure-function relationships of the HIV-1 envelope V3 loop tropism determinant: evidence for two distinct conformations," Aids 7, 639-46 (1993); Linsley, P.S., et al., "Effects of anti-gpl 20 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1," Journal of Virology 62, 3695-702 (1988)); Rini, J.M., et al., "Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen," PNAS (U.S.A.) 90, 6325-9 (1993)).
An Xaa-boroPro molecule, e.g., Lys-boroPro, will be coupled to one of the peptides from the HIV-1 GP 120 protein by using the coupling methodology as described above for coupling Lys-boroPro to the PLP peptide. Of course, any of the other non-boronyl reactive groups described herein can be used in place of the boronyl group to obtain an alternative embodiment.
Different spacer linkers of varying sizes can be evaluated to determine the optimal length for inducing the association between CD26 and CD4. EXA PLE 6
HETEROBIVALENT COMPOUNDS DESIGNED TO
INDUCE ASSOCIATION BETWEEN CD26 AND
OTHER T CELL SURFACE RECEPTORS
Mctcrobivalcnl compounds containing Xaa-boroPro designed to induce association between the CD26 receptor and other T cell surface receptors, e.g., granulocyte colony stimulating factor, can be prepared.
For example, cytokine granulocyte colony stimulating factor (G-CSF or granulocyte macrophage colony stimulating factor, GM-CSF), is produced by T cells and macrophages and binds to its own receptor on the T cell surface (the granulocyte colony stimulating factor receptor). A bivalent, heterodimeric form of granulocyte colony stimulating factor could enhance the potency of granulocyte colony stimulating factor by stimulating growth or differentiation or both in cells of myelomonocytic lineage. This compound can be prepared by using standard coupling methodology to couple granulocyte colony stimulating factor to Lys-boroPro. Also, different spacer linkers of varying sizes will be evaluated to determine the optimal length for inducing the association between CD26 receptor and the receptor for colony stimulating factor.
Stem cell factor (c-kit ligand) is essential in stem cell development and binds to the stem cell factor receptor on T cells. In B cell development, CD44 binding probably has no direct signaling function, but instead promotes the binding of a receptor known as c-kit. Lymphoid progenitor cells and early pro-B cells bind to hyaluronic acid on stromal cells via CD44, promoting the binding of their surface c-kit tyrosine kinase to stem cell factor (SCF) on the stromal cell surface, activating the kinase and inducing proliferation. A bivalent, heterodimeric form of stem cell factor, e.g., by coupling to Lys-boroPro, should enhance the potency of SCF.
Lys-boroPro-SCF, heterodimeric compound can be prepared by using methodologies similar to that described above. Different spacer linkers of varying sizes will be tested to determine the optimal length for inducing the association between CD26 receptor and the receptor for stem cell factor. EXAMPLE 7
SYNTHESIS OF A BIVALENT COMPOUND
LINKED TO AN ALL D-AMINO ACID PEPTIDE SYNTHESIZED
IN REVERSE TO PREVENT NATURAL PROTEOLYSIS
This example is designed to synthesize a bivalent compound linked to different peptides, whereby the peptides arc resistant to natural proteolysis, e.g., Xaa-boroPro coupled to "protected" PLP. In this Example, "protected" means the peptide has been synthesized in the reverse and has a change in chirality.
Retro-Inverso Isomers Evolution has ensured the almost exclusive occurrence of L-amino acids in naturally occurring proteins. Virtually all proteases therefore cleave peptide bonds between adjacent L-amino acids; thus, artificial proteins or peptides composed of D-amino acids are largely resistant to proteolytic breakdown. This resistance has been attractive to drug designers, but the exclusivity of biological systems for proteins made of L-amino acids means that such proteins cannot interact with the mirror image surface formed by enantiomeric proteins. Thus, an all D-amino acid protein usually has no biological effect or activity.
Linear modified retro-peptide structures have been studied for a long time (Goodman, M., et al., On the Concept of Linear Modified Retro-Peptide Structures, Accounts of Chemical Research, 12(1), 1-7 (January, 1979)) and the term "retro-isomer" was designated to include an isomer in which the direction of the sequence is reversed compared with the parent peptide. By "retro-inverso isomer" is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity. More recently, Jameson et al. reportedly engineered an analogue of the hairpin loop of the CD4 receptor by combining these two properties: reverse synthesis and a change in chirality (Jameson el al., A rationally designed CD4 analogue inhibits experimental allergic encephalomyelitis, Nature, 368, 744-746 (1994) and Brady, L. ct al., Reflections on a Peptide, Nature, 368, 692-693 (1994)). The net result of combining D-enantiomers and reverse synthesis is that the positions of carbonyl and amino groups in each amide bond are cxchanged, while the position of the side-chain groups at each alpha carbon is preserved. Jameson et al. reportedly demonstrated an increase in biological activity for their reverse D- peptide, which contrasts to the limited activity in vivo of its conventional all-L enantiomer (owing to its susceptibility to proteolysis). A partially modified retro-inverso pseudopeptide has been reported for use as a non- natural ligand for the human class I histocompatibility molecule, HLA-A2 (Guichard et al., Partially Modified Rctro-Invcrso Pscudopcptidcs as a Non-Natural Ligands for the Human Class I Histocompatibility Molecule HLA-A2, J. Med. Chem. 39, 2030-2039 (1996)). The authors report that such non-natural ligands had increased stability and high MHC-binding capacity.
Retroinverso peptides for incorporation in the compounds of the invention (e.g., as P2 peptides in the compound shown in formula I of the Summary of the Invention) are prepared for peptides of known sequence in the following manner. A peptide having a known sequence (e.g., a tumor antigen peptide) is selected as a model peptide for designing and synthesizing a retroinverso peptide analog. The analog is synthesized using D-amino acids by attaching the amino acids in a peptide chain such that the sequence of amino acids in Ihe retroinverso peptide analog is exactly opposite of that in the selected peptide which serves as the model. To illustrate, if the peptide model is a tumor antigen such as the MAGE-1 peptide Al (formed of L-amino acids) having the sequence, ADPTGHSY, the retroinverso peptide analog of this tumor antigen (formed of D-amino acids) would have the sequence, YSHGTPDA. The procedures for synthesizing a chain of D-amino acids to form the retroinverso peptides are known in the art and are illustrated in the above-noted references.
Since an inherent problem with native peptides is degradation by natural proteases, the heterobivalent or heteromultivalent compounds of this invention will be prepared to include the "retro-inverso isomer" of the desired peptide. Protecting the peptide from natural proteolysis should therefore increase the effectiveness of the specific heterobivalent or heteromultivalent compound.
The protocol given in Example 4, Section 1 , for synthesizing PLP 139-151-KbP can be used, with some modifications as necessary, to synthesize the retro-inverso containing heterodivalent compounds described above. The primary modifications are (1) to synthesize the peptide in reverse and (2) to use D-amino acids for starting materials.
The biological activity of the retro-inverso heterodimer-Lys-boroPro linked to retroinverso PLP 139-151 can be compared to the control heterodimer-Lys-boroPro linked to conventional PLP 139-151. Activation of T cells can be monitored as described previously in Example 4, Section 1.
A higher biological activity is predicted for the retro-inverso containing heterodimer when compared to the non-retro-inverso containing analog owing to protection from degradation by native proteinases.
EXAMPLE 8
OPTIMAL LINKER MOLECULE DETERMINATION
To determine the optimal linker molecule segment capable of inducing the association of one cell surface CD26 receptor with another cell surface CD26 receptor or other rcccr3tor, a scries of bivalent, dimeric compounds, with varying length spacer segments (linker molecules), is prepared. A wide assortment of dicarboxylic spacer linker molecules are commercially available (see below for discussion). This includes linker molecules which have various internal heteroatoms and other functional groups, in addition to the terminal carboxylic groups, e.g., cthylene glycobissuccinatc.
The corresponding span of the binding sites for monoclonal antibodies varies owing to individual flexibility but is typically about 30 A to about l OOA. Thus, most linker molecules designed for constructing the homobivalent and heterobivalent compounds of this invention will fall within the general range of about 20 A to about 300 A; and more specifically, from about 30 A to about 100 A. For example, the spacer linker consisting of six consecutive Ala residues (as used above) was chosen because it was the smallest length spacer believed to be sufficient to crosslink (-30 A).
Using the linker cthylene glycobissuccinatc (instead of adipic acid) provides a bivalent compound with a spacer of about twice the length of the adipoyl moiety. Also, the internal heteroatoms may confer improved water solubility over a straight chain hydrocarbon of similar length. A. Background on Applicable Linker Molecules
Chemical cross-linkers are valuable tools for scientists and are discussed in numerous books and catalogues, e.g., Pierce Catalog and Handbook, Rockford, HI. These reagents arc used to assist in the determination of near-neighbor relationships in proteins, three-dimensional structures of proteins, enzyme-substrate orientation, solid-phase immobilization, hapten-carrier protein conjugation and molecular associations in cell membranes. They are also useful for preparing antibody-enzyme conjugates, immunotoxins and other labeled protein reagents.
Conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits. The number of components should be kept low or to a minimum. Like many applications, it is necessary to maintain the native structure of the protein complex, so cross-linking agents often employ functional groups that couple to amino acid side chains of peptides. Bifunctional reagents are classified on the basis of the following: P. , Functional groups and chemical specificity
2. Length of cross-bridge
3. Whether the cross-linking groups are similar (homobifunctional) or different (hcterobifunctional)
4. Whether the groups react chemically or photochemically 5. Whether the reagent is clcavablc
6. Whether the reagent can be radio-labeled or tagged with another label.
Reactive groups that can be targeted using a cross-linker include primary amines, suifhydryls, carbonyls, carbohydrates and carboxylic acids (to be discussed below). In addition, any reactive group can be coupled nonselcctively using a cross-linker such as photorcactive phenyl azides.
Cross-linkers are available with varying lengths of spacer arms or bridges. These bridges connect the two reactive ends. The most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites for cross-linking, different lengths of bridges are required for the interaction. Normally, a cross-linker with a short spacer arm (4-8 A) is used and the degree of cross-linking is determined. If this is unsuccessful, a cross-linker with a longer spacer arm is used. Shorter spacer arms are often used in intramolecular cross-linking studies. Intermolecular cross-linking is favored with a crosslinker containing a longer space arm.
Many factors must be considered to determine optimum cross-linker-to-protcin molar ratios. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is normally desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal to ensure that the biological activity of the protein is retained. It is also important to consider the number of reactive groups on the surface of the protein. If there are numerous target groups, a lower cross-linkcr-to-protein ratio can be used. For a limited number of potential targets, a higher cross-linker-to-protein ratio may be required. This translates into more cross-linker per gram for a small molecular weight protein.
Conformational changes of proteins associated with a particular interaction may also be analyzed by performing cross-linking studies before and after the interaction. A comparison is made by using different arm-length cross-linkers and analyzing the success of conjugation. The use of cross-linkers with different reactive groups and/or spacer arms may be desirable when the conformation of the protein changes such that hindered amino acids become available for cross-linking. Conjugation reagents contain at least two reactive groups. Homobifunctional cross- linkers can contain at least two identical reactive groups, and hetcrobifunctional reagents contain two or more different reactive groups. Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources.
B. Homobifunctional Cross-linkers
Homobifunctional cross-linkers have at least two identical reactive groups and more often arc used in a one-step reaction procedure in which the compounds to be coupled are mixed, and the cross-linker is added to the solution. This cross-linking method may result in self-conjugation, intramolecular cross-linking and/or polymerization. 1. Primary Amine-Reactive Groups
There are two major types of homobifunctional imidoesters and homobifunctional N-hydroxysuccinimidyl (NHS) esters. Commercially available homobifunctional imidoesters range in size from about 8 A to about 1 1.9 A. Commercially available homobifunctional N-hydroxysuccinimidyl esters range in size from about 6.2 A to about 16.1 A.
Because primary amines are commonly found in proteins, homobifunctional NHS ester cross-linkers are the most commonly used conjugation reagents. Both yield stable derivatives.
2. Sulfhyclryl-Reactive Group
Maleimides, alkyl and aryl halides, α-haloacyls and pyridyl disulfides are thiol reactive groups. These reagents react faster with sulfhydryls, making them thiol-selective. Maleimides, alkyl and aryl halides, and α-haloacyls react with sulfhydryls to form thiol either bonds. Pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable.
3. Nonselective Group
A nonselective homobifunctional is useful for conjugating functional groups, such as hydroxyls for which specific cross-linkers are not available.
