US20090208537A1 - Anti-Neovasculature Preparations for Cancer - Google Patents
Anti-Neovasculature Preparations for Cancer Download PDFInfo
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- US20090208537A1 US20090208537A1 US11/772,811 US77281107A US2009208537A1 US 20090208537 A1 US20090208537 A1 US 20090208537A1 US 77281107 A US77281107 A US 77281107A US 2009208537 A1 US2009208537 A1 US 2009208537A1
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001109—Vascular endothelial growth factor receptors [VEGFR]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
Definitions
- TuAA tumor-associated antigens
- TuAA include proteins normally expressed selectively by the tissue from which the cancer derives (differentiation antigens), proteins that are associated with a different stage of development (oncofetal and cancer-testis antigens), proteins that are created by aberrant chromosomal rearrangement, or proteins that are derived from oncogenic viruses.
- differentiation antigens proteins normally expressed selectively by the tissue from which the cancer derives
- oncofetal and cancer-testis antigens proteins that are associated with a different stage of development
- proteins that are created by aberrant chromosomal rearrangement or proteins that are derived from oncogenic viruses.
- CTL cytotoxic T lymphocytes
- the anti-angiogenesis approach takes advantage of the need of tumors to recruit a blood supply to support their continued growth. To accomplish this, tumors secrete angiogenic factors that promote the growth of new blood vessels.
- the anti-angiogenesis approach aims to disrupt a tumor's supply of nutrients to cause it to die, or at least limit its growth. Attempts at this approach have sought chemotherapeutic drugs used directly against a variety of anti-angiogenic factors and angiogenesis.
- compositions designed to stimulate cellular immune responses targeting tumor-associated neovasculature TuNV.
- the compositions stimulate a CTL response.
- Such compositions may include one or more epitopes of the target antigen.
- One aspect of this embodiment specifically includes a housekeeping epitope, another specifically includes an immune epitope or epitope cluster, and another aspect specifically combines housekeeping and immune epitopes.
- Embodiments of the invention relate to the use of prostate specific membrane antigen (PSMA) as the target antigen of the composition. Aspects of this embodiment include various epitopes derived from PSMA provided directly as polypeptide, or as a nucleic acid capable of conferring expression of the epitope. Other embodiments relate to the use of other TuNV-associated antigens.
- PSMA prostate specific membrane antigen
- compositions are directed against both the TuNV and against TuAA expressed by the cancerous cells, by combining immunogens derived from both sources into a single formulation or method or treatment.
- compositions of this invention can be accomplished using adoptive transfer of immunized T cells into SCID mice bearing microvasculature formed from implanted human dermal microvascular endothelial cells (HDMEC).
- HDMEC human dermal microvascular endothelial cells
- Preclinical evaluation can also be accomplished through the use of HLA-transgenic mice immunized with compositions comprised of epitopes conserved between mice and humans.
- Embodiments of the invention relate to methods of evaluating cell-mediated immunity.
- the methods can include the steps of implanting vascular cells into an immunodeficient mammal; establishing an immune response in the mammal; and assaying a characteristic to determine cell-mediated immunity in the mammal.
- the cell-mediated immunity can be directed against a neovasculature antigen, for example.
- the neovasculature antigen can be preferentially expressed by tumor-associated neovasculature, for example, and in preferred embodiments can be prostate specific membrane antigen (PSMA), vascular endothelial growth factor receptor 2 (VEGFR2), and the like.
- PSMA prostate specific membrane antigen
- VEGFR2 vascular endothelial growth factor receptor 2
- the establishing step can be achieved, for example, by adoptive transfer of T-cells to the mammal, by contacting the mammal with an antigen, and the like.
- the cell-mediated immunity can be mediated by cytotoxic T lymphocytes.
- the vascular cells can be vascular endothelial cells, such as, for example, human dermal microvascular endothelial cells (HDMEC), telomerase-transformed endothelial cells, and the like.
- the immunodeficient mammal can be a mouse, such as for example a SCID mouse.
- the characterizing step can include assessing a parameter, such as for example, vessel formation, vessel destruction, vessel density, proportion of vessels carrying blood of the host mammal, and the like.
- the methods can further include the step of implanting tumor cells or tumor tissue in the mouse.
- the characterizing step can include assessing a parameter, such as, for example, tumor presence, tumor growth, tumor size, rapidity of tumor appearance, dose of vaccine required to inhibit or prevent tumor establishment, tumor vascularization, a proportion of necrotic tissue within the tumor, and the like.
- the methods can further include the steps of providing a first population of mammals and a second populations of mammals; establishing cell-mediated immunity in the first population; differentially establishing cell-mediated immunity in the second population; and comparing a result obtained from the first population of mammals to a result obtained from the second population of mammals.
- the cell-mediated immunity of the first population can include, for example, naive immunity, immunity to an irrelevant epitope, and the like.
- inventions relate to methods of evaluating cell-mediated immunity, including immunity directed against a neovasculature antigen.
- the methods can include the steps of implanting or injecting MHC-transgenic tumor cells into an MHC-transgenic mammal; establishing an immune response in the mammal; and assaying a characteristic to determine cell-mediated immunity in the mammal.
- the MHC-transgenic mammal can be an HLA-transgenic mammal, such as, for example an HLA-A2 transgenic mammal.
- the mammal can be a mouse.
- the cell-mediated immunity can be established by vaccination, which in preferred embodiments can take place prior to, concurrent with, or subsequent to transfer of the tumor cells, for example.
- the cell-mediated immunity can be mediated by cytotoxic T lymphocytes.
- the neovasculature antigen can be preferentially expressed by tumor-associated neovasculature and can also be a tumor-associated antigen.
- the antigen can be the ED-B domain of fibronectin.
- the characterizing step can include, for example, assessing a parameter, including tumor presence, tumor growth, tumor size, rapidity of tumor appearance, dose of vaccine required to inhibit or prevent tumor establishment, tumor vascularization, a proportion of necrotic tissue within the tumor, and the like.
- the methods can further include the steps of providing a first population of mammals and a second populations of mammals; establishing cell-mediated immunity in the first population; differentially establishing cell-mediated immunity in the second population; and comparing a result obtained from the first population of mammals to a result obtained from the second population of mammals.
- the cell-mediated immunity of the first population can include naive immunity, immunity to an irrelevant epitope, and the like.
- Still further embodiments relate to methods of treating neoplastic disease, including the step of immunizing a mammal to induce a cellular immune response directed against an antigen differentially expressed by tumor-associated neovasculature.
- the differentially expressed antigen can be a protein, such as, for example prostate specific membrane antigen, vascular endothelial growth factor receptor 2 (VEGFR2), and the like.
- the antigen can be the ED-B domain of fibronectin.
- the immunization can be carried out, for example, with at least one peptide derived from the sequence of the protein, with a nucleic acid capable of conferring expression of the protein or peptides, and the like.
- the at least one peptide can include a housekeeping epitope, for example, and in preferred embodiments can be co-C-terminal with the housekeeping epitope.
- the methods can further include at least one additional peptide, wherein the at least one additional peptide includes an immune epitope.
- the methods can include an additional step wherein the mammal is treated with an anti-tumor therapy active directly against cancerous cells.
- the anti-tumor therapy can be immunization against a tumor-associated antigen.
- the cellular immune response can include a CTL response.
- the immunogenic compositions can include at least one immunogen corresponding to an antigen expressed by tumor-associated neovasculature, wherein the composition can induce a cellular immune response.
- the immunogen can be one that is not associated with a cell conspecific with the recipient.
- the antigen can be a protein, such as, for example prostate specific membrane antigen, vascular endothelial growth factor receptor 2 (VEGFR2), and the like. In other preferred embodiments the antigen can be the ED-B domain of fibronectin.
- the immunogen can include at least one peptide.
- the compositions can include a nucleic acid capable of conferring expression of the antigen, and wherein the antigen is a protein or a peptide.
- compositions can include at least one peptide that includes a housekeeping epitope, and in preferred embodiments the at least one peptide can be co-C-terminal with the housekeeping epitope. Also, the compositions can additionally include at least one peptide that includes an immune epitope.
- the compositions can include at least one immunogen corresponding to a tumor-associated antigen. In preferred embodiments the cellular immune response can include a CTL response.
- Embodiments relate to methods of anti-tumor vaccine design.
- the methods can include the steps of identifying an antigen differentially expressed by tumor-associated neovasculature; and incorporating a component of the antigen into a vaccine.
- the component can include, for example, a polypeptide fragment of the antigen, a nucleic acid encoding the antigen or a fragment of the antigen, and the like.
- the methods can include implanting a vascular cell and a tumor cell into an immunodeficient mammal.
- the tumor cell and the vascular cell can be implanted adjacent to one another.
- the vascular cell can be a vascular endothelial cell, such as for example HDMEC.
- the vascular endothelial cell can be telomerase-transformed.
- the immunodeficient mammal can be a mouse, such as, for example, a SCID mouse.
- the research models can include an immunodeficient mammal.
- the mammal can include an implanted vascular cell and an implanted tumor cell.
- the vascular cell and the tumor cell can be implanted adjacent to one another.
- FIG. 2 shows binding curves for HLA-A2:PSMA 168-177 and HLA-A2:PSMA 288-297 with controls.
- FIG. 4 shows binding curves for HLA-A2:PSMA 461-469 , HLA-A2:PSMA 460-469, and HLA-A2:PSMA 663-671 , with controls.
- FIG. 5 shows the results of a ⁇ (gamma)-IFN-based ELISPOT assay detecting PSMA 463-471 -reactive HLA-A1 + CD8 + T cells.
- FIG. 6 shows blocking of reactivity of the T cells used in FIG. 10 by anti-HLA-A1 mAb, demonstrating HLA-A1-restricted recognition.
- FIG. 7 shows a binding curve for HLA-A2:PSMA 663-671 , with controls.
- FIG. 8 shows a binding curve for HLA-A2:PSMA 662-671 , with controls.
- FIG. 9 shows epitope specific lysis by CTL from HHD-A2 mice immunized with ED-B 29-38 peptide.
- Embodiments of the invention disclosed herein provide compositions, methods of composition or vaccine design, and methods of treatment related to the generation of a cellular immune response, preferably, a T cell response and, more preferably, a CTL response, directed against the neovasculature of tumors. Such methods and compositions are particularly useful in the treatment and prevention of cancer. Other embodiments relate to composition evaluation models.
- compositions Compositions, Composition Design, and Treatment Using the Compositions
- Embodiments of the invention relate to immunogenic compositions, including vaccines, for the generation of a cellular immune response, particularly a T cell response and specifically a CTL response, directed against tumor neovasculature (TuNV).
- TuNV tumor neovasculature
- TuNV tumor neovasculature
- TuNV tumor neovasculature
- TuNV Tuma virus
- Suitable TuNV antigens can include those that are expressed generally in neovasculature or preferentially by TuNV, for example.
- compositions can include, for example, an epitopic peptide or peptides.
- Immune epitopes may be provided embedded in epitope clusters and protein fragments. Housekeeping epitopes can be provided with the proper C-terminus.
- compositions can include nucleic acids capable of conferring expression of these epitopes on pAPC, for example.
- compositions can be administered directly to the lymphatic system of a mammal being treated.
- This can be applied to both polypeptide and nucleic acid based compositions.
- Administration methods of this type, and related technologies, are disclosed in U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, and a Continuation-in-Part thereof, filed on Feb. 2, 2001; U.S. patent application Ser. No. 09/776,232, both entitled “A METHOD OF INDUCING A CTL RESPONSE,” which are incorporated by reference in their entirety.
- destruction of the blood vessels in a tumor by action of a composition of the invention can eliminate all of the cells in a tumor.
- small tumors including micrometastases, are typically unvasculaturized.
- unvascularized tumors that instead apparently rely on blood flow through channels penetrating the tumor mass have been reported (Maniotis, A. J., et al. Am. J. Pathol. 155: 739-752, 1999).
- the compositions are generally effective as tumor control agents that may not eradicate all cancer cells. Accordingly, the invention provides tools for eliminating tumors, controlling tumor growth, reducing tumor burden, improving overall clinical status, and the like.
- compositions can be desirable to combine these compositions with other treatments that target the cancerous cells directly. Additionally there is evidence that the vasculature in tumors can be mosaic in nature consisting of both endothelial and cancer cells (Chang, Y. S., et al. Proc. Natl. Acad. Sci. USA 97:14608-14613, 2000). Thus, in some embodiments of the invention a course of composition treatment can be followed by administration of a bio- or chemotherapeutic agent. In a particularly preferred embodiment, treatment can include administration of a TuAA directed composition concurrent or subsequent to administration of the anti-TuNV composition.
- suitable TuNV antigens for the compositions can include those that are expressed generally in neovasculature or preferentially by TuNV, for example.
- a variety of techniques for discovery of TuAA are known in the art. Examples of these techniques include, without limitation, differential hybridization and subtractive hybridization, including use of microarrays; expression cloning; SAGE (serial analysis of gene expression); SEREX (serological identification of antigens by recombinant expression cloning); in situ RT-PCT; immunohistochemistry (as was the case for PSMA); EST analysis; variously using bulk, sectioned, and/or microdissected tissue; and the like.
- Utilization of these and other methods provides one of skill in the art the techniques necessary to identify genes and gene products contained within a target cell that may be used as antigens of immunogenic compositions.
- the techniques are applicable to TuAA discovery regardless of whether the target cell is a cancer cell or an endothelial cell. Any identified antigen can be scrutinized for epitopes, which can be used in embodiments of the invention.
- compositions targeting endothelial cells can be comprised of peptides, or nucleic acids conferring expression of the peptides, corresponding to the digestion products of the housekeeping proteasome (i.e. housekeeping epitopes).
- IFN- ⁇ (gamma) secreted by activated cells of the immune system, can induce expression of the immunoproteasome in the target cells.
- the immunoproteasome is constitutive in professional antigen presenting cells (pAPC).
- the immunogenic compositions can include TuNV antigens and epitopes, for example.
- the epitopes can include one or more housekeeping epitopes and/or one or more immune epitopes.
- Specific epitopes useful in compositions can be identified using the methods disclosed in U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed on Apr. 28, 2000.
- peptide sequences that are known or predicted to bind to some MHC restriction element can be compared to fragments produced by proteasomal digestion in order to identify those that are co-C-terminal.
- Examples of useful epitopes for the embodiments of the invention are disclosed in a U.S. Provisional Patent Application No. 60/363,210, entitled “EPITOPE SEQUENCES,” attorney docket number CTLIMM.027PR; filed on Mar. 7, 2002, and two U.S. Provisional Patent Applications, each entitled “EPITOPE SEQUENCES;” Application Ser. No. 60/282,211, filed on Apr. 6, 2001 and 60/337,017, filed on Nov. 7, 2001. Each of these applications is incorporated herein by reference in its entirety.
- PSMA is one example of a TuAA that can be targeted in some embodiments.
- PSMA is expressed in the neovasculature of most tumor types, but not by the vascular endothelium of normal tissues (Chang, S. M. et al., Cancer Res. 59(13):3192-8,1999; Clin Cancer Res. 10:2674-81, 1999).
- PSMA is a membrane antigen, and as such, it may be possible to attack PSMA-expressing TuNV with monoclonal antibody (mAb).
- mAb monoclonal antibody
- the effectiveness of mAb in the treatment of cancer has proved to be more difficult than initially anticipated.
- antigens are discovered to be associated with the TuNV, it is likely that many of them will prove not to be expressed at the vasculature surface, making them inaccessible to mAb attack.
- T cells particularly CTL
- MHC major histocompatability complex
- fibronectin preferably the ED-B domain.
- Fibronectin is subject to developmentally regulated alternative splicing, with the ED-B domain being encoded by a single exon that is used primarily in oncofetal tissues. Matsuura, H. and S. Hakomori Proc. Natl. Acad. Sci. USA 82:6517-6521, 1985; Carnemolla, B. et al. J. Cell Biol. 108:1139-1148, 1989; Loridon-Rosa, B. et al. Cancer Res. 50:1608-1612, 1990; Nicolo, G. et al. Cell Differ. Dev.
- the ED-B domain is also expressed in fibronectin of the neovasculature Kaczmarek, J. et al. Int. J. Cancer 59:11-16, 1994; Castellani, P. et al. Int. J. Cancer 59:612-618, 1994; Neri, D. et al. Nat. Biotech. 15:1271-1275, 1997; Karelina, T. V. and A. Z. Eisen Cancer Detect. Prev. 22:438-444, 1998; Tarli, L. et al. Blood 94:192-198, 1999; Castellani, P. et al. Acta Neurochir. ( Wien ) 142:277-282, 2000.
- the ED-B domain As an oncofetal domain, the ED-B domain is commonly found in the fibronectin expressed by neoplastic cells, in addition to being expressed by the TuNV. Therefore, CTL-inducing compositions targeting the ED-B domain can exhibit two mechanisms of action: direct lysis of tumor cells, and disruption of tumor blood supply through destruction of the TuNV.
- compositions based on it, or similarly expressed antigens can be effective against other conditions associated with inappropriate angiogenesis. Further, as CTL activity can decay rapidly after withdrawal of the composition, interference with normal angiogenesis can be minimal.
- vascular endothelial growth factor receptor 2 VEGFR2
- U.S. Pat. No. 6,342,221 includes discussion of VEGF and VEGFR2, and is hereby incorporated by reference in its entirety.
- any other antigen or protein associated with vascular cells can be a target for the immunogenic compositions, including those that are presently known and those yet to be identified.
- compositions designed based upon the preceding considerations are effective against the various targets.
- additional evaluation can be easily performed at any time, but preferably in a pre-clinical setting.
- such evaluation can be used in order to further aid in composition design.
- Other embodiments of the invention relate to methods of evaluating the immunogenic compositions.
- the compositions of the present invention can be easily evaluated by one of skill in the art using animal models for composition evaluation. For example, following the routine procedures below, one of skill in the art can evaluate TuNV compositions quickly and efficiently. Thus, using the models or guidance described herein, one of skill in the art can evaluate any TuNV composition for any TuNV antigen with little or no experimentation.
- Further embodiments relate to methods of making the animal research models. Other embodiments relate to the research model animals. These embodiments are set forth more fully below.
- Some embodiments relate to a model system for studying the mechanisms of human microvessel formation.
- the model system can be used for preclinical evaluation of compositions.
- the model involves the subcutaneous implantation of telomerase-transformed human dermal microvascular endothelial cells (HDMEC) mixed with MATRIGEL (Becton Dickinson) into SCID mice.
- HDMEC human dermal microvascular endothelial cells
- MATRIGEL Becton Dickinson
- T cells activated by the compositions of this invention can be adoptively transferred, for example, into such implanted mice, and the ability of the T cells to destroy, or prevent the formation of, such human microvessels can be assessed.
- the mouse can be directly vaccinated and evaluated.
- the model system can be further adapted for testing compositions effective in non-human species by substituting DMEC from other species and species-matched telomerase, and by using analogous reagents to those described below for the human system.
- the MHC-restriction elements presenting the epitopes of the composition being tested are shared by the HDMEC line implanted into the mice.
- the T cells can be derived from in vitro immunization of human T cells, or by immunization of HLA-transgenic mice (procedures for which are well known in the art and examples of which are provided in the above incorporated patent applications).
- Use of T cells generated in HLA-transgeneic mice allows matching of genetic backgrounds between the adoptively transferred T cells and the host, thereby reducing the possibility of allogeneic or xenogeneic reactions that might complicate interpretation of the results.
- the donor T cells human or murine
- the donor T cells can be subjected to one or more rounds of in vitro stimulation to enrich for the desired population or establish a clone, and thereby similarly avoid undesired reactivities.
- T cells can constitute a clone or a polyclonal population recognizing one or more epitopes.
- on the order of 10 5 to 10 8 cells are transferred for adoptive transfer experiments in mice.
- Clones and otherwise more enriched populations generally require the transfer of fewer cells.
- Transfer of the T cells can take place shortly before, concurrent with, or subsequent to implantation or establishment of the HDMEC.
- Parameters that can be assessed to evaluate effectiveness of the compositions include vessel formation, changes in vessel density, and ability to carry mouse blood (as described in Yang et al.), and the like.
- Assessment can be carried out as early as one week, and at least as long as 6 weeks, after implantation of telomerase-transformed HDMEC, preferably after 2 weeks; and from a day to more than 6 weeks after T cell transfer, preferably after 1 to 3 weeks.
- assessment can include comparison of mice receiving T cells reactive with the target antigen with mice receiving naive (including sham-immunized), or irrelevant epitope-reactive T cells.
- tumor cells can be implanted along with the HDMEC. This can result in inducing expression of antigens preferentially expressed by TuNV. In one example, this can be accomplished by implanting a block of tumor tissue adjacent to the HDMEC-containing MATRIGEL implant, injecting tumor cells at the site of the implant, implanting tumor cell-containing MATRIGEL adjacent to the HDMEC-containing MATRIGEL implant, incorporating both tumor cells and HDMEC into the same MATRIGEL implant or by any other suitable method. As discussed above, in some embodiments, tumor cells can be implanted along with vascular cells. The animals so made, can be used as research models. Additional variations will be apparent to one of skill in the art.
- HLA-transgenic strains allow another approach, namely vaccination of the model animal to combat a syngeneic tumor.
- the ED-B domain of fibronectin provides such an opportunity, as it is a marker of angiogenesis and has identical amino acid sequence in both humans and mice (Nilsson, F. et al. Cancer Res. 61:711-716, 2001).
- spontaneous tumor tissue from a strain of HLA-A2 transgenic mice has been isolated and propagated. Epitope discovery and selection, and composition design and delivery for CTL inducing compositions are discussed above.
- the tumor cell line, M1 is derived from a spontaneous salivary glandular cystadenocarcinoma.
- the M1 tumor cell line and methods of using the same is disclosed in U.S. Provisional Application No. 60/363,131, filed on Mar. 7, 2001, entitled “AN HLA-TRANSGENIC MURINE TUMOR CELL LINE,” which is hereby incorporated by reference in its entirety.
- the tumor cell line can arise in individuals of the HHD-A2 transgenic mouse strain of S. Pascolo et al. (J. Exp. Med. 185:2043-2051, 1997).
- mice express a single monochain class I molecule comprising human ⁇ (beta)2-microglobulin, and ⁇ 1 (alpha-1), and ⁇ 2 (alpha-2) domains of HLA-A2.1 with the balance of the molecule derived from the murine class I molecule H2D b .
- Blocks of tumor can be transplanted into new individuals where the tumor will re-grow, commonly within 1-3 weeks, with 3 mm blocks growing to 3 cm.
- tumor tissue can be disaggregated and the tumor cells grown in vitro. Upon harvest, the tumor cells can be injected subcutaneously into the neck or abdomen (2.5 ⁇ 10 6 cells for 1-3 successive days), to result in a visible tumor in approximately 5-12 weeks for early passage cells.
- the initial tumor consistently occurs in the vicinity of the salivary glands, but secondary tumors can also occur in a variety of locations, including kidney, lung, liver, and abdominal muscle.
- compositions can be administered before, concurrent with, or subsequent to establishment of the tumor, depending on the expected mechanism of the composition.
- therapeutic compositions intended to be used with some sort of debulking technique e.g. surgery
- concurrent administration can be appropriate. The better established the tumor is before treatment begins, the more stringent the test.
- a peptide, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA 163-192, (SEQ ID NO. 3), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA 168-190 (SEQ ID NO. 4) was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer.
- peptide After side chain deprotection and cleavage from the resin, peptide first dissolved in formic acid and then diluted into 30% Acetic acid, was run on a reverse-phase preparative HPLC C4 column at following conditions: linear AB gradient ( 5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fraction at time 16.642 min containing the expected peptide, as judged by mass spectrometry, was pooled and lyophilized. The peptide was then subjected to proteasome digestion and mass spectrum analysis essentially as described above. Prominent peaks from the mass spectra are summarized in Table 1.
- Binding of the candidate epitope PSMA 168-177 , GMPEGDLVYV, (SEQ ID NO. 6) to HLA-A2.1 was assayed using a modification of the method of Stauss et al., (Proc Natl Acad Sci USA 89(17):7871-5 (1992)). Specifically, T2 cells, which express empty or unstable MHC molecules on their surface, were washed twice with Iscove's modified Dulbecco's medium (IMDM) and cultured overnight in serum-free AIM-V medium (Life Technologies, Inc., Rockville, Md.) supplemented with human ⁇ 2-microglobulin at 3 ⁇ g/ml (Sigma, St.
- IMDM Iscove's modified Dulbecco's medium
- peptide at 800, 400, 200, 100, 50, 25, 12.5, and 6.25 ⁇ g/ml. in a 96-well flat-bottom plate at 3 ⁇ 10 5 cells/200 ⁇ l/well. Peptide was mixed with the cells by repipeting before distributing to the plate (alternatively peptide can be added to individual wells), and the plate was rocked gently for 2 minutes. Incubation was in a 5% CO 2 incubator at 37° C.
- W6/32 (Sigma) can be used as the anti-class I HLA monoclonal antibody
- the cells washed with staining buffer and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat F(ab′) antimouse-IgG (Sigma) for 30 min at 4° C. and washed 3 times as before.)
- the cells were resuspended in 0.5 ml staining buffer.
- the analysis of surface HLA-A2.1 molecules stabilized by peptide binding was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.). If flow cytometry is not to be performed immediately the cells can be fixed by adding a quarter volume of 2% paraformaldehyde and storing in the dark at 4° C.
- this epitope exhibits significant binding at even lower concentrations than the positive control peptides.
- the known A2.1 binder FLPSDYFPSV (HBV 18-27 ; SEQ ID NO: 107) was also used as a positive control.
- An HLA-B44 binding peptide, AEMGKYSFY (SEQ ID NO: 109), was used as a negative control.
- the fluorescence obtained from the negative control was similar to the signal obtained when no peptide was used in the assay.
- Positive and negative control peptides were chosen from Table 18.3.1 in Current Protocols in Immunology p. 18.3.2, John Wiley and Sons, New York, 1998.
- PSMA 281-310 Another peptide, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG, PSMA 281-310 , (SEQ ID NO. 18), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA 283-307 (SEQ ID NO. 19), was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer.
- peptide in ddH2O was run on a reverse-phase preparative HPLC C18 column at following conditions: linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile.
- HLA-A*0201 binding studies were preformed with PSMA 288-297 , GLPSIPVHPI, (SEQ ID NO. 21) essentially as described in Example 1.1 above. As seen in FIG. 2 , this epitope exhibits significant binding at even lower concentrations than the positive control peptides.
- HLA-A*0201 binding studies were preformed, essentially as described in Example 1.1 above, with PSMA 460-469 , YTLRVDCTPL, (SEQ ID NO.33). As seen in FIG. 4 , this epitope was found to bind HLA-A2.1 to a similar extent as the known A2.1 binder FLPSDYFPSV (HBV 18-27 ; SEQ ID NO: 108) used as a positive control. Additionally, PSMA 461-469 , (SEQ ID NO. 32) binds nearly as well.