An example of a nonselective homobifunctional cross-linker is BASED (Product #21564 Pierce Co.). This cross-linker has a long spacer arm and 2 aromatic rings which makes it very hydrophilic with a limited solubility in aqueous systems. This cross-linker also has a large diffusion capacity and should permeate membranes before conjugation initiates.
C. Heterobi functional Cross-linkers Heterobifunclional cross-linkers possess two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation. Heterobifunctional reagents frequently are used when modification of amines is problematic. Amines may sometimes be found at the active sites of macromolecules, and the modification of these may lead to the loss of activity. Other moieties such as sulfhydryls, carboxyls, phenols and carbohydrates may be more appropriate targets. A two-step strategy allows for the coupling of a protein that can tolerate the modification of its amines to a protein with other accessible groups. 5 A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation are commercially available. The majority of commercially available heterobifunctional cross-linkers contain an amine-reactive functional group. Cross-linkers that are amine-reactive at one end and sulfhydryl-rcactivc at the other end arc quite common. Commercially available heterofunctional cross-linkers range in size from about 0
I o A (EDC, Pierce Co.) to about 15.6 A.
When using heterobifunctional reagents, the most labile group should be reacted first to ensure effective cross-linking and avoid unwanted polymerization. Most heterobifunctional cross-linkers are sulfhydryl-reactive NHS-esters. The sulfhydryl reactive groups are usually maleimides, pyridyl disulfides and α-haloacetyls.
15 Carbodifmidcs are carboxyl and aminc reactive.
Heterobifunctional reagents, in which the reactivity can be controlled and that contain one group that is spontaneously non-reactive, have distinct advantages. This allows for specific attachment of the labile group first; the second reaction can then be initiated when appropriate. A selection of heterobifunctional reagents that contain at least one 0 photoafilnity group are commercially available. This selection includes iodinatable and cleavable reagents that react nonspecifically at the azido group and with amines, sulfhydryls, carbohydrates and carbonyls. Often a bifunctional photoactivatable crosslinker has a better chance of forming a covalent cross-link than a bifunctional chemically reactive cross-linker. The high reactivity of the photochemical reagent allows for formation 5 of a conjugate that may not be possible with a group-specific reagent. However, the yield resulting from a pholoreactive cross-linker is low, and yields of less than 10% should be considered acceptable. D. Reactivities of Different Chemical Groups 1. Imidoester Cross-linkers
Imidoester homobifunctional cross-linkers were among the first used to immobilize proteins onto solid-phase supports. They were used extensively for the study of protein structure and molecular associations in membranes. Although these cross-linkers are still used in protein subunit studies and solid-phase immobilization, they have been steadily replaced by the more stable, more efficient homobifunctional NHS-ester cross-linkers. Homobifunctional imidoesters maintain the net electronic charge on protein after crosslinking. Spacer arm lengths range from about 8.6 A to about 1 1.9 A. Imidoester cross- linkers react rapidly with amines at alkaline pH, but they have short half lives.
Imidoesters are also very useful for protein-protein cross-links. These cross-linkers can penetrate cell membranes and cross-link proteins within the membrane to study membrane composition, structure and protein-protein and protein-lipid interactions. Imidoesters are also useful for oligomer formation. For example, cross-linking proteins to form oligomers may reveal if a bivalent, dimeric or trimcric form of the protein is responsible for activity.
2. N-Hydroxysuccinimide-Esters fNHS-estersl
NHS-esters yield stable products upon reaction with primary or secondary amines. Coupling is efficient at physiological pH, NHS-ester cross-linkers arc also more stable in solution than their imidate counterparts. Homobifunctional NHS-ester conjugations are commonly used to cross-link amine-containing proteins in either one-step or two-step reactions.
Primary amines are the principle targets for NHS-esters. Accessible α-amine groups present on the N-termini of proteins react with NHS-esters and form amides. However, because α-amincs on a protein are not always available, the reaction with side chains of amino acids become important. While five amino acids have nitrogen in their side chains, only the e-amines react significantly with NHS-esters. A covalent amide bond is formed when the NHS-ester cross-linking agent reacts with primary amines, releasing N- hydroxysuccinimidc. 3. Coupling through Sulfhydryl Groups
Coupling through sulfhydryl groups is advantageous because it can be site-directed, yield cleavable products and allow for sequential coupling. A protein in a complex mixture can be specifically labeled if it is the only one with a free sulfhydryl group on its surface. a. Maleimides
The maleimide group is specific for sulfhydryl groups when the pH of the reaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reaction of the maleimides with sulfhydryls is 1000-fold faster than with amines. Maleimides do not react with tyrosines, histidines or methionines. b. Haloacetyls
The most common used α-Haloacetyls react with sulfhydryl groups at physiological pH. The reaction of the iodoacetyl group with a sulfhydryl proceeds by nucleophilic substitution of iodine, with a thiol producing a stable thioether linkage. Selectivity for sulfhydr l .groups is ensured by using only a slight excess of the iodoacetyl group over the number of sulfhydryl groups at pH 8.3. In the absence of free sulfhydryls, or if a gross excess of iodoacetyl group is used over the number of sulfhydryls, the iodoacetyl group can react with other amino acids. c. Pyridyl Disulfides Pyridyl disulfides react with sulfhydryls groups to form a disulfide bond. Pyridine- 2-thionc is released as a by-product of this reaction. These reagents can be used as cross- linkers and to introduce sulfhydryl groups into proteins.
4. Coupling Through Carboxyl Groups: Carbodiimidcs Carbodiimides couple carboxyls to primary amines or hydrazides, resulting in formation of amide or hydrazone bonds. Carbodiimides are unlike other conjugation reactions in that no cross-bridge is formed between the molecules being coupled. Carboxy termini of proteins can be targeted, as well as glutamic and aspartic acid side chains. In the presence of excess cross-linker, polymerization is likely to occur because proteins contain carboxyls and amines. No cross-bridge is formed, and the amide bond is the same as a peptide bond, so reversal of the cross-linking is impossible without destruction of the protein. EDC (Pierce Co.) reacts with carbocyclic acid group and activates the carboxyl group, allowing it to be coupled to the amino group (R4NH2) in the reaction mixture.
5. Nonselective Labeling: Arylazides
A photoaffinity reagent is a compound that is chemically inert but becomes reactive when exposed to ultraviolet or visible light. Arylazides are photoaffinity reagents that are photolyzed at wavelengths between 250-460 nm, forming a reactive aryl nitrene. The aryl nitrcnc reacts nonsclcctivcly to form a covalent bond. Reducing agents must be used with caution because they can reduce the azido group.
6. Nonselective Labeling a. Arginine Specific Cross-linkers
Glyoxals are useful compounds for targeting the guanidinyl portion of arginine residues. Glyoxals will target arginines at mildly alkaline pH. There is some cross- reactivity (the greatest at higher pH) with lysines. b. Carbonyl Specific Cross-Linkers Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH 5-7.
The reaction with hydrazides is faster lhan with amines, making this useful for site-specific cross-linking. Carbonyls do not readily exist in proteins; however, mild oxidation of sugar moieties using sodium metapcriodate will convert vicinal hydroxyls to aldehydes or ketones.
7. Applications for Use of Cross-linkers a. Cell Surface Cross-linking To ensure cell-surface specific cross-linking for identification of surface receptors or their ligands, it is best to use membrane-impermeable cross-linkers. In the past, researchers used water-insoluble cross-linkers and carefully controlled the amount of cross- linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and subsequent reaction with membrane proteins. Many references cite the use of membrane-permeable cross-linkers for cell surface cross-linking. b. Subunit Cross-linking and Protein Structural Studies Cross-linkers can be used to study the structure and composition of proteins in biological samples. Some proteins are difficult to study because they exist in different conformations under varying pH or salt conditions. One way to avoid conformational changes is to cross-link the subunits together. Amine-, carboxyl- or sulfydryl-reactive reagents are employed for identification of particular amino acids or for the determination of the number, location and size of subunits in a protein. Short-to-medium spacer arm cross-linkers are selected when intramolecular cross-linking is performed. If the spacer arm is too long, intermolecular cross-linking can occur. Carbodiimides that result in no spacer arm, along with short length conjugating reagents are commercially available. c. Subunit Cross-linkers and Protein Structural Studies Cross-linkers are widely used for identification of near-neighbor protein relationships, ligand-receptor identification and interactions, and enzyme substrate orientations. The cross-linkers chosen for these applications are usually longer than those used for subunit cross-linking. Homobifunctional, amine-reactive NHS-esters or imidates and heterobifunctional, amine-reactive photoactivatable phenyl azides are the most commonly-used cross-linkers for these procedures. Occasionally, a sulfhydryl- and amine- reactive cross-linker may be employed if one of the two proteins or molecules is known to contain sulfhydryls. Cleavable or noncleavable cross-linkers typically are used. Because the distances between two molecules are not always known, the optimum length of the spacer arm of the cross-linker may be determined by the use of a panel of similar cross- linkers with different lengths. NHS-ester, phenylazides are very useful for this type of cross-linking because they usually result in some successful, if not efficient, cross-linking. Cross-linkers can be used to determine whether a particular protein is located on the surface or integral to the membrane. These studies are possible because water-soluble cross-linkers are membrane-impermeable, while water-insoluble cross-linkers are membrane permeable. d. Cell Membrane Structural Studies
Cell membrane structural studies require reagents of varying hydrophobicity to determine the location and the environment within a cell's lipid bilayer. Fluorescent tags are used to locate proteins, lipids or other molecules inside and outside the membrane. Various cross-linkers with differing spacer arm lengths can be used to cross-link proteins to associated molecules within the membrane to determine the distance between molecules. Successful cross-linking with shorter cross-linkers is a strong indication that two molecules are interacting in some manner. Failure to obtain cross-linking with a panel of shorter cross-linkers, while obtaining conjugation with the use of longer reagents, generally indicates that the molecules arc located in die same part of the membrane but are not interacting. Homobifunctional NHS-esters, imidates or heterobifunctional NHS-esters, photoactivatablc, phenyl azides are commonly used for these procedures.
8. Immunotoxins Specific antibodies can be covalently linked to toxic molecules and then used to target antigens on cells. Often these antibodies are specific for tumor associated antigens. Immunotoxins are brought into the cell by surface antigens and, once internalized, they proceed to kill the ceil by ribosome inactivation or other means. The type of cross-linker used to niaj c an immunotoxin can affect its ability to locate and kill the appropriate cells. For immunotoxins to be effective, the conjugate must be stable in vivo. In addition, once the immunotoxin reaches its target, it is important that the antibody be separable from the toxin to allow the toxin to kill the cell. Thio-cleavable, disulfide-containing conjugates have been shown to be more cytotoxic to tumor cells than nonclcavable conjugates of ricin A immunotoxins. Cells are able to break the disulfide bond in the cross-linker, allowing the release of the toxin within the targeted cell.
9. Carrier Protein-Hapten/Peptide/Polypeptide Conjugates for Use as Immunogens
Companies, e.g., Pierce Co., offer products in this area of immunological research. Easy-to-use kits are available for coupling ligands using several different chemistries. There arc many cross-linkers used for the production of these conjugates, and the best choice is dependent on the reactive groups present on the haptcn and the ability of the hapten-carrier conjugate to function successfully as an immunogen after its injection. Carbodiimides are good choices for producing peptide carrier conjugates because both proteins and peptides usually contain several carboxyls and primary amines.
Other heterobifunctional cross-linkers can also be used to make immunogen conjugates. Often peptides are synthesized with terminal cysteines to allow for their attachment to supports or to carrier proteins through a part of the molecule that is not important for activity or recognition. Suifhydryl-reactive, heterobifunctional cross-linkers can be coupled to carrier proteins through their other functional group and then can be linked to peptides through terminal cysteines. This method can be very efficient and yield an immunogen that is capable of eliciting a good response upon injection.
10. Solid-Phase Immobilization
Proteins, peptides and other molecules can be immobilized on solid-phase matrices for use as affinity supports or for sample analysis. The matrices may be agarose, beaded polymers*, polystyrene plates or balls, porous glass or glass slides, and nitrocellulose or other membrane materials. Some supports can be activated for direct coupling to a ligand. Other supports arc made with nuclcophilcs or other functional groups that can be linked to proteins or other ligands using cross-linkers.