- the wells of a nitrocellulose-backed microtiter plate were coated with capture antibody by incubating overnight at 4° C. using 50 ⁇ l/well of 4 ⁇ g/ml murine anti-human ⁇ -IFN monoclonal antibody in coating buffer (35 mM sodium bicarbonate, 15 mM sodium carbonate, pH 9.5). Unbound antibody was removed by washing 4 times 5 min. with PBS. Unbound sites on the membrane then were blocked by adding 200 ⁇ l/well of RPMI medium with 10% serum and incubating 1 hr. at room temperature.
- Antigen stimulated CD8 + T cells in 1:3 serial dilutions, were seeded into the wells of the microtiter plate using 100 ⁇ l/well, starting at 2 ⁇ 10 5 cells/well.
- Primary antigen stimulation was essentially as described in Scheibenbogen, C. et al. Int. J. Cancer 71:932-936, 1997; which is incorporated herein by reference in its entirety.
- PSMA 462-471 SEQ ID NO. 36 was added to a final concentration of 10 ⁇ g/ml and IL-2 to 100 U/ml and the cells cultured at 37° C. in a 5% CO 2 , water-saturated atmosphere for 40 hrs.
- Substrate was prepared by dissolving a 20 mg tablet of 3-amino 9-ethylcoarbasole in 2.5 ml of N, N-dimethylformamide and adding that solution to 47, 5 ml of 0.05 M phosphate-citrate buffer (pH 5.0). 25 ⁇ l of 30% H 2 O 2 was added to the substrate solution immediately before distributing substrate at 100 ⁇ l/well and incubating the plate at room temperature. After color development (generally 15-30 min.), the reaction was stopped by washing the plate with water. The plate was air dried and the spots counted using a stereomicroscope.
- FIG. 5 shows the detection of PSMA 463-471 (SEQ ID NO. 35)-reactive HLA-A1 + CD8 + T cells previously generated in cultures of HLA-A1 + CD8 + T cells with autologous dendritic cells plus the peptide. No reactivity is detected from cultures without peptide (data not shown). In this case it can be seen that the peptide reactive T cells are present in the culture at a frequency between 1 in 2.2 ⁇ 10 4 and 1 in 6.7 ⁇ 10 4 . That this is truly an HLA-A1-restricted response is demonstrated by the ability of anti-HLA-A1 monoclonal antibody to block ⁇ -IFN production; see FIG. 6 .
- FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA 653-687 Another peptide, FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA 653-687 , (SEQ ID NO. 37) containing an A2 epitope cluster from prostate specific membrane antigen, PSMA 660-681 (SEQ ID NO. 38), was synthesized by MPS (purity>95%) and subjected to proteasome digestion and mass spectrum analysis as described above. Prominent peaks from the mass spectra are summarized in Table 7.
- N-terminal addition of authentic sequence to epitopes can generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (R)MMNDQLMFL (SEQ ID NOS. 39 and (40)) with HLA-A*02, where the 10-mer retains substantial predicted binding potential.
- HLA-A*0201 binding studies were preformed, essentially as described in Example 1.1 above, with PSMA 663-671 , (SEQ ID NO. 39) and PSMA 662-671 , RMMNDQLMFL (SEQ NO. 67). As seen in FIGS. 4 , 7 and 8 , this epitope exhibits significant binding at even lower concentrations than the positive control peptide (FLPSDYFPSV (HBV 18-27 ); SEQ ID NO. 108). Though not run in parallel, comparison to the controls suggests that PSMA 662-671 (which approaches the Melan A peptide in affinity) has the superior binding activity of these two PSMA peptides.
- compositions as disclosed herein A multi-center clinical study is carried out using compositions as disclosed herein.
- the results of the study show the compositions to be useful and effective for debulking solid tumors and for generally inducing anti-angiogenic activity.
- HHD1 transgenic A*0201 mice (Pascolo, S., et al. J. Exp. Med. 185:2043-2051, 1997) were anesthetized and injected subcutaneously at the base of the tail, avoiding lateral tail veins, using 100 ⁇ l containing 100 nmol of PSMA 288-297 (SEQ ID NO. 21) and 20 ⁇ g of a HTL epitope peptide in PBS emulsified with 50 ⁇ l of IFA (incomplete Freund's adjuvant).
- PSMA 288-297 SEQ ID NO. 21
- IFA incomplete Freund's adjuvant
- mice Using spleens from 2 na ⁇ ve mice for each group of immunized mice, un-immunized mice were sacrificed and their carcasses placed in alcohol. Using sterile instruments, the top dermal layer of skin on the mouse's left side (lower mid-section) was cut through, exposing the peritoneum. The peritoneum was saturated with alcohol, and the spleen aseptically extracted. The spleens were placed in a petri dish with serum-free media. Splenocytes were isolated by using sterile plungers from 3 ml syringes to mash the spleens. Cells were collected in a 50 ml conical tubes in serum-free media, rinsing dish well.
- mice Three days after LPS stimulation of the blast cells and the same day as peptide loading, the primed mice were sacrificed (at 14 days post immunization) to remove spleens as above. 3 ⁇ 10 6 splenocytes were co-cultured with 1 ⁇ 10 6 LPS blasts/well in 24-well plates at 37° C., with 5% CO 2 in DMEM media supplemented with 10% FCS, 5 ⁇ 10 ⁇ 5 M ⁇ (beta)-mercaptoethanol, 100 ⁇ g/ml streptomycin and 100 IU/ml penicillin. Cultures were fed 5% (vol/vol) ConA supernatant on day 3 and can be transferred on day 7. An aliquot of the CTL are also tested in a standard chromium release assay to ensure activity.
- telomerase-transformed HDMEC in 10 ⁇ l of EGM-2-VM medium (Clonetics, San Diego, Calif.) are mixed with 0.5 ml of MATRIGEL (Becton Dickinson) on ice. The mixture is injected subcutaneously, through a 25 gauge needle, along the ventral midline of the thorax of SCID mice.
- MATRIGEL Becton Dickinson
- 1 ⁇ 10 7 T cells (target epitope-reactive or sham-immunized) in 0.2 ml are injected intravenously (alternatively they can be injected intraperitoneally).
- M1 tumor cells grown in complete RPMI plus 10% serum were harvested and washed with PBS by centrifugation.
- the cells were suspended in PBS at 5 ⁇ 10 6 cells/ml and 0.5 ml of the suspension (early passage) was injected subcutaneously into the abdomen.
- a nucleotide sequence encoding an HLA-A2-restricted fibronectin ED-B domain-derived housekeeping epitope, for example ED-B 29-38 is inserted into an appropriate vaccine vector (e.g. pVAX1 (Invitrogen Inc, Carlsbad, Calif.) or one of the vectors described in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2001, and incorporated by reference above.
- an appropriate vaccine vector e.g. pVAX1 (Invitrogen Inc, Carlsbad, Calif.) or one of the vectors described in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2001, and incorporated by reference above.
- HHD-A2 mice are injected intranodally in the inguinal lymph node with 0, 2, 10, 50, 100, and 200 ⁇ g of vector in PBS every other day over 8 days (4 injections) alternating sides for each injection (single dosage per mouse or group of mice). Injection series are started the day of tumor cell injection, at 2 weeks before, and at 4 and 10 weeks after.
- Effectiveness of the vaccine is expressed as the proportion of vaccinated animals that fail to develop a tumor in the same time frame, the relative size of tumors at the same time point, the delay in time before tumors appear in the vaccinated animals, and the dose and number of composition cycles needed to inhibit or prevent the establishment of tumor.
- mice are vaccinated on the day of tumor cell inoculation, 1 and 2 weeks before, and 3 or 4 days after injections of 1 ⁇ 10 6 cells. Effectiveness of vaccination is assessed at approximately 10 days after tumor cell inoculation.
- HHD-A2 mice were immunized with ED-B29-38 (SEQ ID NO. 103) in complete Freund's adjuvants and spleen cells were harvested and re-stimulated in vitro using standard methodology. The resulting CTL were able to specifically lyse peptide pulsed T2 cells, which are HLA-A2+ ( FIG. 9 ).
- Table 9 discloses epitopes and epitope clusters from PSMA and ED-B that can be useful in construction of compositions according to the present invention.
- SEQ ID NOS.* SEQ ID NO IDENTITY SEQUENCE 1 PSMA protein Accession number**: NP_004467 2 PSMA cDNA Accession number: NM_004476 3 PSMA 163-192 AFSPQGMPEGDLVYVNYARTEDFFKLERDM 4 PSMA 168-190 GMPEGDLVYVNYARTEDFFKLER 5 PSMA 169-177 MPEGDLVYV 6 PSMA 168-177 GMPEGDLVYV 7 PSMA 168-176 GMPEGDLVY 8 PSMA 167-176 QGMPEGDLVY 9 PSMA 169-176 MPEGDLVY 10 PSMA 171-179 EGDLVYVNY 11 PSMA 170-179 PEGDLVYVNY 12 PSMA 174-183 LVYVNYARTE 13 PSMA 177-185 VNYARTEDF 14 PSMA 176-185 YVNYARTEDF 15 PSMA 178-186 NYARTEDFF 16 PSMA 179-186 YARTEDFF 17 PSMA 181-189
- SEQ ID NOS. 5-17, 20-27, 29-36, 39-88, and 92-105 can be useful as epitopes in the various embodiments of the invention.
- Any of SEQ ID NOS. 3, 4, 18, 19, 28, 37, 38, 89 and 90 can be useful as sequences containing epitopes or epitope clusters, as described in various embodiments of the invention.
- **All accession numbers used here and throughout can be accessed through the NCBI databases, for example, through the Entrez seek and retrieval system on the world wide web.
- PSMA LOCUS NM_004476 2653 bp mRNA PRI 01-NOV-2000 DEFINITION Homo sapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1), mRNA. ACCESSION NM_004476 VERSION NM_004476.1 GI: 4758397 KEYWORDS . SOURCE human.
- ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 2653) AUTHORS Israeli, R.
Abstract
Description
- The present application is a divisional of and claims priority under 35 U.S.C. § 120 to U.S. Non-Provisional Patent Application Ser. No. 10/094,699, filed Mar. 7, 2002, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 60/274,063, filed on Mar. 7, 2001, entitled “ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER,” both of which are hereby incorporated by reference in their entirety.
- The treatment of cancer has remained challenging despite the advances in biomedicine. In recent years two approaches have been described showing much promise: therapeutic vaccines and anti-angiogenesis.
- Therapeutic vaccines rely on the observation that cancerous tissues generally express certain antigens preferentially, collectively tumor-associated antigens (TuAA). TuAA include proteins normally expressed selectively by the tissue from which the cancer derives (differentiation antigens), proteins that are associated with a different stage of development (oncofetal and cancer-testis antigens), proteins that are created by aberrant chromosomal rearrangement, or proteins that are derived from oncogenic viruses. These TuAA, or fragments of them, are then used as immunogens in vaccines intended to stimulate cellular immunity, particularly cytotoxic T lymphocytes (CTL), capable of killing the tumor cells.
- The anti-angiogenesis approach takes advantage of the need of tumors to recruit a blood supply to support their continued growth. To accomplish this, tumors secrete angiogenic factors that promote the growth of new blood vessels. The anti-angiogenesis approach aims to disrupt a tumor's supply of nutrients to cause it to die, or at least limit its growth. Attempts at this approach have sought chemotherapeutic drugs used directly against a variety of anti-angiogenic factors and angiogenesis.
- The invention disclosed herein is directed to compositions designed to stimulate cellular immune responses targeting tumor-associated neovasculature (TuNV). In one embodiment of the invention the compositions stimulate a CTL response. Such compositions may include one or more epitopes of the target antigen. One aspect of this embodiment specifically includes a housekeeping epitope, another specifically includes an immune epitope or epitope cluster, and another aspect specifically combines housekeeping and immune epitopes.
- Embodiments of the invention relate to the use of prostate specific membrane antigen (PSMA) as the target antigen of the composition. Aspects of this embodiment include various epitopes derived from PSMA provided directly as polypeptide, or as a nucleic acid capable of conferring expression of the epitope. Other embodiments relate to the use of other TuNV-associated antigens.
- In other embodiments of the invention, compositions are directed against both the TuNV and against TuAA expressed by the cancerous cells, by combining immunogens derived from both sources into a single formulation or method or treatment.
- Preclinical evaluation of the compositions of this invention can be accomplished using adoptive transfer of immunized T cells into SCID mice bearing microvasculature formed from implanted human dermal microvascular endothelial cells (HDMEC). Preclinical evaluation can also be accomplished through the use of HLA-transgenic mice immunized with compositions comprised of epitopes conserved between mice and humans.
- Embodiments of the invention relate to methods of evaluating cell-mediated immunity. The methods can include the steps of implanting vascular cells into an immunodeficient mammal; establishing an immune response in the mammal; and assaying a characteristic to determine cell-mediated immunity in the mammal. The cell-mediated immunity can be directed against a neovasculature antigen, for example. The neovasculature antigen can be preferentially expressed by tumor-associated neovasculature, for example, and in preferred embodiments can be prostate specific membrane antigen (PSMA), vascular endothelial growth factor receptor 2 (VEGFR2), and the like. The establishing step can be achieved, for example, by adoptive transfer of T-cells to the mammal, by contacting the mammal with an antigen, and the like. The cell-mediated immunity can be mediated by cytotoxic T lymphocytes. The vascular cells can be vascular endothelial cells, such as, for example, human dermal microvascular endothelial cells (HDMEC), telomerase-transformed endothelial cells, and the like. The immunodeficient mammal can be a mouse, such as for example a SCID mouse. The characterizing step can include assessing a parameter, such as for example, vessel formation, vessel destruction, vessel density, proportion of vessels carrying blood of the host mammal, and the like.
- The methods can further include the step of implanting tumor cells or tumor tissue in the mouse. The characterizing step can include assessing a parameter, such as, for example, tumor presence, tumor growth, tumor size, rapidity of tumor appearance, dose of vaccine required to inhibit or prevent tumor establishment, tumor vascularization, a proportion of necrotic tissue within the tumor, and the like.
- The methods can further include the steps of providing a first population of mammals and a second populations of mammals; establishing cell-mediated immunity in the first population; differentially establishing cell-mediated immunity in the second population; and comparing a result obtained from the first population of mammals to a result obtained from the second population of mammals. The cell-mediated immunity of the first population can include, for example, naive immunity, immunity to an irrelevant epitope, and the like.
- Other embodiments relate to methods of evaluating cell-mediated immunity, including immunity directed against a neovasculature antigen. The methods can include the steps of implanting or injecting MHC-transgenic tumor cells into an MHC-transgenic mammal; establishing an immune response in the mammal; and assaying a characteristic to determine cell-mediated immunity in the mammal. The MHC-transgenic mammal can be an HLA-transgenic mammal, such as, for example an HLA-A2 transgenic mammal. In preferred embodiments the mammal can be a mouse. The cell-mediated immunity can be established by vaccination, which in preferred embodiments can take place prior to, concurrent with, or subsequent to transfer of the tumor cells, for example. In preferred embodiments the cell-mediated immunity can be mediated by cytotoxic T lymphocytes. The neovasculature antigen can be preferentially expressed by tumor-associated neovasculature and can also be a tumor-associated antigen. Preferably, the antigen can be the ED-B domain of fibronectin. The characterizing step can include, for example, assessing a parameter, including tumor presence, tumor growth, tumor size, rapidity of tumor appearance, dose of vaccine required to inhibit or prevent tumor establishment, tumor vascularization, a proportion of necrotic tissue within the tumor, and the like. The methods can further include the steps of providing a first population of mammals and a second populations of mammals; establishing cell-mediated immunity in the first population; differentially establishing cell-mediated immunity in the second population; and comparing a result obtained from the first population of mammals to a result obtained from the second population of mammals. The cell-mediated immunity of the first population can include naive immunity, immunity to an irrelevant epitope, and the like.
- Still further embodiments relate to methods of treating neoplastic disease, including the step of immunizing a mammal to induce a cellular immune response directed against an antigen differentially expressed by tumor-associated neovasculature. The differentially expressed antigen can be a protein, such as, for example prostate specific membrane antigen, vascular endothelial growth factor receptor 2 (VEGFR2), and the like. In other preferred embodiments, the antigen can be the ED-B domain of fibronectin. The immunization can be carried out, for example, with at least one peptide derived from the sequence of the protein, with a nucleic acid capable of conferring expression of the protein or peptides, and the like. The at least one peptide can include a housekeeping epitope, for example, and in preferred embodiments can be co-C-terminal with the housekeeping epitope. The methods can further include at least one additional peptide, wherein the at least one additional peptide includes an immune epitope. The methods can include an additional step wherein the mammal is treated with an anti-tumor therapy active directly against cancerous cells. The anti-tumor therapy can be immunization against a tumor-associated antigen. Preferably, the cellular immune response can include a CTL response.
- Other embodiments relate to immunogenic compositions. The immunogenic compositions can include at least one immunogen corresponding to an antigen expressed by tumor-associated neovasculature, wherein the composition can induce a cellular immune response. The immunogen can be one that is not associated with a cell conspecific with the recipient. The antigen can be a protein, such as, for example prostate specific membrane antigen, vascular endothelial growth factor receptor 2 (VEGFR2), and the like. In other preferred embodiments the antigen can be the ED-B domain of fibronectin. The immunogen can include at least one peptide. The compositions can include a nucleic acid capable of conferring expression of the antigen, and wherein the antigen is a protein or a peptide. The compositions can include at least one peptide that includes a housekeeping epitope, and in preferred embodiments the at least one peptide can be co-C-terminal with the housekeeping epitope. Also, the compositions can additionally include at least one peptide that includes an immune epitope. The compositions can include at least one immunogen corresponding to a tumor-associated antigen. In preferred embodiments the cellular immune response can include a CTL response.
- Embodiments relate to methods of anti-tumor vaccine design. The methods can include the steps of identifying an antigen differentially expressed by tumor-associated neovasculature; and incorporating a component of the antigen into a vaccine. The component can include, for example, a polypeptide fragment of the antigen, a nucleic acid encoding the antigen or a fragment of the antigen, and the like.
- Further embodiments relate to methods of making a research model. The methods can include implanting a vascular cell and a tumor cell into an immunodeficient mammal. The tumor cell and the vascular cell can be implanted adjacent to one another. The vascular cell can be a vascular endothelial cell, such as for example HDMEC. In preferred embodiments the vascular endothelial cell can be telomerase-transformed. The immunodeficient mammal can be a mouse, such as, for example, a SCID mouse.
- Other embodiments relate to research models. The research models can include an immunodeficient mammal. The mammal can include an implanted vascular cell and an implanted tumor cell. The vascular cell and the tumor cell can be implanted adjacent to one another.
-
FIGS. 1A , B, and C show results of N-terminal pool sequencing of a T=60 min. time point aliquot of the PSMA163-192 proteasomal digest. -
FIG. 2 shows binding curves for HLA-A2:PSMA168-177 and HLA-A2:PSMA288-297 with controls. -
FIG. 3 shows results of N-terminal pool sequencing of a T=60 min. time point aliquot of the PSMA281-310 proteasomal digest. -
FIG. 4 shows binding curves for HLA-A2:PSMA461-469, HLA-A2:PSMA460-469, and HLA-A2:PSMA663-671, with controls. -
FIG. 5 shows the results of a γ (gamma)-IFN-based ELISPOT assay detecting PSMA463-471-reactive HLA-A1+ CD8+ T cells. -
FIG. 6 shows blocking of reactivity of the T cells used inFIG. 10 by anti-HLA-A1 mAb, demonstrating HLA-A1-restricted recognition. -
FIG. 7 shows a binding curve for HLA-A2:PSMA663-671, with controls. -
FIG. 8 shows a binding curve for HLA-A2:PSMA662-671, with controls. -
FIG. 9 shows epitope specific lysis by CTL from HHD-A2 mice immunized with ED-B 29-38 peptide. - Embodiments of the invention disclosed herein provide compositions, methods of composition or vaccine design, and methods of treatment related to the generation of a cellular immune response, preferably, a T cell response and, more preferably, a CTL response, directed against the neovasculature of tumors. Such methods and compositions are particularly useful in the treatment and prevention of cancer. Other embodiments relate to composition evaluation models.
- Embodiments of the invention relate to immunogenic compositions, including vaccines, for the generation of a cellular immune response, particularly a T cell response and specifically a CTL response, directed against tumor neovasculature (TuNV). “Tumor neovasculature” is broadly meant to include any vasculature found in or around tumor masses, vasculature which supports or is necessary for tumor growth, and the like. It should be noted, and one of skill in the art will appreciate, that although the discussion herein refers generally to the tumors and tumor neovasculature, the embodiments of the present invention also can be applied to other conditions or disease states associated with inappropriate angiogenesis.
- Until now the design of anti-tumor vaccines has concentrated on antigens expressed by the malignant cells themselves. However, larger tumors are complex structures and not simply a homogeneous mass of cells. All cells, particularly rapidly growing cells, need a supply of nutrients (oxygen, glucose, amino acids, etc.), as well as a means of removal of metabolic wastes, in order to remain metabolically active and intact. This is normally accomplished by the flow of blood and lymph through the various organs of the body. At a cellular level, the tissues of the body are permeated by a fine network of capillaries—tiny vessels through which nutrients and waste products can be exchanged with the surrounding cells by diffusion. Diffusion is effective over relatively short distances. The capillary beds are so extensive that generally cells are at most located only a few cells away from a capillary. If a tumor merely grew by propagation of its malignant cells, soon those cells in the interior of the mass would be unable to sustain themselves. In fact, the interiors of unvascularized tumors often contain necrotic tissue. Thus, in order to grow unchecked, tumors secrete factors that promote the in-growth of new blood vessels, namely TuNV. Since the TuNV expresses antigens differentiating it from other tissues, cancer can be treated with therapeutic compositions directed against the TuNV, instead of directly targeting the cancerous cells themselves. Suitable TuNV antigens can include those that are expressed generally in neovasculature or preferentially by TuNV, for example.
- In some embodiments of the invention the compositions can include, for example, an epitopic peptide or peptides. Immune epitopes may be provided embedded in epitope clusters and protein fragments. Housekeeping epitopes can be provided with the proper C-terminus. In other embodiments of the invention the compositions can include nucleic acids capable of conferring expression of these epitopes on pAPC, for example.
- In preferred embodiments, the compositions can be administered directly to the lymphatic system of a mammal being treated. This can be applied to both polypeptide and nucleic acid based compositions. Administration methods of this type, and related technologies, are disclosed in U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, and a Continuation-in-Part thereof, filed on Feb. 2, 2001; U.S. patent application Ser. No. 09/776,232, both entitled “A METHOD OF INDUCING A CTL RESPONSE,” which are incorporated by reference in their entirety.
- In a preferred embodiment, destruction of the blood vessels in a tumor by action of a composition of the invention can eliminate all of the cells in a tumor. However, small tumors, including micrometastases, are typically unvasculaturized. Additionally, unvascularized tumors that instead apparently rely on blood flow through channels penetrating the tumor mass have been reported (Maniotis, A. J., et al. Am. J. Pathol. 155: 739-752, 1999). Thus in other embodiments, the compositions are generally effective as tumor control agents that may not eradicate all cancer cells. Accordingly, the invention provides tools for eliminating tumors, controlling tumor growth, reducing tumor burden, improving overall clinical status, and the like. In some embodiments, it can be desirable to combine these compositions with other treatments that target the cancerous cells directly. Additionally there is evidence that the vasculature in tumors can be mosaic in nature consisting of both endothelial and cancer cells (Chang, Y. S., et al. Proc. Natl. Acad. Sci. USA 97:14608-14613, 2000). Thus, in some embodiments of the invention a course of composition treatment can be followed by administration of a bio- or chemotherapeutic agent. In a particularly preferred embodiment, treatment can include administration of a TuAA directed composition concurrent or subsequent to administration of the anti-TuNV composition.
- As mentioned above, suitable TuNV antigens for the compositions can include those that are expressed generally in neovasculature or preferentially by TuNV, for example. A variety of techniques for discovery of TuAA are known in the art. Examples of these techniques include, without limitation, differential hybridization and subtractive hybridization, including use of microarrays; expression cloning; SAGE (serial analysis of gene expression); SEREX (serological identification of antigens by recombinant expression cloning); in situ RT-PCT; immunohistochemistry (as was the case for PSMA); EST analysis; variously using bulk, sectioned, and/or microdissected tissue; and the like. Utilization of these and other methods provides one of skill in the art the techniques necessary to identify genes and gene products contained within a target cell that may be used as antigens of immunogenic compositions. The techniques are applicable to TuAA discovery regardless of whether the target cell is a cancer cell or an endothelial cell. Any identified antigen can be scrutinized for epitopes, which can be used in embodiments of the invention.
- The endothelial cells making up the lining of the vasculature can express housekeeping proteasomes. Thus, compositions targeting endothelial cells can be comprised of peptides, or nucleic acids conferring expression of the peptides, corresponding to the digestion products of the housekeeping proteasome (i.e. housekeeping epitopes). IFN-γ (gamma), secreted by activated cells of the immune system, can induce expression of the immunoproteasome in the target cells. Generally, the immunoproteasome is constitutive in professional antigen presenting cells (pAPC). Thus, it can be helpful to include immune epitopes or epitope clusters in CTL-inducing compositions to ensure that there are CTL able to recognize the target cell regardless of the state that the target cell is in. This can be particularly true with endothelial cells, which readily assume antigen presentation functions. These concepts are more fully explained in U.S. patent application Ser. No. 09/560,465, filed on Apr. 28, 2000; U.S. patent application Ser. No. 10/005,905, filed on Nov. 7, 2001; and a continuation thereof, U.S. application Ser. No. 10/026,066 filed on Dec. 7, 2001, attorney docket number CTLIMM.21CP1C, each of which is entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” and each of which is hereby incorporated by reference in its entirety.
- As discussed above, the immunogenic compositions, including in preferred embodiments, vaccines, can include TuNV antigens and epitopes, for example. The epitopes can include one or more housekeeping epitopes and/or one or more immune epitopes. Specific epitopes useful in compositions can be identified using the methods disclosed in U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed on Apr. 28, 2000. For example, peptide sequences that are known or predicted to bind to some MHC restriction element can be compared to fragments produced by proteasomal digestion in order to identify those that are co-C-terminal.
- Examples of useful epitopes for the embodiments of the invention, including epitopes of ED-B and PSMA, are disclosed in a U.S. Provisional Patent Application No. 60/363,210, entitled “EPITOPE SEQUENCES,” attorney docket number CTLIMM.027PR; filed on Mar. 7, 2002, and two U.S. Provisional Patent Applications, each entitled “EPITOPE SEQUENCES;” Application Ser. No. 60/282,211, filed on Apr. 6, 2001 and 60/337,017, filed on Nov. 7, 2001. Each of these applications is incorporated herein by reference in its entirety.