Immobilization of the compounds of the invention to solid-supports can be accomplished using routine coupling chemistries. In general, the compounds of the invention arc immobilized by including in the compounds an accessible first functional group (e.g., an alcohol group) and contacting the compound with a solid-support containing a complementary second functional group (e.g., carboxyl groups) under conditions and for a period of time sufficient to permit the first and the second functional groups to react with one another to form a covalent bond (e.g., ester bond). By "accessible" in reference to a functional group, it is meant that the functional group is in a form which is reactive and is not stcrically precluded from reacting with the solid-support.
The functional groups for immobilizing the compounds of the invention to a solid- support can be introduced into the peptide binding moieties or the linker portions of these compounds. For example, amino acids that include functional groups in their side chains (e.g., aspartale, glutamatc, cystcine residues) can be incorporated into the peptide binding moiety during synthesis and positioned at a sufficient distance from the reactive group which binds to the target protein to avoid unwanted steric hindrance by the solid-support in the reaction between the compound and its target protein. Alternatively, the compounds of the invention can be immobilized via a functional group in the linker molecule to a solid- support. Thus, for example, the linkers which arc used in this aspect of the invention can include, in addition to the first and the second linker reactive groups for binding to the first and the second peptide binding moieties, a further functional group for binding to the solid support. Trifunctional molecules of this type are illustrated in Example 1 1. To prevent side reactions, it is preferred that the linker reactive groups that are used for coupling the linker molecule to the peptide binding moieties be different from functional groups that are used for coupling the linker to the solid-support. Such functional groups can be introduced into the linker molecules at any time during or after the synthesis of these molecules. Thus, in general, the same types of functional groups, protection/deprotection reactions and reagents, and reaction conditions that are established in the art for using linker molecules to couple, e'g,, proteins or peptides to one another or to solid supports can be used for immobilizing the compounds of the invention to a solid support.
1 1. Protein-Protein Conjugates
One of the most widely used applications for cross-linkers is the production of protein-protein conjugates. Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate an enzyme, fiuorophore or other molecule to a protein that has affinity for one of the components in the biological system being studied. Antibody-enzyme conjugates (primary or secondary antibodies) are among the most common protein-protein conjugates used. Secondary antibodies are relatively inexpensive and arc commercially available. Listed below is a representative sampling of commercially available cross-linkers, e.g., from Pierce Catalog and Handbook, Rockford, HI. The table also identifies which group the linker is reactive towards, e.g., sulfhydryls, carboxyls. REACTIVITY OF COMMERCIALLY AVAILABLE CROSS-LINKERS
CO
Figure imgf000080_0001
s
Figure imgf000081_0001
Figure imgf000081_0002
C
Figure imgf000082_0001
Figure imgf000083_0001
EXAMPLE 9
PREVENTION OF CYCLIZATION BY DESIGNING A COMPOUND
CONTAINING AN OLEFIN GROUP
In this Example, Xaa-boroPro analogs containing an olefin group, e.g., a fluoroolefin will be constructed to prevent cyclization and increase biological activity. The biological activity of his olefin containing compound will be tested by the methods described previously in Example 2, section III, e.g., comparing the production of IL-2 using an olefin containing compound versus using a compound that does not contain an olefin.
1. Identification of Active (Open') and Inactive (Cyclic) Species of Monomeric Compounds as Related to Inhibitory Activity of Soluble CD26 (DP IV)
The inventors have previously shown that synthetic diastereomeric monomeric compounds, e.g., L-Ala-D,L-boroPro and L-Pro-D,L-boroPro, were potent inhibitors of the catalytic activity of soluble DP IV (CD26). They also encountered a problem because these monomerfcinhibitors lost some of their inhibitory activity rapidly in aqueous solution at a pH of about neutral. For example, Ala-boroPro lost DP IV inhibitory activity with a half- life of about 5 minutes and pro-boroPro lost activity with a half life of about one hour. It was determined that the inhibitors did not undergo degradation in aqueous solutions of neutral pH and higher, but did undergo a cyclization reaction.
In aqueous solution at all pH values, the inhibitors exist as a slowly equilibrating mixture of two conformations: an open chain structure which is inhibitory (active species), and a cyclic structure which is non-inhibitory (inactive species). See Fig. 10 which is a diagram showing the structures of the open and cyclizcd forms of Xaa-boroPro inhibitors (conformational equilibrium of Xaa-boroProline inhibitors). The open, active, inhibitory chain species is favored at low pH while the cyclized structure is favored at high pH. The reaction is fully reversible: the open chain becomes predominate at low pH. The open chain to cyclic species reaction involves a trans to cis iso erization of the proline and the formation of a new N-B bond. The cyclized structure is the boron analog of a di etόpipcrazinc, a product often seen in peptide chemistry. Cyclization liberates one equivalent of H+ thereby explaining the requirement for base in the cyclization reaction and acid in the opening reaction. The cyclic structure is quite stable in aqueous solutions of high pH.
Prolonged incubation at high pH never leads to the complete disappearance of DP IV inhibitory activity for any of the Xaa-boroPro compounds examined. This observation was the first evidence that the active inhibitor was in a conformation equilibrium with a non-inhibitory species rather than undergoing an irreversible inactivation. The half life for the reformation of the open chain species from the cyclic structure is surprisingly low. Thus, the loss of inhibitory activity in aqueous solution was due to a pH dependent conformational equilibrium rather than a degradation reaction. The fact that the inhibitory activity does not go to zero for any of the Xaa-boroPro inhibitors, even after prolonged incubation, together with the fact that the reverse reaction, i.e., cyclic to open chain is slow, suggested that it should be possible to measure the equilibrium constant for the conformation equilibrium by measuring the apparent Ki at equilibrium and comparing it with the true Ki. It has been demonstrated that the ratio of [cyclic] : [open] forms, at neutral pH, is
156:1 for Pro-boroPro and 1 130: 1 for Val -boroPro (W.G. Gutheil and W.W. Bachovchin, Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Depeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). This means that less than 1 % Pro-boroPro and less than 0.1% of Val-boroPro exists as the open chain, inhibitory species, at equilibrium at pH 7.0. Nevertheless, under these conditions the inhibitors behave as though they had Ki's of 2.5 nM and 1.8 nM respectively. This apparent Ki of the "fully inactivated" species is still substantially better than, (- 1000-fold) that of other inhibitors of DP IV thus far reported.
2. Background Information on Olefin Containinp Compounds
Previously, fluoroolefin peptide isostercs have been used as tools for controlling peptide conformations (Boros, L.G., et al Fluoroolefin Peptide Isostcres - Tools for Controlling Peptide Conformations, Tetrahedron Letters, 35(33), 6033-6036 (1994)). Fluoroolefin containing dipeptide isosteres have also been shown to be effective inhibitors of dipeptidyl peptidase IV (CD26) (Welch, J.T. et al Fluoroolefin Containing Dipeptide Isostercs as Inhibitors of Dipeptidyl Peptidase IV (CD26), Tetrahedron, 52(1), 291-304 (1995)).
3. Prevent Cyclization to Increase Biological Activity of Bivalent or
Multivalent Compound The inventors predict that biological bioavailability (biological function) for the compounds taught in this invention could be significantly increased (approximately 100- 1000 times) by preventing peptide conformational changes, e.g., intermolecular cyclization, by constructing a bivalent or multivalent compound containing an olefin group (a carbon atom double bonded to another carbon atom; see Fig. 1 ID), e.g., fluoroolefin. Figs. 1 1A- 1 ID are diagrams of different examples of bivalent compounds containing an olefin group and are not meant to limit the scope of the invention. Fig. 1 ID is a fluoroolefin isostere of Xaa-boroProline. A fluoroolefin mimics a peptide bond but prevents cis trans isomerization and therefore prevents cyclization. Thus, if cyclization can be blocked, the inventors*prcdict that the bioavailability of the compounds taught herein can be increased by approximately 100 - 1000 fold.
The methods used to synthesize fluoroolcfins are described below. See also, Livia G. Boros, et al "Fluoroolefin peptide isostercs - tools for controlling peptide conformations," Tetrahedron Letters, Vol. 35, No. 33, pp. 6033 and 6036, 1994; and John T. Welch and Jian Lin, "Fluoroolefin containing dipeptide isosteres as inhibitors of dipeptidyl peptides IV (CD26)", Tetrahedron, Vol. 52, No. 1, pp. 291-304, 1996 for alternative procedures that can be adapted to prepare the compounds of the invention.
The procedures for forming a particularly preferred fluorolefin compound of the invention are provided. However, it is contemplated that any of the compounds of the invention can be designed and constructed to incorporate an olefin group using these procedures. Accordingly, it will be appreciated that the procedures can be used to prepare olcfin-containing boronyl compounds of the invention in which the olefin is unsubstituted or substituted with a halogen other than fluorine. Further, it will be appreciated that although a compound having a boronyl reactive group is illustrated, a reactive group other than a boronyl group can be prepared by using an analog of compound (a) in which an alternative reactive group is substituted for BC13 in the reaction. The synthesis of alternative non-boronyl group, olefin-containing compounds can be performed using the synthesis procedures identifed above in reference to the Ferring published PCT application. The synthesis protocol involves three reactions. In the first reaction, compound (a) is converted to compound (b) using the reaction conditions set forth in Welch, J. et al., Tetrahedron 52:291-304 (1996). In the second reaction, compound (b) is converted to compound (c) using the reaction conditions set forth in Tilley, J.W. et al., J. Med. Chem. 34:1125-1 136 (1991). In the third reaction, compound (c) is converted to the fluorolefin- derivative compound using the reaction conditions set forth in Lipshultz, B.H. et al., J. Org. Chem. 54:4975 (1989).
Figure imgf000087_0001
Figure imgf000087_0002
(b)
Figure imgf000087_0003
Figure imgf000087_0004
Thc fluoroolefin analogs of Xaa-boroPros will be compared to Xaa-boroPros by measuring the potency of inhibition of CD26 proteinase activity. The immunodulatory effects of these fluoroolefin containing analogs will be evaluated by in vivo experiments using animal models and in vitro experiments using cell culture. The cell culture experiments will monitor cytokine production by cells of lymphoid origin, proliferation of cells of lymphoid origin, or both.
EXAMPLE 10
SYNTHESIS OF FLUORESCENTLY LABELLED
MONOMERIC DERIVATIVES OF LYS-BOROPRO
Fiuorescently labelled derivatives of monomeric LysboroPro can be prepared and used to determine if monomeric Lys-boroPro induces internalization of CD26 into cells. Fluorescence microscopy can be used to monitor intercellular trafficking of CD26.
One labelling approach is directly couple the side chain amino group of monomeric Z-Lys-bordPro-diester to a functional group on a fluorescent molecule such as the isothiocyanate of fluorescein isothiocynate (FITC). Lys-boroPro can be linked to FITC either directly or through a spacer linker molecule.
Another labelling approach is to link the side chain amino group of Lys-boroPro to biotin, again either directly or through a spacer and then use the avidin, strcpavidinbiotin systems for detection. Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. This protein contains four identical subunits having a combined molecular mass of 67,000-68,000 daltons. Each subunit binds one molecule of biotin, and studies have shown that tryptophan and lysine arc involved in the binding site for biotin. The sequencing of the subunit indicates it consists of 128 amino acids. Avidin has an isoelectric point of 10- 10.5 and is very soluble in water and salt solutions. Avidin is stable over a wide range of pMs and temperatures. Extensive chemical modification has little effect on the activity of avidin, making it useful for detection and protein purification.
Streptavidin is another biotin-binding protein, and it is isolated from Sireptomyces avidinii. The molecular weight of streptavidin is about 60,000. Unlike avidin, streptavidin has no carbohydrate and has an acidic isoelectric point of 5. Streptavidin is much less soluble in water than avidin and can be crystallized from water or 505 isopropanol.
Advantages of this approach over the above approach include: (1) the biotin-avidin system is well developed, well described and has been successfully utilized for the type of work proposed herein in many other systems; (2) improved flexibility because a large number of reagents are commercially available for use in this system: (3) improved sensitivity because this system provides for amplification of the signal from the biotinylated moiety (e.g., the biotinylated Lys-boroPro can be rendered fluorescent by reaction with either avidin (or streptavidin) conjugated either to a fluorochro e such as FITC or conjugated to an enzyme such as horseradish peroxidase). Avidin-FITC conjugate has many FITC groups per avidin molecule compared to only one of the first approach described above. The avidin-enzyme conjugate approach provides a large amplification owing to the many molecules of substrate converted to detectable product per avidin molecule. N jydroxysuccinmide biotin (NHS-biotin, available from Pierce Company) reacts readily with free primary amines to form a biotin conjugate linked with a peptide bond. First, NHS-biotin, and NHS-LC-biotin (which has a 22.4 A spacer arm to separate biotin from the amino group of the molecule to which it is to be conjugated) is linked to the side chain amino group of Lysine of Lys-boroPro, and these molecules are characterized as to their ability to inhibit DP IV and their ability to serve as a detection tool for Lys-boroPro- protein complexes using the avidin detection systems.