- PSMA is one example of a TuAA that can be targeted in some embodiments. PSMA is expressed in the neovasculature of most tumor types, but not by the vascular endothelium of normal tissues (Chang, S. M. et al., Cancer Res. 59(13):3192-8,1999; Clin Cancer Res. 10:2674-81, 1999). PSMA is a membrane antigen, and as such, it may be possible to attack PSMA-expressing TuNV with monoclonal antibody (mAb). However, the effectiveness of mAb in the treatment of cancer has proved to be more difficult than initially anticipated. Moreover, as other antigens are discovered to be associated with the TuNV, it is likely that many of them will prove not to be expressed at the vasculature surface, making them inaccessible to mAb attack.
- T cells, particularly CTL, on the other hand, survey the expression of internal components of the cell through the process of major histocompatability complex (MHC)-restricted antigen presentation. The parameters for determining the effectiveness of T cell-activating vaccines and compositions against self-antigens are subtle. Some of the critical features and parameters relating to appropriate epitope selection are disclosed in U.S. patent application Ser. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed on Apr. 28, 2001; U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed on Apr. 28, 2001; and U.S. patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,” filed on Apr. 28, 2001. Features of DNA vaccine design promoting epitope synchronization are disclosed in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2001 and U.S. Provisional Application No. 60/336,968 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN,” filed on Nov. 7, 2001. Particularly effective means of vaccine delivery are described in U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, and a Continuation-in-Part thereof, U.S. patent application Ser. No. 09/776,232, filed on Feb. 2, 2001, both entitled “A METHOD OF INDUCING A CTL RESPONSE.” Each of the above-mentioned references is incorporated herein by reference in its entirety.
- Another example of a TuNV antigen that can be used in embodiments is fibronectin, preferably the ED-B domain. Fibronectin is subject to developmentally regulated alternative splicing, with the ED-B domain being encoded by a single exon that is used primarily in oncofetal tissues. Matsuura, H. and S. Hakomori Proc. Natl. Acad. Sci. USA 82:6517-6521, 1985; Carnemolla, B. et al. J. Cell Biol. 108:1139-1148, 1989; Loridon-Rosa, B. et al. Cancer Res.50:1608-1612, 1990; Nicolo, G. et al. Cell Differ. Dev. 32:401-408, 1990; Borsi, L. et al. Exp. Cell Res. 199:98-105, 1992; Oyama, F. et al. Cancer Res. 53:2005-2011, 1993; Mandel, U. et al. APMIS 102:695-702, 1994; Farnoud, M. R. et al. Int. J. Cancer 61:27-34, 1995; Pujuguet, P. et al. Am. J. Pathol. 148:579-592, 1996; Gabler, U. et al. Heart 75:358-362, 1996; Chevalier, X. Br. J. Rheumatol. 35:407-415, 1996; Midulla, M. Cancer Res. 60:164-169, 2000.
- The ED-B domain is also expressed in fibronectin of the neovasculature Kaczmarek, J. et al. Int. J. Cancer 59:11-16, 1994; Castellani, P. et al. Int. J. Cancer 59:612-618, 1994; Neri, D. et al. Nat. Biotech. 15:1271-1275, 1997; Karelina, T. V. and A. Z. Eisen Cancer Detect. Prev. 22:438-444, 1998; Tarli, L. et al. Blood 94:192-198, 1999; Castellani, P. et al. Acta Neurochir. (Wien) 142:277-282, 2000. As an oncofetal domain, the ED-B domain is commonly found in the fibronectin expressed by neoplastic cells, in addition to being expressed by the TuNV. Therefore, CTL-inducing compositions targeting the ED-B domain can exhibit two mechanisms of action: direct lysis of tumor cells, and disruption of tumor blood supply through destruction of the TuNV.
- It should be noted that expression of the fibronectin ED-B domain has been reported in both tumor-associated and normal neovasculature (Castellani, P. et al. Int. J. Cancer 59:612-618, 1994). Thus, compositions based on it, or similarly expressed antigens, can be effective against other conditions associated with inappropriate angiogenesis. Further, as CTL activity can decay rapidly after withdrawal of the composition, interference with normal angiogenesis can be minimal.
- Other targets for the immunogenic compositions include growth factor receptors, including those associated with vascular cells. One such example is the vascular endothelial growth factor receptor 2 (VEGFR2). U.S. Pat. No. 6,342,221 includes discussion of VEGF and VEGFR2, and is hereby incorporated by reference in its entirety. One of skill in the art will appreciate that any other antigen or protein associated with vascular cells can be a target for the immunogenic compositions, including those that are presently known and those yet to be identified.
- Compositions designed based upon the preceding considerations are effective against the various targets. However, additional evaluation can be easily performed at any time, but preferably in a pre-clinical setting. For example, such evaluation can be used in order to further aid in composition design. Other embodiments of the invention relate to methods of evaluating the immunogenic compositions. The compositions of the present invention can be easily evaluated by one of skill in the art using animal models for composition evaluation. For example, following the routine procedures below, one of skill in the art can evaluate TuNV compositions quickly and efficiently. Thus, using the models or guidance described herein, one of skill in the art can evaluate any TuNV composition for any TuNV antigen with little or no experimentation. Further embodiments relate to methods of making the animal research models. Other embodiments relate to the research model animals. These embodiments are set forth more fully below.
- Some embodiments relate to a model system for studying the mechanisms of human microvessel formation. For example, in some embodiments, the model system can be used for preclinical evaluation of compositions. The model involves the subcutaneous implantation of telomerase-transformed human dermal microvascular endothelial cells (HDMEC) mixed with MATRIGEL (Becton Dickinson) into SCID mice. Subcutaneous implantation of telomerase-transformed HCMEC is described in Yang, J. et al. Nature Biotech 19:219-224, 2001, which is hereby incorporated by reference in its entirety. T cells activated by the compositions of this invention can be adoptively transferred, for example, into such implanted mice, and the ability of the T cells to destroy, or prevent the formation of, such human microvessels can be assessed. In other embodiments, the mouse can be directly vaccinated and evaluated. Also, in still further embodiments, the model system can be further adapted for testing compositions effective in non-human species by substituting DMEC from other species and species-matched telomerase, and by using analogous reagents to those described below for the human system.
- The MHC-restriction elements presenting the epitopes of the composition being tested, preferably, are shared by the HDMEC line implanted into the mice. The T cells can be derived from in vitro immunization of human T cells, or by immunization of HLA-transgenic mice (procedures for which are well known in the art and examples of which are provided in the above incorporated patent applications). Use of T cells generated in HLA-transgeneic mice allows matching of genetic backgrounds between the adoptively transferred T cells and the host, thereby reducing the possibility of allogeneic or xenogeneic reactions that might complicate interpretation of the results. However, depending on the mouse strains available, this might require cross-breeding to get the HLA-transgene and SCID phenotype on the same genetic background. In the alternative, the donor T cells (human or murine) can be subjected to one or more rounds of in vitro stimulation to enrich for the desired population or establish a clone, and thereby similarly avoid undesired reactivities.
- Techniques for in vitro immunization are know in the art, for example, Stauss et al., Proc. Natl. Acad. Sci. USA 89:7871-7875, 1992; Salgaller et al. Cancer Res. 55:4972-4979, 1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et al., J. Immunother. 22:279-287, 1999. Once generated, whether in vivo or in vitro, sufficient numbers of such T cells can be obtained by expansion in vitro through stimulation with the compositions of this invention and/or cytokines (see for example Kurokawa, T. et al., Int. J. Cancer 91:749-746, 2001) or other mitogens. These T cells can constitute a clone or a polyclonal population recognizing one or more epitopes. In preferred embodiments, on the order of 105 to 108 cells are transferred for adoptive transfer experiments in mice. (See for example Drobyski, W. R. et al. Blood 97:2506-2513, 2001; Seeley B. M. et al. Otolaryngol. Head Neck Surg. 124:436-441, 2001; Kanwar, J. R. et al. Cancer Res. 61:1948-1956, 2001). Clones and otherwise more enriched populations generally require the transfer of fewer cells.
- Transfer of the T cells can take place shortly before, concurrent with, or subsequent to implantation or establishment of the HDMEC. Parameters that can be assessed to evaluate effectiveness of the compositions include vessel formation, changes in vessel density, and ability to carry mouse blood (as described in Yang et al.), and the like. Assessment can be carried out as early as one week, and at least as long as 6 weeks, after implantation of telomerase-transformed HDMEC, preferably after 2 weeks; and from a day to more than 6 weeks after T cell transfer, preferably after 1 to 3 weeks. Generally, assessment can include comparison of mice receiving T cells reactive with the target antigen with mice receiving naive (including sham-immunized), or irrelevant epitope-reactive T cells.
- Relevant antigens can be expressed generally in neovasculature or preferentially by TuNV. Expression can be confirmed by a variety of techniques known in the art, including immunohistochemistry and RT-PCR. For example, tumor cells can be implanted along with the HDMEC. This can result in inducing expression of antigens preferentially expressed by TuNV. In one example, this can be accomplished by implanting a block of tumor tissue adjacent to the HDMEC-containing MATRIGEL implant, injecting tumor cells at the site of the implant, implanting tumor cell-containing MATRIGEL adjacent to the HDMEC-containing MATRIGEL implant, incorporating both tumor cells and HDMEC into the same MATRIGEL implant or by any other suitable method. As discussed above, in some embodiments, tumor cells can be implanted along with vascular cells. The animals so made, can be used as research models. Additional variations will be apparent to one of skill in the art.
- For antigens that are conserved, in sequence and/or expression profile, between human and the model species, HLA-transgenic strains allow another approach, namely vaccination of the model animal to combat a syngeneic tumor. The ED-B domain of fibronectin provides such an opportunity, as it is a marker of angiogenesis and has identical amino acid sequence in both humans and mice (Nilsson, F. et al. Cancer Res. 61:711-716, 2001). Moreover, spontaneous tumor tissue from a strain of HLA-A2 transgenic mice has been isolated and propagated. Epitope discovery and selection, and composition design and delivery for CTL inducing compositions are discussed above.
- The tumor cell line, M1, is derived from a spontaneous salivary glandular cystadenocarcinoma. The M1 tumor cell line and methods of using the same is disclosed in U.S. Provisional Application No. 60/363,131, filed on Mar. 7, 2001, entitled “AN HLA-TRANSGENIC MURINE TUMOR CELL LINE,” which is hereby incorporated by reference in its entirety. The tumor cell line, can arise in individuals of the HHD-A2 transgenic mouse strain of S. Pascolo et al. (J. Exp. Med. 185:2043-2051, 1997). These mice express a single monochain class I molecule comprising human β (beta)2-microglobulin, and α1 (alpha-1), and α2 (alpha-2) domains of HLA-A2.1 with the balance of the molecule derived from the murine class I molecule H2Db. Blocks of tumor can be transplanted into new individuals where the tumor will re-grow, commonly within 1-3 weeks, with 3 mm blocks growing to 3 cm. Alternatively, tumor tissue can be disaggregated and the tumor cells grown in vitro. Upon harvest, the tumor cells can be injected subcutaneously into the neck or abdomen (2.5×106 cells for 1-3 successive days), to result in a visible tumor in approximately 5-12 weeks for early passage cells. After the cells have become better adapted to growth in vitro, single injections of 1×106 to 1×107 cells lead to visible tumor in ten days. Generally, the initial tumor consistently occurs in the vicinity of the salivary glands, but secondary tumors can also occur in a variety of locations, including kidney, lung, liver, and abdominal muscle.
- To evaluate the efficacy of a composition, it can be administered before, concurrent with, or subsequent to establishment of the tumor, depending on the expected mechanism of the composition. For therapeutic compositions intended to be used with some sort of debulking technique (e.g. surgery), concurrent administration can be appropriate. The better established the tumor is before treatment begins, the more stringent the test.
- Both animal evaluation models have been described for the testing of human compositions. However, application to veterinary compositions is analogous, requiring only the substitution of species-matched endothelial cells, MHC, TuAA, etc.
- All patents, patent applications, and publications referred to herein are hereby incorporated by reference in their entirety.
- The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.
- A preclinical study was carried out using the already identified antigens PSMA and ED-B disclosed herein. The results of the study revealed excellent candidate epitopes. See table 9 below.
- A peptide, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA163-192, (SEQ ID NO. 3), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA168-190 (SEQ ID NO. 4) was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer. After side chain deprotection and cleavage from the resin, peptide first dissolved in formic acid and then diluted into 30% Acetic acid, was run on a reverse-phase preparative HPLC C4 column at following conditions: linear AB gradient ( 5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fraction at time 16.642 min containing the expected peptide, as judged by mass spectrometry, was pooled and lyophilized. The peptide was then subjected to proteasome digestion and mass spectrum analysis essentially as described above. Prominent peaks from the mass spectra are summarized in Table 1.
-
TABLE 1 PSMA163-192 Mass Peak Identification. CALCU- SEQ LATED ID MASS NO. PEPTIDE SEQUENCE (MH+) 110 163-177 AFSPQGMPEGDLVYV 1610.0 111 178-189 NYARTEDFFKLE 1533.68 112 170-189 PEGDLVYVNYARTEDFFKLE 2406.66 113 178-191 NYARTEDFFKLERD 1804.95 114 170-191 PEGDLVYVNYARTEDFFKLERD 2677.93 115 178-192 NYARTEDFFKLERDM 1936.17 116 163-176 AFSPQGMPEGDLVY 1511.70 117 177-192 VNYARTEDFFKLERDM 2035.30 118 163-179 AFSPQGMPEGDLYYVNY 1888.12 119 180-192 ARTEDFFKLERDM 1658.89 120 163-183 AFSPQGMPEGDLVYVNYARTE 2345.61 121 184-192 DFFKLERDM 1201.40 122 176-192 YVNYARTEDFFKLERDM 2198.48 123 167-185 QGMPEGDLVYVNYARTEDF 2205.41 124 178-186 NYARTEDFF 1163.22 Boldface sequences correspond to peptides predicted to bind to MHC, see Table 2. - One aliquot at one hour of the proteasomal digestion was subjected to N-terminal amino acid sequence analysis by an ABI 473A Protein Sequencer (Applied Biosystems, Foster City, Calif.). Determination of the sites and efficiencies of cleavage was based on consideration of the sequence cycle, the repetitive yield of the protein sequencer, and the relative yields of amino acids unique in the analyzed sequence. That is if the unique (in the analyzed sequence) residue X appears only in the nth cycle a cleavage site exists n-i residues before it in the N-terminal direction. In addition to helping resolve any ambiguity in the assignment of mass to sequences, these data also provide a more reliable indication of the relative yield of the various fragments than does mass spectrometry.
- For PSMA163-192 (SEQ ID NO. 3) this pool sequencing supports a single major cleavage site after V177 and several minor cleavage sites, particularly one after Y179. Reviewing the results presented in
FIGS. 1A-C reveals the following: -
- S at the 3rd cycle indicating presence of the N-terminus of the substrate.
- Q at the 5th cycle indicating presence of the N-terminus of the substrate.
- N at the 1st cycle indicating cleavage after V177.
- N at the 3rd cycle indicating cleavage after V175. Note the fragment 176-192 in Table 1.
- T at the 5th cycle indicating cleavage after V177.
- T at the 1st-3rd cycles, indicating increasingly common cleavages after R181, A180 and Y179. Only the last of these correspond to peaks detected by mass spectrometry; 163-179 and 180-192, see Table 1. The absence of the others can indicate that they are on fragments smaller than were examined in the mass spectrum.
- K at the 4th, 8th, and 10th cycles indicating cleavages after E183, Y179, and V177, respectively, all of which correspond to fragments observed by mass spectroscopy. See Table 1.
- A at the 1st and 3rd cycles indicating presence of the N-terminus of the substrate and cleavage after V177, respectively.
- P at the 4th and 8th cycles indicating presence of the N-terminus of the substrate.
- G at the 6th and 10th cycles indicating presence of the N-terminus of the substrate.
- M at the 7th cycle indicating presence of the N-terminus of the substrate and/or cleavage after F185.
- M at the 15th cycle indicating cleavage after V177.
- The 1st cycle can indicate cleavage after D191, see Table 1.
- R at the 4th and 13th cycle indicating cleavage after V177.
- R at the 2nd and 11th cycle indicating cleavage after Y179.
- V at the 2nd, 6th, and 13th cycle indicating cleavage after V175, M169 and presence of the N-terminus of the substrate, respectively. Note fragments beginning at 176 and 170 in Table 1.
- Y at the 1st, 2nd, and 14th cycles indicating cleavage after V175, V177, and presence of the N-terminus of the substrate, respectively.
- L at the 11th and 12th cycles indicating cleavage after V177, and presence of the N-terminus of the substrate, respectively, is the interpretation most consistent with the other data. Comparing to the mass spectrometry results we see that L at the 2nd, 5th, and 9th cycles is consistent with cleavage after F186, E183 or M169, and Y179, respectively. See Table 1.
- Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further analysis. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include a predicted HLA-A1 binding sequence, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 2.
-
TABLE 2 Predicted HLA binding by proteasomally generated fragments I. SEQ ID NO II. PEPTIDE HLA SYFPEITHI NIH 5 & (6) (G)MPEGDLVYV A*0201 17(27) (2605) B*0702 20 <5 B*5101 22 314 7 & (8) (Q)GMPEGDLVY A1 24(26) <5 A3 16(18) 36 B*2705 17 25 9 MPEGDLVY B*5101 15 NP† 10 & (11) (P)EGDLVYVNY A1 27(15) 12 A26 23(17) NP 12 LVYVNYARTE A3 21 <5 13 & (14) (Y)VNYARTEDF A26 (20) NP B*08 15 <5 B*2705 12 50 15 NYARTEDFF A24 NP† 100 Cw*0401 NP 120 16 YARTEDFF B*08 16 <5 17 RTEDFFKLE A1 21 <5 A26 15 NP †No prediction - Binding of the candidate epitope PSMA168-177, GMPEGDLVYV, (SEQ ID NO. 6) to HLA-A2.1 was assayed using a modification of the method of Stauss et al., (Proc Natl Acad Sci USA 89(17):7871-5 (1992)). Specifically, T2 cells, which express empty or unstable MHC molecules on their surface, were washed twice with Iscove's modified Dulbecco's medium (IMDM) and cultured overnight in serum-free AIM-V medium (Life Technologies, Inc., Rockville, Md.) supplemented with human β2-microglobulin at 3μg/ml (Sigma, St. Louis, Mo.) and added peptide, at 800, 400, 200, 100, 50, 25, 12.5, and 6.25 μg/ml. in a 96-well flat-bottom plate at 3×105 cells/200 μl/well. Peptide was mixed with the cells by repipeting before distributing to the plate (alternatively peptide can be added to individual wells), and the plate was rocked gently for 2 minutes. Incubation was in a 5% CO2 incubator at 37° C. The next day the unbound peptide was removed by washing twice with serum free RPMI medium and a saturating amount of anti-class I HLA monoclonal antibody, fluorescein isothiocyanate (FITC)-conjugated anti-HLA A2, A28 (One Lambda, Canoga Park, Calif.) was added. After incubation for 30 minutes at 4° C., cells were washed 3 times with PBS supplemented with 0.5% BSA, 0.05%(w/v) sodium azide, pH 7.4-7.6 (staining buffer). (Alternatively W6/32 (Sigma) can be used as the anti-class I HLA monoclonal antibody the cells washed with staining buffer and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat F(ab′) antimouse-IgG (Sigma) for 30 min at 4° C. and washed 3 times as before.) The cells were resuspended in 0.5 ml staining buffer. The analysis of surface HLA-A2.1 molecules stabilized by peptide binding was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.). If flow cytometry is not to be performed immediately the cells can be fixed by adding a quarter volume of 2% paraformaldehyde and storing in the dark at 4° C.
- As seen in
FIG. 2 , this epitope exhibits significant binding at even lower concentrations than the positive control peptides. The Melan-A peptide used as a control in this assay (and throughout this disclosure), ELAGIGILTV (SEQ ID NO: 106), is actually a variant of the natural sequence (EAAGIGILTV; SEQ ID NO: 107)) and exhibits a high affinity in this assay. The known A2.1 binder FLPSDYFPSV (HBV18-27; SEQ ID NO: 107) was also used as a positive control. An HLA-B44 binding peptide, AEMGKYSFY (SEQ ID NO: 109), was used as a negative control. The fluorescence obtained from the negative control was similar to the signal obtained when no peptide was used in the assay. Positive and negative control peptides were chosen from Table 18.3.1 in Current Protocols in Immunology p. 18.3.2, John Wiley and Sons, New York, 1998. - Another peptide, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG, PSMA281-310, (SEQ ID NO. 18), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA283-307 (SEQ ID NO. 19), was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer. After side chain deprotection and cleavage from the resin, peptide in ddH2O was run on a reverse-phase preparative HPLC C18 column at following conditions: linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fraction at time 17.061 min containing the expected peptide as judged by mass spectrometry, was pooled and lyophilized. The peptide was then subjected to proteasome digestion and mass spectrum analysis essentially as described above. Prominent peaks from the mass spectra are summarized in Table 3.
-
TABLE 3 PSMA281-310 Mass Peak Identification. CAL- SEQ CULATED ID MASS NO. PEPTIDE SEQUENCE (MH+) 125 281-297 RGIAEAVGLPSIPVHPI* 1727.07 126 286-297 AVGLPSIPVHPI** 1200.46 127 287-297 VGLPSIPVHPI 1129.38 128 288-297 GLPSIPVHPI † 1030.25 129 298-310 GYYDAQKLLEKMG‡ 1516.5 130 298-305 GYYDAQKL§ 958.05 131 281-305 RGIAEAVGLPSIPVHPIGYYDAQKL 2666.12 132 281-307 RGIAEAVGLPSIPVHPIGYYDAQKLLE 2908.39 133 286-307 AVGLPSIPVHPIGYYDAQKLLE¶ 2381.78 134 287-307 VGLPSIPVHPIGYYDAQKLLE 2310.70 135 288-307 GLPSIPVHPIGYYDAQKLLE# 2211.57 136 281-299 RGIAEAVGLPSIPVHPIGY 1947 137 286-299 AVGLPSIPVHPIGY 1420.69 138 287-299 VGLPSIPVHPIGY 1349.61 139 288-299 GLPSIPVHPIGY 1250.48 140 287-310 VGLPSIPVHPIGYYDAQKLLEKMG 2627.14 141 288-310 GLPSIPVHPIGYYDAQKLLEKMG 2528.01 Boldface sequences correspond to peptides predicted to bind to MHC, see Table 4. *By mass alone this peak could also have been 296-310 or 288-303. **By mass alone this peak could also have been 298-307. Combination of HPLC and mass spectrometry show that at some later time points this peak is a mixture of both species. †By mass alone this peak could also have been 289-298. ≠By mass alone this peak could also have been 281-295 or 294-306. §By mass alone this peak could also have been 297-303. ¶By mass alone this peak could also have been 285-306. #By mass alone this peak could also have been 288-303. None of these alternate assignments are supported N-terminal pool sequence analysis. - One aliquot at one hour of the proteasomal digestion (see Example 3
part 3 above) was subjected to N-terminal amino acid sequence analysis by an ABI 473A Protein Sequencer (Applied Biosystems, Foster City, Calif.). Determination of the sites and efficiencies of cleavage was based on consideration of the sequence cycle, the repetitive yield of the protein sequencer, and the relative yields of amino acids unique in the analyzed sequence. That is if the unique (in the analyzed sequence) residue X appears only in the nth cycle a cleavage site exists n-1 residues before it in the N-terminal direction. In addition to helping resolve any ambiguity in the assignment of mass to sequences, these data also provide a more reliable indication of the relative yield of the various fragments than does mass spectrometry. - For PSMA281-310 (SEQ ID NO. 18) this pool sequencing supports two major cleavage sites after V287 and I297 among other minor cleavage sites. Reviewing the results presented in
FIG. 3 reveals the following: -
- S at the 4th and 11th cycles indicating cleavage after V287 and presence of the N-terminus of the substrate, respectively.
- H at the 8th cycle indicating cleavage after V287. The lack of decay in peak height at
positions - D at the 2nd, 4th, and 7th cycles indicating cleavages after Y299, I297, and V294, respectively. This last cleavage is not observed in any of the fragments in Table 4 or in the alternate assignments in the notes below.
- Q at the 6th cycle indicating cleavage after I297.
- M at the 10th and 12th cycle indicating cleavages after Y299 and I297, respectively.
- Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include a predicted HLA-A1 binding sequence, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 4.
-
TABLE 4 Predicted HLA binding by proteasomally generated fragments: PSMA281-310 III. SEQ ID NO. IV. PEPTIDE HLA SYFPEITHI NIH 20 & (21) (G)LPSIPVHPI A*0201 16(24) (24) B*0702/B7 23 12 B*5101 24 572 Cw*0401 NP† 20 22 & (23) (P)IGYYDAQKL A*0201 (16) <5 A26 (20) NP B*2705 16 25 B*2709 15 NP B*5101 21 57 Cw*0301 NP 24 24 & (25) (P)SIPVHPIGY A1 21(27) <5 A26 22 NP A3 16 <5 26 IPVHPIGY B*5101 16 NP 27 YYDAQKLLE A1 22 <5 †No prediction
As seen in Table 4, N-terminal addition of authentic sequence to epitopes can often generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (G)LPSIPVHPI with HLA-A*0201, where the 10-mer can be used as a vaccine useful with several MHC types by relying on N-terminal trimming to create the epitopes for HLA-B7,-B*5101, and Cw*0401. - HLA-A*0201 binding studies were preformed with PSMA288-297, GLPSIPVHPI, (SEQ ID NO. 21) essentially as described in Example 1.1 above. As seen in
FIG. 2 , this epitope exhibits significant binding at even lower concentrations than the positive control peptides. - Another peptide, SSIEGNYTLRVDCTPLMYSLVHLTKEL, PSMA454-481, 5 (SEQ ID NO. 28) containing an epitope cluster from prostate specific membrane antigen, was synthesized by MPS (purity>95%) and subjected to proteasome digestion and mass spectrum analysis as described above. Prominent peaks from the mass spectra are summarized in Table 5.