Each of the compounds prepared as described above can be purified to homogeneity using HPLC and its identity can be confirmed by NMR spectroscopy, amino acid composition, or mass .spectroscopy as deemed necessary.
EXAMPLE 11
SYNTHESIS OF FLUORESCENTLY LABELLED BIVALENT AND MULTIVALENT DERIVATIVES OF LYS-boroPRO
Fluorescently labelled dimeric and multimeric derivatives containing preferred P'R1 monomers (e.g., Lys-boroPro) can be prepared and used to determine if bivalent and trivalent derivatives of, e.g., Lys-boroPro, can induce aggregation and internalization of cells surface CD26.
Producing such molecules can be accomplished in several ways. One way is to use a trifunctional molecule of the type illustrated below. With appropriate chemical methodology, the carboxy late groups can be coupled to Lys-boroPro molecules while the amino group can be coupled to an FITC molecule, or other fiuorophore, or more preferably, to biotin via a suitable length spacer. Trifunctional molecules of the type shown below are available or can be synthesized relatively easily.
O-C-(CH2)n-CM-(CH2)π-C-O-
II I II
O +NH3 O
Trimeric forms of Lys-boroPro also are possible through straightforward extension of the approach described above for the bivalent forms. Another approach takes advantage of the multjmeric nature of the biotin-avidin system. Avidin has four subunits, each of which has a biotin binding site for biotin. If fluorescent trimeric inliibitors are needed, biotin can be coupled to Lys-boroPro through a suitable length spacer (the NHS-LC-biotin should have an adequate length spacer arm 22.4 A). The effect of various amounts of avidin, both on T cell activation and on cell surface CD26 aggregation can be determined using FITC-conjugated avidin. This approach likely yields a mixture of divalent, trivalent, and tetravalent inliibitors.
The compounds prepared as described above can be purified by HPLC and their structure can be confirmed by NMR spectroscopy, amino acid composition, or mass spectroscopy as necessary.
EXAMPLE 12 SYNTHESIS OF LYS-BOROPRO LINKED TO INSOLUBLE SUPPORTS
Lys-BoroPro linked to insoluble supports may be useful for three reasons. The first is for determining the effect of such solid phase immobilization of Lys-boroPro on T cell proliferation for comparison with analogous experiments with solid phase immobilized anti-CD26 mAbs. Solid phase immobilized Lys-boroPro derivatives should induce aggregation of cell surface CD26, but differ from soluble, multimeric inhibitors in that they should prevent internal ization, or at least internal ization of inhibitor bound CD26. The second use is for determining if Lys-boroPro binds to proteins other than CD26 to an appreciable extent. The third use is to produce an affinity column for producing purified CD26 from various sources.
The bi and multivalent compounds of the invention, as well as P'R1 monomers (e.g., Lys-boroPro), can be immobilized on solid supports in many ways, each with certain advantages. Initially, solid state immobilized avidin together with biotinylated Lys- boroPro can be tested because this approach offers substantial flexibility. Solid state immobilized avidin, e.g., linked to agarosc, is commercially available (Pierce Chemical Co.) with avidin and can be obtained in both multimeric and in monomeric forms. The monomeric form is designed to allow for the removal and recovery of biotinylated proteins from the resin and therefore may be preferred for the second and third purposes described above. There are, however, other ways to provide for the removal and recovery of biotinylated Lys-boroPro protein conjugates. For example, (i) high concentration of biotin may compete with and displace the biotinylated Lys-boroPro from the solid state immobilized avidin, (ii) free Lys-boroPro may displace the biotinylated inhibitor from the proteins, (iii) a biotin derivative with a cleavable group in the spacer arm can be used in preparing the biotinylated Lys-boroPro, (iv) lowering the pH to -4.0 will dramatically lower the affinity of Lys-boroPro for the active site of DP IV and thus should allow elution of the protein from the resin leaving behind biotinylated Lys-boroPro attached to avidin (G.R. Flentke, et al., Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991); Bachovchin, W.W. et al., Inhibition of IgAl proteinases from Neisseria gonorrhoea and Hemophilus influenzae by peptide prolyl boronic acids, J. of Biol. Chem. 265, 3738-43 (1990)). Diols, such as pinancdiol or pinacol, can be added to the elution buffer to bind to and thus tie up the hydroxyl groups on the boronyl group. Each of the compounds prepared as described above can be purified to homogeneity using HPLC and its identity can be confirmed by NMR spectroscopy, amino acid composition, or mass spectroscopy as deemed necessary.
EXAMPLE 13 CD26 (DP IV) AFFINITY STUDIES
Since the purification of CD26 has remained a major problem, employing an affinity column as described below should be of substantial benefit. The fact that the linked homobivalent, dimeric molecule, KbP2-Adipate, was shown to be a potent inhibitor at lower concentrations, e.g., 10*10M, (See Example 1, 1, B(l) above)) suggests that an immobilized compound of the invention can be used for affinity purification of CD26. Derivatizing the e-amino group of the Lys-boroPro, without sacrificing affinity, should help in developing an affinity column specific for the purification of CD26 from various sources, e.g., cell lines transfected with CD26 (DP IV) genes.
EXAMPLE 14 MEASURING STANDARD CD26 (DP IV) ACTIVITY
Assays to measure CD26 (DP IV) activity can be performed on both the homobivalent compounds, e.g., Lys-boroPro coupled to another, and the heterobivalent compounds, e.g., Lys-boroPro coupled to a peptide specific for a T cell surface receptor, e.g., moth cytochrome C peptide. Methods for quantitatively measuring the interaction of small peptidomimctic inhibitors with CD26 or DP IV, as well as for the interaction of CD26 with larger ligands, e.g., the HIV Tat protein, have been developed (W.G. Gutheil and W.W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993); Gutheil, W.G., and W., B.W. Kinlsq, A Matlab Program for Fitting Kinetics Data with Numerically Integrated Rate Equations and Its Application to the Analysis of Slow, Tight Binding Data, Analytical Biochemistry 223, 13-20 (1 94); Gutheil, W.G.-, et al., HIV- 1 Tat Binds to DP IV (CD26): A possible Mechanism for Tat's Immunosuppressive Activity, Proc. Natl. Acad. Sci. U.S.A. 1, 6594-6598 (1994)). These methods use the chromatogenic substrate Ala-Pro-p-nitroanilide (AppNA) and fluorescent substrate Ala- Pro-7-amino-4-trifluorornethyl coumarin (AP-AFC). AppNA and AP-AFC are commercially available (e.g., Enzyme Systems Products, Dublin, CA).
EXAMPLE 15
IMMUNOLOGICAL STUDIES ON HOMOBIVALENT AND HETEROBIVALENT COMPOUNDS
1. T-cell Functional Assays
The ability of bivalent Lys-boroPro molecules designed to induce association of cell surface CD26 can be measured in an antigen specific T cell response as previously described (G.R. Flentkc, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inliibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991); M. Subramanyam, et al., "Mechanism of IIIV-1 Tatjnduced inhibition of antigen-specific T cell responsiveness," J. Immunol. 150, 2544-2553 (1993)).
Briefly, in the human system, peripheral blood mononuclear cells (PBMC) are cultured with suboptimal doses of anti-CD3 mAbs, in the presence or absence of the CD26 associating agents. Alternatively, a recall antigen response is measured to a suboptimal concentration of tetanus toxoid or Candida antigen, plus or minus the CD26 associating agents (M. Subramanyam, et al., "Mechanism of HIV- 1 Tat Induced inhibition of antigen- specific T cell responsiveness," J. Immunol. 150, 2544-2553 (1993)).
In the murine system, the cytochrome C system is used for measuring the response in the 2B4 T cell hybridoma (G.R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function," PNAS (USA) 88, 1556-1559 (1991)). Cellular activation is measured by the production of IL-2 by the T cell hybridoma, and the level of IL-2 produced is quantified in a biological assay, using HT-2 indicator cells. 2. Internalization of CD26/Drug Delivery
Because antibody mediated crosslinking of CD26 induces internalization, it is likely that homobivalent compounds of Lys-boroPro also have this biological activity. Receptor internalization can be determined by employing FITC labelled bivalent Lys-boroPro and performing flow cytometric analysis (N.H. Dang, Y. Torimoto, K. Sugita, J.F. Daley, P. Schow, C. Prado, S.F. Sehlossman, and C. Morimoto. Cell surface modulation of CD26 by anti-lF7 monoclonal antibody: Analysis of surface expression and human T cell activation, Journal of Immunology 145, 3963-3971 (1990)). To compare cell membrane versus cytoplasmic staining, the cells are analyzed (i) with intact membranes, allowing only cell surface staining, and (ii) after permeabilization of the membrane with saponin, which allows the antibody to cross the membrane, although the membrane stays structurally intact (de Caestecker, M.P., Telfer, B.A., Hutchinson, I.V., and Ballardie, F.W., "The detection of intcrcytoplasmic interlcukin l et, interleukin lβ and tumour necrosis factor α expression in human monocytes using two colour immunofluorescence flow cytometry," J. Immunol. Methods 4, 1 1 -20 ( 1992). Once it is demonstrated that the CD26 molecule becomes internalized after incubation with a bivalent or multivalent derivative of Lys-boroPro, a larger molecule is coupled to the bivalent molecule, preferentially an enzyme that can be easily detected, e.g., luciferase, alkaline phosphate or β-galactosidase. Expression of these proteins can be measured using a cellular extract. Kits are commercially available for this determination (e.g., Novacasatra Laboratories Ltd., Newcastle upon Tyne, UK).
Furthermore, mAbs are commercially available (e.g., Southern Biotech, Birmingham Alabama) for these enzymes, enabling detection of protein expression in single cells by flow cytometry. As above, saponin can be used to permeabilize the cells, enabling entry of the mAbs. This method is very sensitive and allows the simultaneous analysis of other cell surface or cytoplasmic proteins.
Uses and Advantages
As described herein, the invention provides a number of uses and advantages. The low molecular weight, synthetic bivalent or multivalent compounds of the invention arc designed to induce the association between association-activated receptors and, therefore, offer considerable potential for regulating biological systems. Thus, this new class of bioiogically active compounds, or compositions thereof, are useful for inducing the association between association-activated receptors on human T cells and for the treatment of a wide variety of T-cell mediated diseases in animals; for example, autoimmune disease. Generally, these bivalent or multivalent homo- or hetero-compounds or compositions thereof are useful as immune response modulating therapeutics (1) to treat disease conditions characterized by immunosuppression, e.g., AIDS or AIDS-related complex, other virally or environmentally induced conditions and certain congenital immune deficiencies; (2) to increase an immune function which has been impaired by the use of i munosuppressive drugs, (3) to treat systemic lupus erythematosis, rheumatoid arthritis, and multiple sclerosis.
For example, the compounds of this invention can be used to deliver a member of the superantigen family to stimulate T cells. Superantigens comprise a class of disease- associated, immunostimulatory molecules that bind class II MHC molecules and stimulate large numbers of T cells (Jardetzky, T.S., et al. "Three Dimensional Structure of a Human Class II I- istocompatiblity Molecule Complexd with Superantigen", Nature 368, 711-718 (April, 1994)). Members of the superantigen family include toxins from S. aureus and other bacteria, as well as viral superantigens from mouse mammary tumor virus. The toxicity of the bacterial superantigens is thought to be mediated by their potent T-cell stimulating activities, leading to lymphokine release, respiratory distress and shock. Superantigens have also been implicated in rabies, rheumatoid arthiritis, and mouse and human AIDS.
These bivalent or multivalent homo- or hetero-compounds or compositions thereof can be used to stimulate the growth of hemapopoietic cells in culture. Such cells can be transplanted into mammals, e.g., humans, to stcngthen or boost the hematopoietic, immune system, or both. These compounds also can be used to treat patients suffering from disease or from deficiency of hematopoietic cells such as AIDS patients, patients undergoing chemotherapy and/or patients under radiotherapy for hcmatological or other cancers, and patients undergoing bone marrow transplants.
When administered to mammals, e.g., humans, the compounds of the invention may enhance the ability of the immune system to regenerate cells that arc immunosuppressed, e.g., CD4 and T cells. Thus, the bivalent compounds of this invention, can be administered to mammals, e.g., humans, in an effective amount alone or in combination with a pharmaceutically acceptable carrier, excipient, or diluent, in unit dosage form. Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions to administer these compounds to patients suffering from immunosuppression or an immune deficiency or presymptomatic of AIDS. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intrcapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene polyoxyproplyene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for these bivalent compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems or iiposomes. Formulations of inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxethylene-9-lauryl ether, glycocholatc and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
These bivalent homo- or heterbivalent compounds or compositions thereof can be used to control the T cell activation process and thus be used to prevent unwanted immune responses.
The homobivalent compounds designed to induce the association between one CD26 T cell receptor and another CD26 T cell receptor can be useful in stimulating the proliferation of CD4+ cells. These compounds could, therefore, help restore CD4+ cells numbers in immunosuppressed patients, e.g., AIDS patients, and thereby reverse the decline in immune function. The homobivalent compounds or compositions thereof (CD26 T cell receptor associated with another CD26 T cell receptor) of the invention can be used to enhance recall-antigen specific immune responses. Lymphocytes from most HIV-infcctcd individuals exhibit a qualitative defect in their ability to respond to recall antigens (A.S. Fauci. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 617-722 (1988)). This defect is exhibited early after infection and long before CD4+ T cell number decline. Accordingly, it is believed that these compounds will be useful in treating AIDS. Thus, the stimulatory activities should improve lymphocyte function in HIV infected individuals by ameliorating the defective recall antigen responses which show up early after infection, and by improving CD4+T cell numbers.
Since the compounds of the invention and, in particular, the compounds of claim 1 have high affinity and specificity for CD26, they can be useful for the selective delivery of other therapeutic agents to, and into CD4+ T cells. Thus, the compounds of the invention can be used to deliver pharmacological agents inside CD4+ cells that normally are unable to penetrate such cells. For example, many highly potent inhibitors of the HIV protease have been developed, which despite their high affinities for the HIV proteinase, are limited in blocking HIV in vivo owing to their inability to get inside the CD4+ cells. These HIV proteinases hibitors inhibitors can be linked to the spacer arm of a bivalent CD26 ligand and delivered into CD26+ lymphocytes, which are the cells the virus primarily invades. Even if a drug is capable of entering CD4+ cells, the compounds of the invention can be used to concentrate the drug in CD26 cells, thereby maximizing the desired pharmacological activity while minimizing unwanted toxic side effects on other cells. This delivery vehicle therefore provides a mechanism to prevent or reduce AZT resistance by providing higher concentrations of AZT in CD4+ cells and lower concentrations elsewhere. Thus, the CD26 internalization activity of the compounds disclosed herein can be used to provide a vehicle for delivering and concentrating other therapeutic agents into CD4+T cells.
The heterobivalent compounds designed to induce the association between a CD26 T cell receptor and a different T cell receptor, e.g., the T cell receptor TCR/CD3, can be useful in stimulating a cell-mediated immune response against specific antigens.
Heterobivalent compounds or compositions thereof comprising a CD26 inhibitor and an antigenic peptide stimulate a cellular immune response against an antigenic peptide as compared to an antibody-mediated immune response. This stimulation of cell-mediated response against specific antigens can be useful in patients with AIDS because these patients have high concentrations of anti-HIV antibodies. Thus, the CD26-TCR association-inducing activity can be useful for stimulating a cell mediated immune response to specific antigens. This biological activity should prove especially useful in vaccine development, particularly for AIDS because a cellular or T„l immune response is the appropriate response for HIV-1 and is apparently lacking in AIDS patients.
Such heterobivalent compounds can be useful in the development and manufacture of peptide-based vaccines and therapeutic agents for the treatment of allergies and autoimmune diseases.
Because the response stimulated is cell mediated and specific for the antigen chosen, such bifunctional agents can be useful in the development of vaccines for modulating a cell mediated immune response, especially for the development of a vaccine for treating AIDS.
The heterobivalent compounds designed to induce the association between a CD26 T cell receptor and a different T cell receptor, e.g., CD4, can be useful in activating T cell function. This heterobivalent compound or compositions thereof can be used to selectively deliver agents to CD4+ CD26+ cells, or they can be useful in blocking HIV entry into CD4+ cells.
The heterobivalent compounds or compositions thereof, e.g., as shown in claim 17, can be used to deliver a toxin, e.g., ricin A immunotoxin or AZT, within a CD26+ T cell. A toxin of choice can be coupled to a dimeric compound having high affinity for the CD26 T cell surface receptor. Also, a toxin can be coupled to a peptide having specificity for a specific T cell receptor. Either way, once the dimeric compound associates or binds to the CD26 T cell surface receptor, the toxin is internalized within the T cell, thereby delivering this toxin to a T cell.
The compounds or compositions thereof can be administered alone or in combination with one another, or in combination with other therapeutic agents. For example, treatment with one of the bivalent compounds can be combined with more traditional therapies for treating disorders of the immune. When administered, the pharmaceutical preparations of the invention are applied in pharmaceutical ly-acceptable amounts and in pharmaceutically-acceptably compositions. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutical ly-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimcrosal. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the crosslinking compound, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed arc water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
A variety of administration routes are available for treating a subject. The particular mode of delivery selected will depend, of course, upon the particular crosslinking compound selected, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes. Such modes of administration also include obtaining T cells or bone marrow cells, stem cells or early lineage progenitor cells from a patient and contacting the isolated cells with the crosslinking compounds of the invention ex vivo, followed by reintrodUcing the treated cells to the patient. The treated cells can be reintroduced to the patient in any manner known in the art for administering viable cells.
As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic and other treatment because of the convenience to the patient as well as the dosing schedule.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. The methods include the step of bringing the crosslinking compounds of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the crosslinking compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the crosslinking compound of the invention. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the crosslinking compounds described above, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactoncs, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075, 10 Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the crosslinking compound is contained in a form within a matrix such as those described in U.S. Patent Nos.
4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, as used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 10 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
The crosslinking compounds described herein are administered in effective amounts. An effective amount is a dosage of the crosslinking compound sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. For example, an effective amount for stimulating T cell activation would be an amount sufficient to increase to a statistically significant extent T cell activation as for example, measured by proliferation or by increased T cell activity. An effective amount for stimulating a desired immune response also can be measured, for example, by determining a change in the immune function in a subject (e.g., increased B cell response, increased cytotoxic T cell response, stimulation of bone marrow proliferation, or an ability to slow, halt, or prevent an infection or cancer). An effective amount for treating an autoimmune disorder or allergic disorder would be that amount sufficient to lessen or inhibit altogether the immune or allergic response associated with the disorder so as to slow or halt the development of or the progression of the disorder. Thus, it will be understood that the crosslinking compounds of the invention can be used to treat an autoimmune disorder (e.g., transplant rejection) prophylactically in subjects at risk of developing an immune response (e.g., recipients prior to transplant). As used in the claims, "inhibit" embraces all of the foregoing. Likewise, an effective amount for treating an immune system disorder is that amount which can slow or halt altogether the symptoms associated with the immune system disorder so as to prevent the disorder, slow its progression, or halt the progression of the immune system disorder. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
Generally, doses of active compounds will be from about 0.001 mg/kg per day to 1000 mg/kg per day. It is expected that doses range of 0.01 to 100 mg/kg will be suitable, preferably orally and in one or several administrations per day. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
Other embodiments arc within the following claims. What is claimed is:
SEQUENCE LISTING
{!) GENERAL INFORMATION
(i) APPLICANT: Trustees of Tufts College
(ii) TITLE OF THE INVENTION: MULTIVALENT COMPOUNDS FOR CROSSLINKING RECEPTORS AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 27
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Wolf, Greenfield & Sacks, P.C.
(B) STREET: 600 Atlantic Avenue
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02210
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 28-JUN-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/671,756
(B) FILING DATE: 28- UN-1996
(A) APPLICATION NUMBER: 08/837,305
(B) FILING DATE: ll-APR-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Plumer, Elizabeth R
(B) REGISTRATION NUMBER: 36,637
(6) REFERENCE/DOCKET NUMBER: 10254/7007 (ix) TEI_ECOMMUNICATI0N INFORMATION:
(A) TELEPHONE: 617-720-3500
(B) TELEFAX: 617-720-2441
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Glu Ala Asp Pro Thr Gly His Ser Tyr 1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Scr Ala Tyr Gly Glu Pro Arg Lys Leu l 5
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE Q-ARACTERISTICS :
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Glu Val Asp Pro He Gly His Leu Tyr (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Leu Leu Ala Val Leu Tyr Cys Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOI-≤CULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Tyr Met Asn Gly Thr Met Ser Gin Val 1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Tyr Leu Glu Pro Gly Pro Val Thr Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Ala Ala Gly iϊe Gly He Leu Thr Val 1 5
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :
Ser Ser Ser Thr Leu Cys Thr Ser Lys Ala Asp Lys Ser Ser Gly Asn
1 5 10 15
Gin Gly Gly Asn Gly Val Phe He Val Val Asn Ala Trp Tyr Ser 20 25 30
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Glu Asp Leu Thr Ala Gly Tyr Cys Lys Cys Phe Glu Glu Phe Val
1 5 10 15
Leu Ala Ser Arg Cys Lys 20
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Glu Gin Arg Gin Gly He Lys Val Gin Leu He Leu Phe He Leu Arg
1 5 10 ' 15
Ala Leu Met He Asn Thr Ser Ser Ser Asn His He Leu Asp Ser Arg
20 25 30
Asn Val Phe Leu His Thr Gly His Gly Glu Pro Met Val Gin Lys Gin
35 40 45
He Glu Trp Val Leu He Met Glu Leu He Lys Met 50 55 60
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
His Lys Ala Val Phe Arg Ser Glu He Ser Leu Gin Lys Trp Cys Ser 1 5 10 15
Asp Thr Gin Lys Ser Thr 20
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Asp Ser Phe Glu Ser Val Arg Leu Pro Ala Pro Phe Arg Val Asn His
1 5 10 15
Ala Val Glu Trp 20
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
He He Ser Pro Val He Phe Gin He Ala Leu Asp Lys Pro Cys His
1 5 10 15
Gin Ala Glu Val Lys His Leu His His Leu Leu Lys Gin Leu Lys Pro
20 25 30
Ser Glu Lys Tyr Leu Lys He Lys His Leu Leu Leu Lys Arg Glu Arg
35 40 45
Val Asp Leu Ser Lys Leu Gin 50 55
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Arg Ser Lys Thr Leu His His Leu Leu Lys Gin Leu Lys Pro Ser Glu
1 5 10 15
Lys Tyr Leu Lys He Lys His Leu Leu Leu Lys Arg Glu Arg Val Asp
20 25 30
Leu Ser Lys Leu Gin 35
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE C_HARACTERISTICS :
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Pro Pro Gin Thr Gly Glu Lys Tyr Leu Lys He Lys His Leu Leu Leu
1 5 10 15
Lys Arg Glu Arg Val Asp Leu Ser Lys Leu Gin 20 25
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE : peptide
(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 17 :
Asp Ala Asp Thr Tyr Tyr He Leu Pro Arg Lys Val Leu Gin Met Asp
1 5 10 15
Phe Leu Val His Pro Ala 20 -109-
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asp Thr Leu Leu Leu Leu Pro Arg Lys Val Leu Gin Met Asp Phe Leu
1 5 10 15
Val His Pro Ala 20
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Leu His Phe Ala Ser Arg Trp He Phe Leu Phe He Gin Pro Glu Cys
1 5 10 15
Ser Glu Pro Arg 20
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE : peptide
(xi) SEQUENCE DESCRIPTION: SΞQ ID NO : 20 :
in Asp Leu Thr Met Lys Tyr Gin He Phe 1 5 10 (2) INFORMATION FOR SEQ ID NO.21.
(l) SEQUENCE CHARACTERISTICS :
(A) IENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21.
Ala Ala Ala Ala Ala Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 22.
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 13 amino acids
(B) TYPE, amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe 1 5 10
(2) INFORMATION FOR SEQ ID NO: 23:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23.