-
TABLE 5 PSMA454-481 Mass Peak Identification. SEQ ID MS PEAK CALCULATED NO. (measured) PEPTIDE SEQUENCE MASS (MH+) 142 1238.5 454-464 SSIEGNYTLRV 1239.78 143 1768.38 ± 0.60 454-469 SSIEGNYTLRVDCTPL 1768.99 144 1899.8 454-470 SSIEGNYTLRVDCTPLM 1900.19 145 1097.63 ± 0.91 463-471 RVDCTPLMY 1098.32 146 2062.87 ± 0.68 454-471* SSIEGNYTLRVDCTPLMY 2063.36 147 1153 472-481** SLVHNLTKEL 1154.36 148 1449.93 ± 1.79 470-481 MYSLVHNLTKEL 1448.73 Boldface sequence correspond to peptides predicted to bind to MHC, see Table 6. *On the basis of mass alone this peak could equally well be assigned to the peptide 455-472 however proteasomal removal of just the N-terminal amino acid is considered unlikely. If the issue were important it could be resolved by N-terminal sequencing. **On the basis of mass this fragment might also represent 455-464. - Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include predicted HLA-A2.1 binding sequences, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 6.
-
TABLE 6 Predicted HLA binding by proteasomally generated fragments V. SEQ ID NO VI. PEPTIDE HLA SYFPEITHI NIH 29 & (30) (S)IEGNYTLRV A1 (19) <5 31 EGNYTLRV A*0201 16(22) <5 B*5101 15 NP† 32 & (33) (Y)TLRVDCTPL A*0201 20(18) (5) A26 16(18) NP B7 14 40 B8 23 <5 B*2705 12 30 Cw*0301 NP (30) 34 LRVDCTPLM B*2705 20 600 B*2709 20 NP 35 & (36) (L)RVDCTPLMY A2 32(22) 125 (13.5) A3 25 <5 A26 22 NP B*2702 NP (200) B*2705 13(NP) (1000) †No prediction
As seen in Table 6, N-terminal addition of authentic sequence to epitopes can often generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (L)RVDCTPLMY (SEQ ID NOS 35 and (36)) with HLA-B*2702/5, where the 10-mer has substantial predicted halftimes of dissociation and the co-C-terminal 9-mer does not. Also note the case of SIEGNYTLRV (SEQ ID NO 30) a predicted HLA-A*0201 epitope which can be used as a vaccine useful with HLA-B*5101 by relying on N-terminal trimming to create the epitope. - HLA-A*0201 binding studies were preformed, essentially as described in Example 1.1 above, with PSMA460-469, YTLRVDCTPL, (SEQ ID NO.33). As seen in
FIG. 4 , this epitope was found to bind HLA-A2.1 to a similar extent as the known A2.1 binder FLPSDYFPSV (HBV18-27; SEQ ID NO: 108) used as a positive control. Additionally, PSMA461-469, (SEQ ID NO. 32) binds nearly as well. - The wells of a nitrocellulose-backed microtiter plate were coated with capture antibody by incubating overnight at 4° C. using 50 μl/well of 4 μg/ml murine anti-human □-IFN monoclonal antibody in coating buffer (35 mM sodium bicarbonate, 15 mM sodium carbonate, pH 9.5). Unbound antibody was removed by washing 4
times 5 min. with PBS. Unbound sites on the membrane then were blocked by adding 200 μl/well of RPMI medium with 10% serum and incubating 1 hr. at room temperature. Antigen stimulated CD8+ T cells, in 1:3 serial dilutions, were seeded into the wells of the microtiter plate using 100 μl/well, starting at 2×105 cells/well. (Prior antigen stimulation was essentially as described in Scheibenbogen, C. et al. Int. J. Cancer 71:932-936, 1997; which is incorporated herein by reference in its entirety.) PSMA462-471 (SEQ ID NO. 36) was added to a final concentration of 10 μg/ml and IL-2 to 100 U/ml and the cells cultured at 37° C. in a 5% CO2, water-saturated atmosphere for 40 hrs. Following this incubation the plates were washed with 6times 200 μl/well of PBS containing 0.05% Tween-20 (PBS-Tween). Detection antibody, 50 μl/well of 2 g/ml biotinylated murine anti-human □-IFN monoclonal antibody in PBS+10% fetal calf serum, was added and the plate incubated at room temperature for 2 hrs. Unbound detection antibody was removed by washing with 4times 200 μl of PBS-Tween. 100 μl of avidin-conjugated horseradish peroxidase (Pharmingen, San Diego, Calif.) was added to each well and incubated at room temperature for 1 hr. Unbound enzyme was removed by washing with 6times 200 μl of PBS-Tween. Substrate was prepared by dissolving a 20 mg tablet of 3-amino 9-ethylcoarbasole in 2.5 ml of N, N-dimethylformamide and adding that solution to 47, 5 ml of 0.05 M phosphate-citrate buffer (pH 5.0). 25 μl of 30% H2O2 was added to the substrate solution immediately before distributing substrate at 100 μl/well and incubating the plate at room temperature. After color development (generally 15-30 min.), the reaction was stopped by washing the plate with water. The plate was air dried and the spots counted using a stereomicroscope. -
FIG. 5 shows the detection of PSMA463-471 (SEQ ID NO. 35)-reactive HLA-A1+ CD8+ T cells previously generated in cultures of HLA-A1+ CD8+ T cells with autologous dendritic cells plus the peptide. No reactivity is detected from cultures without peptide (data not shown). In this case it can be seen that the peptide reactive T cells are present in the culture at a frequency between 1 in 2.2×104 and 1 in 6.7×104. That this is truly an HLA-A1-restricted response is demonstrated by the ability of anti-HLA-A1 monoclonal antibody to block □-IFN production; seeFIG. 6 . - Another peptide, FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA653-687, (SEQ ID NO. 37) containing an A2 epitope cluster from prostate specific membrane antigen, PSMA660-681 (SEQ ID NO. 38), was synthesized by MPS (purity>95%) and subjected to proteasome digestion and mass spectrum analysis as described above. Prominent peaks from the mass spectra are summarized in Table 7.
-
TABLE 7 PSMA653-687 Mass Peak Identification. SEQ ID MS PEAK CALCULATED NO. (measured) PEPTIDE SEQUENCE MASS (MH+) 149 906.17 ± 0.65 681-687** LPDRPFY 908.05 150 1287.73 ± 0.76 677-687** DPLGLPDRPFY 1290.47 151 1400.3 ± 1.79 676-687 IDPLGLPDRPFY 1403.63 152 1548.0 ± 1.37 675-687 FIDPLGLPDRPFY 1550.80 153 1619.5 ± 1.51 674-687** AFIDPLGLPDRPFY 1621.88 154 1775.48 ± 1.32 673-687* RAFIDPLGLPDRPFY 1778.07 155 2440.2 ± 1.3 653-672 FDKSNPIVLRMMNDQLMFLE 2442.932313.82 156 1904.63 ± 1.56 672-687* ERAFIDPLGLPDRPFY 1907.19 157 2310.6 ± 2.5 653-671 FDKSNPIVLRMMNDQLMFL 2313.82 158 2017.4 ± 1.94 671-687 LERAFIDPLGLPDRPFY 2020.35 159 2197.43 ± 1.78 653-670 FDKSNPIVLRMMNDQLMF 2200.66 Boldface sequence correspond to peptides predicted to bind to MHC, see Table 7. *On the basis of mass alone this peak could equally well be assigned to a peptide beginning at 654, however proteasomal removal of just the N-terminal amino acid is considered unlikely. If the issue were important it could be resolved by N-terminal sequencing. **On the basis of mass alone these peaks could have been assigned to internal fragments, but given the overall pattern of digestion it was considered unlikely. - Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include predicted HLA-A2.1 binding sequences, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 8.
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TABLE 8 Predicted HLA binding by proteasomally generated fragments VII. SEQ ID NO VIII. PEPTIDE HLA SYFPEITHI NIH 39 & (40) (R)MMNDQLMFL A*0201 24 (23) 1360 (722) A*0205 NP† 71 (42) A26 15 NP B*2705 12 50 41 RMMNDQLMF B*2705 17 75 †No prediction - As seen in Table 8, N-terminal addition of authentic sequence to epitopes can generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (R)MMNDQLMFL (SEQ ID NOS. 39 and (40)) with HLA-A*02, where the 10-mer retains substantial predicted binding potential.
- HLA-A*0201 binding studies were preformed, essentially as described in Example 1.1 above, with PSMA663-671, (SEQ ID NO. 39) and PSMA662-671, RMMNDQLMFL (SEQ NO. 67). As seen in
FIGS. 4 , 7 and 8, this epitope exhibits significant binding at even lower concentrations than the positive control peptide (FLPSDYFPSV (HBV18-27); SEQ ID NO. 108). Though not run in parallel, comparison to the controls suggests that PSMA662-671 (which approaches the Melan A peptide in affinity) has the superior binding activity of these two PSMA peptides. - A multi-center clinical study is carried out using compositions as disclosed herein. The results of the study show the compositions to be useful and effective for debulking solid tumors and for generally inducing anti-angiogenic activity.
- HHD1 transgenic A*0201 mice (Pascolo, S., et al. J. Exp. Med. 185:2043-2051, 1997) were anesthetized and injected subcutaneously at the base of the tail, avoiding lateral tail veins, using 100 μl containing 100 nmol of PSMA288-297 (SEQ ID NO. 21) and 20 μg of a HTL epitope peptide in PBS emulsified with 50 μl of IFA (incomplete Freund's adjuvant).
- Using spleens from 2 naïve mice for each group of immunized mice, un-immunized mice were sacrificed and their carcasses placed in alcohol. Using sterile instruments, the top dermal layer of skin on the mouse's left side (lower mid-section) was cut through, exposing the peritoneum. The peritoneum was saturated with alcohol, and the spleen aseptically extracted. The spleens were placed in a petri dish with serum-free media. Splenocytes were isolated by using sterile plungers from 3 ml syringes to mash the spleens. Cells were collected in a 50 ml conical tubes in serum-free media, rinsing dish well. Cells were centrifuged (12000 rpm, 7 min) and washed one time with RPMI. Fresh spleen cells were resuspended to a concentration of 1×106 cells per ml in RPMI-10%FCS (fetal calf serum). 25 g/ml lipopolysaccharide and 7 μg/ml Dextran Sulfate were added. Cell were incubated for 3 days in T-75 flasks at 37° C., with 5% CO2. Splenic blasts were collected in 50 ml tubes pelleted (12,000 rpm, 7 min) and resuspended to 3×107/ml in RPMI. The blasts were pulsed with the priming peptide at 50 μg/ml,
RT 4 hr. mitomycin C-treated at 25 μg/ml, 37° C., 20 min and washed three times with DMEM. - Three days after LPS stimulation of the blast cells and the same day as peptide loading, the primed mice were sacrificed (at 14 days post immunization) to remove spleens as above. 3×106 splenocytes were co-cultured with 1×106 LPS blasts/well in 24-well plates at 37° C., with 5% CO2 in DMEM media supplemented with 10% FCS, 5×10−5 M β(beta)-mercaptoethanol, 100 μg/ml streptomycin and 100 IU/ml penicillin. Cultures were fed 5% (vol/vol) ConA supernatant on
day 3 and can be transferred onday 7. An aliquot of the CTL are also tested in a standard chromium release assay to ensure activity. - 1×106 telomerase-transformed HDMEC in 10 μl of EGM-2-VM medium (Clonetics, San Diego, Calif.) are mixed with 0.5 ml of MATRIGEL (Becton Dickinson) on ice. The mixture is injected subcutaneously, through a 25 gauge needle, along the ventral midline of the thorax of SCID mice. One week later 1×107 T cells (target epitope-reactive or sham-immunized) in 0.2 ml are injected intravenously (alternatively they can be injected intraperitoneally).
- At one and two weeks after transfer remove implants, fix in 10% buffered overnight, embed in paraffin, and section. For immunofluorescence detection of human microvessels using anti-human type IV collagen IgG and fluorescently-labeled secondary antibody, deparifinize and retrieve antigen by microwaving
thin sections 2×7 minutes in 10 mM citric acid, pH 6.0. Vessel density is assessed as a function of the average number of positively stained annular structures observed in five separate, randomly selected 20× fields-of-view, from at least three sections per implant. - M1 tumor cells grown in complete RPMI plus 10% serum were harvested and washed with PBS by centrifugation. The cells were suspended in PBS at 5×106 cells/ml and 0.5 ml of the suspension (early passage) was injected subcutaneously into the abdomen.
- A nucleotide sequence encoding an HLA-A2-restricted fibronectin ED-B domain-derived housekeeping epitope, for example ED-B29-38 (SEQ ID NO. 103), is inserted into an appropriate vaccine vector (e.g. pVAX1 (Invitrogen Inc, Carlsbad, Calif.) or one of the vectors described in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2001, and incorporated by reference above. HHD-A2 mice are injected intranodally in the inguinal lymph node with 0, 2, 10, 50, 100, and 200 μg of vector in PBS every other day over 8 days (4 injections) alternating sides for each injection (single dosage per mouse or group of mice). Injection series are started the day of tumor cell injection, at 2 weeks before, and at 4 and 10 weeks after.
- At approximately 12 weeks after injection of tumor cells visible tumors are observed in the mice receiving the vehicle instead of the vaccine. Effectiveness of the vaccine is expressed as the proportion of vaccinated animals that fail to develop a tumor in the same time frame, the relative size of tumors at the same time point, the delay in time before tumors appear in the vaccinated animals, and the dose and number of composition cycles needed to inhibit or prevent the establishment of tumor.
- The availability of more aggressive later passage M1 cells enables a more compressed experimental schedule. Instead mice are vaccinated on the day of tumor cell inoculation, 1 and 2 weeks before, and 3 or 4 days after injections of 1×106 cells. Effectiveness of vaccination is assessed at approximately 10 days after tumor cell inoculation.