His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Ala Ala Ala
1 5 10 15
Ala Ala Ala
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Ala Ala Ala
1 5 10 15
Ala Ala
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Ala Ala Ala Ala Ala Phe Lys Asp Pro His Gly Leu Trp Lys Gly Leu
1 5 10 15
Ser His
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ala Asp Pro Thr Gly His Ser Tyr 1 5
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Tyr Ser His Gly Thr Pro Asp Ala 1 5

Claims

A compound, having the structure
Y1 Y3
/
D1 - A t ~ A2 ~ A3 - A4 /
02 AS - Aβ ~ A7 ~ A3 — β
\
Y2 Y4
wherein Dl and D2, independently, are selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen "~"is, independently, selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al and A5 are, independently, selected from a group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C: each A2, A3, A4, A6, A7 and A8 are, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, are, independently, selected from the group consisting ol'any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein 0 represents any isotope of oxygen; wherein each Yl , Y2, Y3 and Y4 are. independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiological conditions; and L represents a linker molecule (i) having a molecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A lo about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous.
2. The compound of claim l wherein the following structures
^ Y1
D1 - A t - A2 - A3 - A4 S
I Y2 and
Y3
D2 - AS - A0 - A7 -A3 — B
\
Y4
represent, independently, a binding moiety, wherein R represents the remainder of the 5 molecule.
3. The compound of claim 2 wherein there are 4 atoms positioned between the group consisting of Dl and D2 and B of the binding moiety.
4. The compound of claim 2 wherein the binding moiety is in an L-configuration.
5. The compound of claim 2 wherein Yl , Y2, Y3 and Y4 are hydroλ'yl groups.
10 6. The compound of claim 1 wherein the A4 bonded to the B is in the L-configuration and the A5 bonded to the B is in the L-configuration.
7. The compound of claim 2 wherein the binding moiety is an L-amino acid residue conjugated to B, a boron molecule.
8. The compound of claim 2 wherein the binding moiety is selected from the group \5 consisting of L-Lys-L-boroPro and a derivative of L-Lys-L-boroPro.
9. The compound of claim 1 wherein the linker molecule contains a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfliydryl group, an imidazole group, an alkene group, an acyl halogen group, and CILX, wherein X represents a halogen.
10. The compound of claim 1 wherein the linker molecule is further defined as having the following structure: wherein [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogen and a sulfur atom; [J] is selected from the group consisting of a CH2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p, and q represent an integer from 1 to 50, inclusive.
1 1. The compound of claim 10 wherein [G] is an R group selected from the group consisting of L-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine.
12. The compound of claim 1 wherein the linker molecule is selected from the group consisting of hexanedioic acid (adipic acid), EGS, 1, 4-diaminobutane, 1, 4-dithiobutane, dithiothreitol, lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine.
13. The compound of claim 1 wherein the linker molecule contains al least two amino groups when the binding moieties contain glutamic acid residues.
14. The compound of claim 1 wherein the linker molecule contains at least two amino groups when the binding moieties contain aspartic acid residues.
15. The compound of claim 1 wherein the linker molecule contains at least two sulfhydryi groups when the binding moieties contain cystcine residues.
16. ' The compound of claim 1 wherein the linker molecule span ranges from about 30 A to about 100 A.
17. A c
Figure imgf000118_0001
wherein D is independently selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "~"is, independently, selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from a group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C; each A2, A3 and A4 arc, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl and Y2 are, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under physiological conditions; L represents a linker molecule (i) having a molecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents an isotope of sulfur and Ph represents any isotope of phosphorous; and P represents a peptide ranging from 3 to 30 amino acids having sufficient sequence homology lo bind to a naturally occurring receptor.
18. The compound of claim 17 wherein the following structures
Y1
R Y2 and
Y3
D2 - A5 - A6 - A7 ~Aβ — β
\
Y4
represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
1 . The compound of claim 18 wherein there are 4 atoms positioned between D and B of the binding moiety.
20. The compound of claim 18 wherein the binding moiety is in an L-configuration.
21. The compound of claim 17 wherein Yl and Y2 are hydroxyl groups.
22. The compound of claim 17 wherein the A4 bonded to the B is in the L- configuration.
23. The compound of claim 18 wherein the binding moiety is an L-amino acid residue conjugated to B, a boron molecule.
24. The compound of claim 18 wherein the binding moiety is selected from the group consisting of L-Lys-L-boroPro and a derivative of L-Lys-L-boroPro.
25. The compound of claim 17 wherein (he linker molecule contains a functional group selected from the group consisting of a carboxylate group, an amino group, a sulfliydryl group, an imidazole group, an alkene group, an acyl halogen group, and C112X, wherein X represents a halogen. -118- 26. The compound of claim 17 wherein the linker molecule is further defined as having the following:
Figure imgf000120_0001
5 wherein [G] contains atoms selected from the group consisting of a carbon, nitrogen, oxygen, hydrogen and a sulfur atom; [JJ is selected from the group consisting of a CH2 molecule, a chain of carbon atoms, a chain of nitrogen atoms, and a chain of oxygen atoms; and m, p and q represent an integer from 1 to 50, inclusive.
27. The compound of claim 26 wherein [G] is an R group selected from the group
I o consisting of L-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine and D-amino acid residues selected from the group consisting of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine and asparagine.
28. The compound of claim 17 wherein the linker molecule is selected from the group 15 consisting of adipic acid, between 2 and 15 consecutive amino acid residues, 1 , 4- diaminobutane, 1 ,4-dithiobutane, and dithiothreitol.
29. The compound of claim 17 wherein the linker molecule span ranges from about 30 A to about 100 A.
30. The compound of claim 17 wherein the peptide ranges from about 7 to 25 amino 20 acids.
31. The compound of claim 17 wherein the peptide is selected from the group consisting of: a) a peptide derived from a human myelin protein. b) a moth cytochrome C peptide; c) a tetanus toxin; d) a HIV- 1 GP 120 peptide; e) myelin basic protein peptide; f) a tumor antigenic peptide; and g) antigenic peptides of infectious agents.
32. The compound of claim 31 wherein the human Myelin protein peptide is selected from the group consisting of a myelin basic protein; a mylene proteolipid protein, and a myelin associated glycoprotein, wherein the myelin proteolipid protein peptide is selected from the group consisting of PLP peptide 139-151 and PLP peptide 190-209, wherein the Moth cytochrome C peptide is peptide MCC 94-103, wherein the myelin basic protein peptide is MBP peptide 1-1 1, and wherein the tetanus toxin peptide is selected from the group consisting of tetanus toxoid peptide and P2 tetanus toxoid peptide.
33. The compound of claim 17 wherein the naturally occurring receptor is a T cell surface receptor or a B cell surface receptor.
34. The compound of claim 33 wherein the cell surface receptor is selected from the group consisting of TCR CD3, CD2, CD4, CD8, CD 10, CD26, CD28, CD40, CD45, B7.1 and B7.2.
35. A compound, having the structure
.Yi
D - A1 - A2 - A3 - A4- / t \ D - A1 - A2 - A3 - A4 -
Y2 \
Y2
- G L
Et — U -E2 - Ph £_ ' EV- W, E2 TO, £_ wherein D is independently selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, wherein H represents any isotope of hydrogen; "~" is, independently, selected from the group consisting of a single bond and a double bond; B represents, independently, any isotope of boron; Al is, independently, selected from a group consisting of a C, a CX moiety and an N, wherein C represents any isotope of carbon, wherein X represents any atom capable of forming a single bond with C: each A2, A3 and A4 are, independently, selected from a group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently, are selected from the group consisting of any atom capable of forming a single bond and any atom capable of forming a double bond with C or N and wherein O represents any isotope of oxygen; wherein each Yl and Y2 are, independently, selected from the group consisting of a hydroxyl moiety and any reactive moiety that converts to a hydroxyl moiety under ' physiological conditions; n represents an integer between 1 and 200, inclusive; wherein El and E3 are distinct reactive species in which: a) R and R' comprise the remainder of the molecules not relevant to this reaction; b) El is attached to R' by a covalent bond which are together designated as El-R'or R'-El ; c) E3 is attached to R by a covalent bond which arc together designated as E3-
R or R-E3; d) R' represents the part of E 1 -R not undergoing a chemical reaction; e) R represents the part of R-E3 not undergoing a chemical reaction; f) El undergoes a chemical reaction with E3 to form the product El'-E3' and a byproduct F, wherein F is selected from the group consisting of 2H4 and
2e', H20, and any other byproduct; g) where H+ is the cation of any isotope of hydrogen and e" is an electron;
h) where H represents any isotope of hydrogen and O represents any isotope of oxygen; I) where El' and E3 are covalently bonded; j) El docs not undergo a chemical reaction with another El ; k) E3 does not undergo a chemical reaction with another E3; and
(1) El and E3 arc selected from the group consisting of a carboxylate, amino, imidazole, sulfliydryl, aldehyde, ester, and any other reactive species; wherein [J]p, E2, [I]q and [G]m together comprise a linker moiety, and wherein [G]m, [Jjp, and [Ijq represent, independently, linker molecules (i) having a molecular weight ranging between about 100 daltons and about 2000 daltons, (ii) having a span ranging from about 20 A to about 300 A, and (iii) containing a chain of atoms selected from the group consisting of a combination of C, O, N, S, and Ph atoms, connected by single bonds or by double bonds in a manner that does not violate the laws of chemistry and wherein S represents any isotope of sulfur and Ph represents any isotope of phosphorous; and wherein m, p and q represent, independently, an integer from 1 to 50, inclusive; and wherein E2 is selected from the group consisting of CX, CH, N, PhYZ, PhU and any other moiety capable of forming covalent bonds with [Jjp, [GJm, and [I],, and wherein: a) C is any isotope of carbon; b) x is any isotope of any atom capable of forming a single bond with carbon; c) I I is any isotope of hydrogen; d) N is any isotope of nitrogen; e) Ph is any isotope of phosphorous; f) Y is any isotope of any atom capable of forming a single bond with phosphorous; g) Z is any isotope of any atom capable of forming a single bond with phosphorous; and h) U is any isotope of any atom capable of forming a double bond with phosphorous.
36. The compound of claim 17 wherein the following structures
Y1
D1 - A1 - A2 - A3 - A4 B
Y2 and
Y3
D2 - A5 - A6 - A7 ~A8—B
\
Y4
represent, independently, a binding moiety, wherein R represents the remainder of the molecule.
37. The compound of claim 35 wherein (a) [G]m is the side chain of a D- or L- isomer of lysine, cystcine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (c) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (d) El and E3 are distinct from each other.
38. The compound of claim 35 wherein (a) [G]m is the side chain of a D- or L- isomer of lysine, cysteine, glutamic acid, aspartic acid, histidine, arginine, glutamine, and asparagine; (b) E2 is selected from the group consisting of 2-carboxybutyl, 2- carboxypropyl, 2-aminobutyl, 2-aminopropyl, and a hydrocarbon chain with an amino or carboxy side chain; (c) [J]p and [I]q represent, independently, hydrocarbon chains; (d) El and E3 are selected from the group consisting of an amino moiety and a carboxylic acid moiety; and (e) El and E3 are distinct from each other.
39. A method for stimulating activation of proliferation of human CD26-bcaring lymphocytes or CD26-bcaring hematopoietic cells, said method comprising contacting said lymphocytes or hematopoietic cells with a proliferation or activation-inducing concentration of the compound of any of claims 1, 17, 35 or 49.
40. The method of claim 39, wherein said contacting is carried out by administering said compound to a human patient suffering from a disease state characterized by inadequate lymphocyte activation or concentration.
41. The method of claim 40, wherein said disease state is caused by HIV infection.
42. The method of claim 40, wherein said compound is administered in conjunction with a second, different agent which stimulates activation or proliferation of said lymphocytes.
43. The method of claim 40, wherein said compound is administered by a route selected from the group consisting of orally, intramuscularly, subcutaneously, and intravenously.
44. The method of claim 39, wherein said contacting of lymphocytes with said compound is carried out in vitro.
45. The method of claim 40, wherein said disease state is a neoplasm, and said CD26- bearing lymphocytes arc cytolytic or helper T cells.
46. The method of claim 40, wherein said patient is suffering from side effects of chemotherapy or radiation therapy, one of which side effects being a consequence of depletion of cells of the immune system, wherein cells of the immune system are selected from the group consisting of cells derived from lymphoid, crythroid and myeloid lineages.
47. The method of claim 40, wherein said patient suffers from kidney failure resulting in depletion of ceils of the immune system.
48. • The method of claim 40, wherein said patient suffers from a bone marrow disorder resulting in immunodeficiency.
49. A compound having the formula I:
[P2(R2)m]n - L - P'R'
wherein P1 presents a first targeting moiety that mimics the substrate binding site of a protease expressed on the surface of a cell involved in immune system modulation; R1 represents a reactive group that reacts with a functional group in the reactive center of Ihe protease;
P2 represents a second targeting moiety that may be the same or different from the first targeting moiety; R2 represents a second reactive group that may be the same or different from the first reactive group;
111=0 or 1 and n=a whole number from 1 to 10, and L represents a linker molecule.
50. The compound of claim 49, wherein P2=P' and R2 is absent or is different from R1.
51. The compound of claim 49, wherein P1 selectively binds to a DP IV on a first cell and P2 selectively binds to a major histocompatibility molecule on an antigen presenting cell.
52. A vaccine comprising the compound of claim 49.
53. A pharmaceutical composition comprising the compound of claims 1, 17, 35 or 49, in a pharmaceutically acceptable carrier.
54. A method for manufacturing a pharmaceutical composition comprising: placing the compound of claims 1 , 17, 35, or 49 in a pharmaceutically acceptable carrier.
55. The method of claim 39, wherein administering comprises obtaining the T cells, bone marrow cells, stem cells or early lineage progenitor cells from the subject, contacting the isolated cells with the compound ex vivo in an amount effective to stimulate the cells, and reintroducing the cells to the subject.
56. A method for treating an autoimmune condition comprising: administering the compound of claims 1, 17, 35, or 49 to a subject in need of such treatment in an amount effective to inhibit the autoimmune condition in the subject.
57. The method of claim 40, wherein said patient suffers from immunodeficiency symptoms resulting from depletion of cells of the immune system.
58. The compound of claim 49, wherein P2 is a retroinverso peptide.
59. The compound of claim 49, wherein P2 is a tumor antigenic peptide.
PCT/US1997/011279 1996-06-28 1997-06-27 Multivalent compounds for cross-linking receptors and uses thereof WO1998000439A2 (en)

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999056753A1 (en) * 1998-05-04 1999-11-11 Point Therapeutics, Inc. Hematopoietic stimulation
WO1999063992A1 (en) * 1998-06-08 1999-12-16 Advanced Medicine, Inc. Novel calcium channel drugs and uses
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
SG83721A1 (en) * 1998-06-08 2001-10-16 Advanced Medicine Inc Beta 2-adrenergic receptor agonists
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6576793B1 (en) 1999-12-08 2003-06-10 Theravance, Inc. β2-adrenergic receptor agonists
US6593497B1 (en) 1999-06-02 2003-07-15 Theravance, Inc. β2-adrenergic receptor agonists
US6683115B2 (en) 1999-06-02 2004-01-27 Theravance, Inc. β2-adrenergic receptor agonists
US6713651B1 (en) 1999-06-07 2004-03-30 Theravance, Inc. β2-adrenergic receptor agonists
WO2005019819A1 (en) 2003-08-20 2005-03-03 Biosite, Inc. Methods and compositions for measuring biologically active natriuretic peptides and for improving their therapeutic potential
US6875737B1 (en) 1996-06-28 2005-04-05 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US6897305B2 (en) 1998-06-08 2005-05-24 Theravance, Inc. Calcium channel drugs and uses
US6979697B1 (en) 1998-08-21 2005-12-27 Point Therapeutics, Inc. Regulation of substrate activity
US7101909B2 (en) 1998-10-12 2006-09-05 Theravance, Inc. Calcium channel drugs and uses
EP1743676A1 (en) * 2003-11-12 2007-01-17 Phenomix Corporation Heterocyclic boronic acid derivatives, dipeptidyl peptidase IV inhibitors
US7317109B2 (en) 2003-11-12 2008-01-08 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7576121B2 (en) 2003-11-12 2009-08-18 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7767828B2 (en) 2003-11-12 2010-08-03 Phenomix Corporation Methyl and ethyl substituted pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7825139B2 (en) 2005-05-25 2010-11-02 Forest Laboratories Holdings Limited (BM) Compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
US8093236B2 (en) 2007-03-13 2012-01-10 Takeda Pharmaceuticals Company Limited Weekly administration of dipeptidyl peptidase inhibitors
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8324383B2 (en) 2006-09-13 2012-12-04 Takeda Pharmaceutical Company Limited Methods of making polymorphs of benzoate salt of 2-[[6-[(3R)-3-amino-1-piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]-benzonitrile
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6825169B1 (en) * 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US6140475A (en) * 1997-04-11 2000-10-31 Altus Biologics Inc. Controlled dissolution crosslinked protein crystals
US6040145A (en) * 1997-05-07 2000-03-21 Tufts University Potentiation of the immune response
FR2828404B1 (en) * 2001-08-10 2005-07-15 Neovacs COMPOSITE SUPERIMMUNOGEN FOR BIFUNCTIONAL VACCINE USE FOR THE TREATMENT OF DISEASES ASSOCIATED WITH STROMAL TISSUE DISORDER
US20060014697A1 (en) * 2001-08-22 2006-01-19 Travis Mickle Pharmaceutical compositions for prevention of overdose or abuse
US6780602B2 (en) * 2001-11-01 2004-08-24 Microbiosystems, Limited Partnership Taxonomic identification of pathogenic microorganisms and their toxic proteins
CA2468192A1 (en) * 2001-11-26 2003-06-05 Trustees Of Tufts College Peptidomimetic inhibitors of post-proline cleaving enzymes
IL158569A0 (en) * 2002-06-29 2004-05-12 Aquanova Ger Solubilisate Tech Isoflavone concentrates and methods for the production thereof
JP2006506442A (en) * 2002-07-09 2006-02-23 ポイント セラピューティクス, インコーポレイテッド Boroproline compound combination therapy
US7598224B2 (en) 2002-08-20 2009-10-06 Biosurface Engineering Technologies, Inc. Dual chain synthetic heparin-binding growth factor analogs
US7166574B2 (en) 2002-08-20 2007-01-23 Biosurface Engineering Technologies, Inc. Synthetic heparin-binding growth factor analogs
US8227411B2 (en) 2002-08-20 2012-07-24 BioSurface Engineering Technologies, Incle FGF growth factor analogs
US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
US20040242568A1 (en) 2003-03-25 2004-12-02 Syrrx, Inc. Dipeptidyl peptidase inhibitors
EP1625122A1 (en) 2003-05-14 2006-02-15 Takeda San Diego, Inc. Dipeptidyl peptidase inhibitors
DE602004009314T2 (en) 2003-08-12 2008-02-07 Lipoxen Technologies Ltd. POLYSIALINSÄUREDERIVATE
US7169926B1 (en) 2003-08-13 2007-01-30 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US20050065148A1 (en) 2003-08-13 2005-03-24 Syrrx, Inc. Dipeptidyl peptidase inhibitors
US7678909B1 (en) 2003-08-13 2010-03-16 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
ATE550656T1 (en) * 2003-08-20 2012-04-15 Alere San Diego Inc METHODS AND COMPOSITIONS FOR MEASURING BIOLOGICALLY ACTIVE NATRIURETIC PEPTIDES AND FOR IMPROVING THEIR THERAPEUTIC POTENTIAL
JP2007505121A (en) 2003-09-08 2007-03-08 武田薬品工業株式会社 Dipeptidyl peptidase inhibitor
US7414028B1 (en) 2004-02-04 2008-08-19 Biosurface Engineering Technologies, Inc. Growth factor analogs
US20060024347A1 (en) * 2004-02-10 2006-02-02 Biosurface Engineering Technologies, Inc. Bioactive peptide coatings
US7528105B1 (en) 2004-02-10 2009-05-05 Biosurface Engineering Technologies Heterodimeric chain synthetic heparin-binding growth factor analogs
US20080227696A1 (en) 2005-02-22 2008-09-18 Biosurface Engineering Technologies, Inc. Single branch heparin-binding growth factor analogs
US7671012B2 (en) 2004-02-10 2010-03-02 Biosurface Engineering Technologies, Inc. Formulations and methods for delivery of growth factor analogs
JP4895826B2 (en) 2004-02-20 2012-03-14 バイオサーフェス エンジニアリング テクノロジーズ,インク. Bone morphogenetic protein-2 positive modulator
US7732446B1 (en) 2004-03-11 2010-06-08 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
KR101071390B1 (en) 2004-03-15 2011-10-07 다케다 야쿠힌 고교 가부시키가이샤 dipeptidyl peptidase inhibitors
US7687638B2 (en) 2004-06-04 2010-03-30 Takeda San Diego, Inc. Dipeptidyl peptidase inhibitors
WO2006019965A2 (en) 2004-07-16 2006-02-23 Takeda San Diego, Inc. Dipeptidyl peptidase inhibitors
US7884185B2 (en) 2004-07-28 2011-02-08 University Of Delaware Hydrogels and uses thereof
US20060063719A1 (en) * 2004-09-21 2006-03-23 Point Therapeutics, Inc. Methods for treating diabetes
US7872124B2 (en) 2004-12-21 2011-01-18 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US20070060529A1 (en) * 2005-09-14 2007-03-15 Christopher Ronald J Administration of dipeptidyl peptidase inhibitors
EP1951740B1 (en) * 2005-11-10 2014-07-16 James Paulson High affinity siglec ligands
CA2629897A1 (en) * 2005-11-14 2007-05-24 Ud Technology Corporation Novel hydrogels and uses thereof
CA2658227A1 (en) 2006-01-17 2007-07-26 Health Research, Inc. Heteroduplex tracking assay
WO2007112347A1 (en) 2006-03-28 2007-10-04 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
WO2007117419A2 (en) * 2006-03-31 2007-10-18 Dara Biosciences, Inc. Methods and compositions relating to post-prolyl cleaving enzyme inhibitors
US7820172B1 (en) 2006-06-01 2010-10-26 Biosurface Engineering Technologies, Inc. Laminin-derived multi-domain peptides
CA2692240C (en) 2006-06-22 2018-03-13 Biosurface Engineering Technologies, Inc. Composition and method for delivery of bmp-2 amplifier/co-activator for enhancement of osteogenesis
WO2009117497A1 (en) * 2008-03-19 2009-09-24 University Of Delaware Delivery of hydrogels as sprays
MX2019014719A (en) 2017-06-14 2020-08-03 Virometix Ag Cyclic peptides for protection against respiratory syncytial virus.
CN113260377A (en) 2018-12-20 2021-08-13 维罗米蒂克斯股份公司 Lipopeptide building blocks and synthetic virus-like particles
US20230355737A1 (en) 2020-09-28 2023-11-09 Dbv Technologies Particle comprising an rsv-f protein for use in rsv vaccination

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5329028A (en) * 1992-08-05 1994-07-12 Genentech, Inc. Carbohydrate-directed cross-linking reagents
WO1995015309A1 (en) * 1993-12-03 1995-06-08 Ferring B.V. Dp-iv-serine protease inhibitors
WO1995029190A1 (en) * 1994-04-22 1995-11-02 Institut Pasteur Multirepresentation of a peptide analogue of the dppiv substrate, especially of the kpr type, to inhibit the entry of hiv in cells

Family Cites Families (66)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE270382C (en)
DE158109C (en)
DE296075C (en)
US3854480A (en) * 1969-04-01 1974-12-17 Alza Corp Drug-delivery system
US3832253A (en) * 1973-03-21 1974-08-27 Baxter Laboratories Inc Method of making an inflatable balloon catheter
US4318904A (en) * 1980-04-25 1982-03-09 Research Corporation Peptide affinity labels for thrombin and other trypsin-like proteases
US4443609A (en) * 1980-05-19 1984-04-17 Petrolite Corporation Tetrahydrothiazole phosphonic acids or esters thereof
US4667014A (en) * 1983-03-07 1987-05-19 Syntex (U.S.A.) Inc. Nonapeptide and decapeptide analogs of LHRH, useful as LHRH antagonists
US4452775A (en) * 1982-12-03 1984-06-05 Syntex (U.S.A.) Inc. Cholesterol matrix delivery system for sustained release of macromolecules
CA1200416A (en) * 1983-05-13 1986-02-11 Societe Des Produits Nestle S.A. Food process
US4582821A (en) * 1983-11-16 1986-04-15 E. I. Du Pont De Nemours And Company Inhibition of cyclic nucleotide independent protein kinases
US4499082A (en) * 1983-12-05 1985-02-12 E. I. Du Pont De Nemours And Company α-Aminoboronic acid peptides
US4636492A (en) * 1984-08-29 1987-01-13 E. I. Du Pont De Nemours And Company Inhibition of viral protease activity by peptide halomethyl ketones
US4652552A (en) * 1984-09-10 1987-03-24 E. I. Du Pont De Nemours And Company Tetrapeptide methyl ketone inhibitors of viral proteases
US4644055A (en) * 1984-12-17 1987-02-17 E. I. Du Pont De Nemours And Company Method for preparing specific inhibitors of virus-specified proteases
US5187157A (en) * 1987-06-05 1993-02-16 Du Pont Merck Pharmaceutical Company Peptide boronic acid inhibitors of trypsin-like proteases
US5250720A (en) * 1987-06-05 1993-10-05 The Dupont Merck Pharmaceutical Company Intermediates for preparing peptide boronic acid inhibitors of trypsin-like proteases
US5242904A (en) * 1987-06-05 1993-09-07 The Dupont Merck Pharmaceutical Company Peptide boronic acid inhibitors of trypsin-like proteases
US4935493A (en) * 1987-10-06 1990-06-19 E. I. Du Pont De Nemours And Company Protease inhibitors
JPH04505547A (en) * 1988-01-04 1992-10-01 イー・アイ・デユポン・ド・ネモアース・アンド・コンパニー Multi-step affinity method for isolating specific cells from cell mixtures
US4963655A (en) * 1988-05-27 1990-10-16 Mayo Foundation For Medical Education And Research Boron analogs of amino acid/peptide protease inhibitors
US5215926A (en) * 1988-06-03 1993-06-01 Cellpro, Inc. Procedure for designing efficient affinity cell separation processes
EP0420913B1 (en) * 1988-06-14 1995-11-15 Cell Med, Inc. Heterofunctional cellular immunological reagents, vaccines containing same and methods for the use of same
IL91307A0 (en) 1988-08-24 1990-03-19 Merck & Co Inc Hiv protease inhibitors and pharmaceutical compositions for the treatment of aids containing them
DE3840452A1 (en) 1988-12-01 1990-06-07 Hoechst Ag (BETA) -amino-boronic acid DERIVATIVES
DE3842197A1 (en) * 1988-12-15 1990-06-21 Hoechst Ag RAPID CLEANER SUBSTRATE FOR THE HIV PROTEASE
US5437994A (en) * 1989-06-15 1995-08-01 Regents Of The University Of Michigan Method for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells
US5635386A (en) * 1989-06-15 1997-06-03 The Regents Of The University Of Michigan Methods for regulating the specific lineages of cells produced in a human hematopoietic cell culture
DE4033062A1 (en) 1990-10-18 1992-04-23 Merck Patent Gmbh AMINOSAEUREDERIVATE
US5462928A (en) * 1990-04-14 1995-10-31 New England Medical Center Hospitals, Inc. Inhibitors of dipeptidyl-aminopeptidase type IV
WO1991016339A1 (en) * 1990-04-14 1991-10-31 New England Medical Center Hospitals, Inc. Inhibitors of dipeptidyl-aminopeptidase type iv
US5635387A (en) * 1990-04-23 1997-06-03 Cellpro, Inc. Methods and device for culturing human hematopoietic cells and their precursors
US5378624A (en) * 1990-04-23 1995-01-03 Cellpro, Incorporated Methods for removing ligands from a particle surface
WO1991017767A1 (en) * 1990-05-21 1991-11-28 New England Medical Center Hospitals, Inc. Method of treating inhibition of dipeptidyl aminopeptidase type iv
GB9017694D0 (en) * 1990-08-13 1990-09-26 Sandoz Ltd Improvements in or relating to organic chemistry
US5288707A (en) * 1990-08-13 1994-02-22 Sandoz Ltd. Borolysine peptidomimetics
JPH04167172A (en) * 1990-10-31 1992-06-15 Nec Corp Vector processor
JPH06504547A (en) * 1990-12-28 1994-05-26 ジョージア・テック・リサーチ・コーポレーション Peptide ketoamides, keto acids and ketoesters
EP0578745A1 (en) * 1991-04-04 1994-01-19 The Upjohn Company Phosphorus containing compounds as inhibitors of retroviruses
IE922316A1 (en) * 1991-07-17 1993-01-27 Smithkline Beecham Corp Retroviral protease inhibitors
US5554728A (en) * 1991-07-23 1996-09-10 Nexstar Pharmaceuticals, Inc. Lipid conjugates of therapeutic peptides and protease inhibitors
WO1993005011A1 (en) * 1991-08-29 1993-03-18 Sandoz Ltd. Novel immunosuppressants
CA2121369C (en) * 1991-10-22 2003-04-29 William W. Bachovchin Inhibitors of dipeptidyl-aminopeptidase type iv
MX9206628A (en) * 1991-11-22 1993-05-01 Boehringer Ingelheim Pharma PROLINABORONATE ESTER AND METHOD FOR ITS PREPARATION.
WO1993016102A1 (en) * 1992-02-06 1993-08-19 Dana-Farber Cancer Institute, Inc. Human cd26 and methods for use
US5296604A (en) * 1992-05-15 1994-03-22 Miles Inc. Proline derivatives and compositions for their use as inhibitors of HIV protease
AU4794393A (en) * 1992-07-31 1994-03-03 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Producing increased numbers of hematopoietic cells by administering inhibitors of dipeptidyl peptidase iv
WO1994009132A1 (en) * 1992-08-21 1994-04-28 Dana-Farber Cancer Institute, Inc. Human cd26 and methods for use
FR2701951B1 (en) 1993-02-24 1995-06-09 Adir NOVEL PEPTIDE DERIVATIVES OF BORONIC ACID, THEIR PREPARATION PROCESS AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.
DK0688336T3 (en) * 1993-03-03 1997-10-20 Sandoz Ltd Peptide boronic acid derivatives with protease inhibitory activity
US5384410A (en) * 1993-03-24 1995-01-24 The Du Pont Merck Pharmaceutical Company Removal of boronic acid protecting groups by transesterification
AU6773994A (en) * 1993-04-23 1994-11-21 Cellpro, Incorporated Methods for enriching fetal progenitor cells from maternal blood
SE9301911D0 (en) * 1993-06-03 1993-06-03 Ab Astra NEW PEPTIDE DERIVATIVES
FR2707170A1 (en) * 1993-06-04 1995-01-13 Pasteur Institut Expression of CD4 and CD26 receptors in recombinant cells, CD26 receptor inhibitors.
WO1995011689A1 (en) * 1993-10-29 1995-05-04 Trustees Of Tufts College Use of inhibitors of dipeptidyl-aminopeptidase to block entry of hiv into cells
NL9400309A (en) * 1993-11-04 1995-06-01 Eurogenetics Nv Ligand which is able to bind to the adenosine deaminase binding site of CD26
US5543396A (en) * 1994-04-28 1996-08-06 Georgia Tech Research Corp. Proline phosphonate derivatives
DE4416963C1 (en) * 1994-05-13 1995-07-13 Boehringer Ingelheim Kg Enantiomerically pure di:aryl-prolinol derivs. prodn.
AU2790895A (en) * 1994-06-10 1996-01-05 Universitaire Instelling Antwerpen Purification of serine protease and synthetic inhibitors thereof
FR2721611B1 (en) 1994-06-22 1996-09-27 Adir New peptide derivatives of boronic acid, their process of preparation and the pharmaceutical compositions which contain them.
ES2162081T3 (en) * 1995-06-07 2001-12-16 Aastrom Biosciences Inc APPARATUS AND PROCEDURE FOR THE MAINTENANCE AND GROWTH OF BIOLOGICAL CELLS.
US5856571A (en) * 1995-06-07 1999-01-05 Cellpro, Incorporated Semicarbazide-containing linker compounds for formation of stably-linked conjugates and methods related thereto
US5965532A (en) 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6100234A (en) 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6040145A (en) 1997-05-07 2000-03-21 Tufts University Potentiation of the immune response
CA2304206A1 (en) 1997-09-29 1999-04-08 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5329028A (en) * 1992-08-05 1994-07-12 Genentech, Inc. Carbohydrate-directed cross-linking reagents
WO1995015309A1 (en) * 1993-12-03 1995-06-08 Ferring B.V. Dp-iv-serine protease inhibitors
WO1995029190A1 (en) * 1994-04-22 1995-11-02 Institut Pasteur Multirepresentation of a peptide analogue of the dppiv substrate, especially of the kpr type, to inhibit the entry of hiv in cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. Wijdenes et al. 'Monoclonal Antibodies (mAb) against GP130 imitating Cytokines which use the gp130 for Signal Trans- duction' in: The 9th International Congress of Immunology, Abstract Book, 23-29 July, 1995, San Francisco, CA, USA XP002051385 *

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6875737B1 (en) 1996-06-28 2005-04-05 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6703238B2 (en) 1997-09-29 2004-03-09 Point Therapeutics, Inc. Methods for expanding antigen-specific T cells
WO1999056753A1 (en) * 1998-05-04 1999-11-11 Point Therapeutics, Inc. Hematopoietic stimulation
US6770628B2 (en) 1998-05-04 2004-08-03 Point Therapeutics, Inc. Hematopoietic stimulation
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US7067489B2 (en) 1998-05-04 2006-06-27 Point Therapeutics, Inc. Hematopoietic stimulation
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6362371B1 (en) 1998-06-08 2002-03-26 Advanced Medicine, Inc. β2- adrenergic receptor agonists
US6541669B1 (en) 1998-06-08 2003-04-01 Theravance, Inc. β2-adrenergic receptor agonists
SG83720A1 (en) * 1998-06-08 2001-10-16 Advanced Medicine Inc Beta 2-adrenergic receptor agonists
SG83721A1 (en) * 1998-06-08 2001-10-16 Advanced Medicine Inc Beta 2-adrenergic receptor agonists
US6897305B2 (en) 1998-06-08 2005-05-24 Theravance, Inc. Calcium channel drugs and uses
SG80038A1 (en) * 1998-06-08 2001-04-17 Advanced Medicine Inc Novel calcium channel drugs and uses
WO1999063992A1 (en) * 1998-06-08 1999-12-16 Advanced Medicine, Inc. Novel calcium channel drugs and uses
US6979697B1 (en) 1998-08-21 2005-12-27 Point Therapeutics, Inc. Regulation of substrate activity
US7101909B2 (en) 1998-10-12 2006-09-05 Theravance, Inc. Calcium channel drugs and uses
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US6949514B2 (en) 1999-05-25 2005-09-27 Point Therapeutics, Inc. Anti-tumor agents
US6683115B2 (en) 1999-06-02 2004-01-27 Theravance, Inc. β2-adrenergic receptor agonists
US6593497B1 (en) 1999-06-02 2003-07-15 Theravance, Inc. β2-adrenergic receptor agonists
US6713651B1 (en) 1999-06-07 2004-03-30 Theravance, Inc. β2-adrenergic receptor agonists
US6576793B1 (en) 1999-12-08 2003-06-10 Theravance, Inc. β2-adrenergic receptor agonists
WO2005019819A1 (en) 2003-08-20 2005-03-03 Biosite, Inc. Methods and compositions for measuring biologically active natriuretic peptides and for improving their therapeutic potential
US7576121B2 (en) 2003-11-12 2009-08-18 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7317109B2 (en) 2003-11-12 2008-01-08 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
EP1743676A1 (en) * 2003-11-12 2007-01-17 Phenomix Corporation Heterocyclic boronic acid derivatives, dipeptidyl peptidase IV inhibitors
US7674913B2 (en) 2003-11-12 2010-03-09 Phenomix Corporation Heterocyclic boronic acid compounds
US7767828B2 (en) 2003-11-12 2010-08-03 Phenomix Corporation Methyl and ethyl substituted pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7884217B2 (en) 2003-11-12 2011-02-08 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7906658B2 (en) 2003-11-12 2011-03-15 Phenomix Corporation Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US7825139B2 (en) 2005-05-25 2010-11-02 Forest Laboratories Holdings Limited (BM) Compounds and methods for selective inhibition of dipeptidyl peptidase-IV
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8324383B2 (en) 2006-09-13 2012-12-04 Takeda Pharmaceutical Company Limited Methods of making polymorphs of benzoate salt of 2-[[6-[(3R)-3-amino-1-piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]-benzonitrile
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
US8093236B2 (en) 2007-03-13 2012-01-10 Takeda Pharmaceuticals Company Limited Weekly administration of dipeptidyl peptidase inhibitors

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