- HHD-A2 mice were immunized with ED-B29-38 (SEQ ID NO. 103) in complete Freund's adjuvants and spleen cells were harvested and re-stimulated in vitro using standard methodology. The resulting CTL were able to specifically lyse peptide pulsed T2 cells, which are HLA-A2+ (
FIG. 9 ). - Table 9 discloses epitopes and epitope clusters from PSMA and ED-B that can be useful in construction of compositions according to the present invention.
-
TABLE 9 SEQ ID NOS.* SEQ ID NO IDENTITY SEQUENCE 1 PSMA protein Accession number**: NP_004467 2 PSMA cDNA Accession number: NM_004476 3 PSMA 163-192 AFSPQGMPEGDLVYVNYARTEDFFKLERDM 4 PSMA 168-190 GMPEGDLVYVNYARTEDFFKLER 5 PSMA 169-177 MPEGDLVYV 6 PSMA 168-177 GMPEGDLVYV 7 PSMA 168-176 GMPEGDLVY 8 PSMA 167-176 QGMPEGDLVY 9 PSMA 169-176 MPEGDLVY 10 PSMA 171-179 EGDLVYVNY 11 PSMA 170-179 PEGDLVYVNY 12 PSMA 174-183 LVYVNYARTE 13 PSMA 177-185 VNYARTEDF 14 PSMA 176-185 YVNYARTEDF 15 PSMA 178-186 NYARTEDFF 16 PSMA 179-186 YARTEDFF 17 PSMA 181-189 RTEDFFKLE 18 PSMA 281-310 RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG 19 PSMA 283-307 IAEAVGLPSIPVHPIGYYDAQKLLE 20 PSMA 289-297 LPSIPVHPI 21 PSMA 288-297 GLPSIPVHPI 22 PSMA 297-305 IGYYDAQKL 23 PSMA 296-305 PIGYYDAQKL 24 PSMA 291-299 SIPVHPIGY 25 PSMA 290-299 PSIPVHPIGY 26 PSMA 292-299 IPVHPIGY 27 PSMA 299-307 YYDAQKLLE 28 PSMA 454-481 SSIEGNYTLRVDCTPLMYSLVHLTKEL 29 PSMA 456-464 IEGNYTLRV 30 PSMA 455-464 SIEGNYTLRV 31 PSMA 457-464 EGNYTLRV 32 PSMA 461-469 TLRVDCTPL 33 PSMA 460-469 YTLRVDCTPL 34 PSMA 462-470 LRVDCTPLM 35 PSMA 463-471 RVDCTPLMY 36 PSMA 462-471 LRVDCTPLMY 37 PSMA 653-687 FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY 38 PSMA 660-681 VLRMMNDQLMFLERAFIDPLGL 39 PSMA 663-671 MMNDQLMFL 40 PSMA 662-671 RMMNDQLMFL 41 PSMA 662-670 RMMNDQLMF 42 PSMA 4-12 LLHETDSAV 43 PSMA 13-21 ATARRPRWL 44 PSMA 53-61 TPKHNMKAF 45 PSMA 64-73 ELKAENIKKF 46 PSMA 69-77 NIKKFLH1NF 47 PSMA 68-77 ENIKKFLH1NF 48 PSMA 220-228 AGAKGVILY 49 PSMA 468-477 PLMYSLVHNL 50 PSMA 469-477 LMYSLVHNL 51 PSMA 463-471 RVDCTPLMY 52 PSMA 465-473 DCTPLMYSL 53 PSMA 507-515 SGMPRISKL 54 PSMA 506-515 FSGMPRISKL 55 PSMA 211-218 GNKVKNAQ 56 PSMA 202-209 IARYGKVF 57 PSMA 217-225 AQLAGAKGV 58 PSMA 207-215 KVFRGNKVK 59 PSMA 211-219 GNKVKNAQL 60 PSMA 269-277 TPGYPANEY 61 PSMA 268-277 LTPGYPANEY 62 PSMA 271-279 GYPANEYAY 63 PSMA 270-279 PGYPANEYAY 64 PSMA 266-274 DPLTPGYPA 65 PSMA 492-500 SLYESWTKK 66 PSMA 491-500 KSLYESWTKK 67 PSMA 486-494 EGFEGKSLY 68 PSMA 485-494 DEGFEGKSLY 69 PSMA 498-506 TKKSPSPEF 70 PSMA 497-506 WTKKSPSPEF 71 PSMA 492-501 SLYESWTKKS 72 PSMA 725-732 WGEVKRQI 73 PSMA 724-732 AWGEVKRQI 74 PSMA 723-732 KAWGEVKRQI 75 PSMA 723-730 KAWGEVKR 76 PSMA 722-730 SKAWGEVKR 77 PSMA 731-739 QIYVAAFTV 78 PSMA 733-741 YVAAFTVQA 79 PSMA 725-733 WGEVKRQIY 80 PSMA 727-735 EVKRQIYVA 81 PSMA 738-746 TVQAAAETL 82 PSMA 737-746 FTVQAAAETL 83 PSMA 729-737 KRQIYVAAF 84 PSMA 721-729 PSKAWGEVK 85 PSMA 723-731 KAWGEVKRQ 86 PSMA 100-108 WKEFGLDSV 87 PSMA 99-108 QWKEFGLDSV 88 PSMA 102-111 EFGLDSVELA 89 ED-B domain of EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI Fibronectin TVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID YDISVITLINGGESAPTTLTQQT 90 ED-B domain of CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIP Fibronectin with EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI flanking sequence TVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID from Fribronectin YDISVITLINGGESAPTTLTQQT AVPPPTDLRFTNIGPDTMRVTW 91 ED-B domain of Accession number: X07717 Fibronectin cds 92 ED-B 4′-5 TIIPEVPQL 93 ED-B 5′-5 DTIIPEVPQL 94 ED-B 1-10 EVPQLTDLSF 95 ED-B 23-30 TPLNSSTI 96 ED-B 18-25 IGLRWTPL 97 ED-B 17-25 SIGLRWTPL 98 ED-B 25-33 LNSSTIIGY 99 ED-B 24-33 PLNSSTIIGY 100 ED-B 23-31 TPLNSSTII 101 ED-B 31-38 IGYRITVV 102 ED-B 30-38 IIGYRITVV 103 ED-B 29-38 TIIGYRITVV 104 ED-B 31-39 IGYRITVVA 105 ED-B 30-39 IIGYRITVVA 106 Melan-A 26-35A>L ELAGIGILTV 107 Melan-A 26-35 EAAGIGILTV 108 HBV 18-27 FLPSDYFPSV 109 HLA-B 44 binder AEMGKYSFY 1This H was reported as Y in the SWISSPROT database. *Any of SEQ ID NOS. 5-17, 20-27, 29-36, 39-88, and 92-105 can be useful as epitopes in the various embodiments of the invention. Any of SEQ ID NOS. 3, 4, 18, 19, 28, 37, 38, 89 and 90 can be useful as sequences containing epitopes or epitope clusters, as described in various embodiments of the invention. **All accession numbers used here and throughout can be accessed through the NCBI databases, for example, through the Entrez seek and retrieval system on the world wide web. -
PSMA LOCUS NM_004476 2653 bp mRNA PRI 01-NOV-2000 DEFINITION Homo sapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1), mRNA. ACCESSION NM_004476 VERSION NM_004476.1 GI: 4758397 KEYWORDS . SOURCE human. ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 ( bases 1 to 2653)AUTHORS Israeli, R. S., Powell, C. T., Fair, W. R. and Heston, W. D. TITLE Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen JOURNAL Cancer Res. 53 (2), 227-230 (1993) MEDLINE 93113576 REFERENCE 2 ( bases 1 to 2653)AUTHORS Rinker-Schaeffer C W, Hawkins A L, Su S L, Israeli R S, Griffin C A, Isaacs J T and Heston W D. TITLE Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11JOURNAL Genomics 30 (1), 105-108 (1995) MEDLINE 96129312 PUBMED 8595888 REFERENCE 3 ( bases 1 to 2653)AUTHORS O'Keefe D S, Su S L, Bacich D J, Horiguchi Y, Luo Y, Powell C T, Zandvliet D, Russell P J, Molloy P L, Nowak N J, Shows T B, Mullins C, Vonder Haar R A, Fair W R and Heston W D. TITLE Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene JOURNAL Biochim. Biophys. Acta 1443 (1-2), 113-127 (1998) MEDLINE 99057588 PUBMED 9838072 REFERENCE 4 ( bases 1 to 2653)AUTHORS Maraj B H, Leek J P, Karayi M, Ali M, Lench N J and Markham A F. TITLE Detailed genetic mapping around a putative prostate- specific membrane antigen locus on human chromosome 11p11.2 JOURNAL Cytogenet. Cell Genet. 81 (1), 3-9 (1998) MEDLINE 98358137 PUBMED 9691167 COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from M99487.1. FEATURES Location/ Qualifiers source 1 . . . 2653 /organism = “Homo sapiens” /db_xref = “taxon: 9606” /chromosome = “11” /map = “11p11.2” /sex = “male” /cell_line = “LNCaP-ATCC” /cell_type = “prostate” /tissue_type = “prostatic carcinoma metastatic lymph node” /tissue_lib = “LNCaP cDNA of Ron Israeli” gene 1 . . . 2653 /gene = “FOLH1” /note = “FOLH; PSM; PSMA” /db_xref = “LocusID: 2346” /db_xref = “MIM: 600934” CDS 262 . . . 2514 /gene = “FOLH1” /note = “folate hydrolase 1 (prostate-specific membrane antigen)” /codon_start = 1 /db_xref = “LocusID: 2346” /db_xref = “MIM: 600934” /evidence = experimental /product = “folate hydrolase (prostate-specific membrane antigen) 1” /protein_id = “NP_004467.1” /db_xref = “GI: 4758398” /translation = “MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIK (SEQ ID NO. 1) SSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPP FSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQ LAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANE YAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFT GNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGA AVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSG MPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFY DPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKT YSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLP DRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQ AAAETLSEVA” misc feature 778 . . . 1029 /note = “PA; Region: PA domain” BASE COUNT 782 a 524 c 640 g 707 t ORIGIN 1 ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg (SEQ ID NO. 2) 61 attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga 121 gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac 181 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 241 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc 301 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt 361 ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact 421 ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc 481 ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca 541 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat 601 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa 661 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat 721 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 841 atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag 901 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac 961 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc 1021 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca 1081 gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1141 gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca 1201 ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt 1261 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca 1321 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt 1381 ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct 1441 gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga 1501 agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1561 tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac 1621 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg 1681 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt 1741 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc 1801 aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc 1861 agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac 1921 agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac 1981 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc 2041 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt 2101 atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt 2161 tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt 2221 gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga 2281 gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct 2341 ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt 2401 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat 2461 gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat 2521 tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2581 atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa 2641 aaaaaaaaaa aaa ED-B domain of Fibronectin LOCUS HSFIBEDB 2823 bp DNA linear PRI 09-AUG-1999 DEFINITION Human fibronectin gene ED-B region. ACCESSION X07717 VERSION X07717.1 GI: 31406 KEYWORDS alternate splicing; fibronectin. SOURCE human. ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 ( bases 1 to 2823)AUTHORS Paolella, G., Henchcliffe, C., Sebastio, G. and Baralle, F. E. TITLE Sequence analysis and in vivo expression show that alternative splicing of ED-B and ED-A regions of the human fibronectin gene are independent events JOURNAL Nucleic Acids Res. 16 (8), 3545-3557 (1988) MEDLINE 88233940 FEATURES Location/ Qualifiers source 1 . . . 2823 /organism = “Homo sapiens” /db_xref = “taxon: 9606” /clone = “MA10” exon 1 . . . 104 /number = 1 /product = “fibronectin” CDS join(<2 . . . 104, 1375 . . . 1647, 2758 . . . >2823) /codon_start = 1 /product = “fibronectin” /protein_id = “CAB52437.1” /db_xref = “GI: 5725425” /translation = “CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSF (SEQ ID NO. 90) VDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID YDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTW” intron 105 . . . 1374 /number = 1 exon 1375 . . . 1647 /note = “ED-B exon” /number = 2 /product = “fibronectin” intron 1648 . . . 2757 /number = 2 exon 2758 . . . 2823 /number = 3 /product = “fibronectin” BASE COUNT 824 a 556 c 528 g 915 t ORIGIN 1 ctgcactttt gataacctga gtcccggcct ggagtacaat gtcagtgttt acactgtcaa (SEQ ID NO. 91) 61 ggatgacaag gaaagtgtcc ctatctctga taccatcatc ccaggtaata gaaaataagc 121 tgctatcctg agagtgacat tccaataaga gtggggatta gcatcttaat ccccagatgc 181 ttaagggtgt caactatatt tgggatttaa ttccgatctc ccagctgcac tttccaaaac 241 caagaagtca aagcagcgat ttggacaaaa tgcttgctgt taacactgct ttactgtctg 301 tgcttcactg ggatgctgtg tgttgcagcg agtatgtaat ggagtggcag ccatggcttt 361 aactctgtat tgtctgctca catggaagta tgactaaaac actgtcacgt gtctgtactc 421 agtactgata ggctcaaagt aatatggtaa atgcatccca tcagtacatt tctgcccgat 481 tttacaatcc atatcaattt ccaacagctg cctatttcat cttgcagttt caaatccttc 541 tttttgaaaa ttggatttta aaaaaaagtt aagtaaaagt cacaccttca gggttgttct 601 ttcttgtggc cttgaaagac aacattgcaa aggcctgtcc taaggatagg cttgtttgtc 661 cattgggtta taacataatg aaagcattgg acagatcgtg tccccctttg gactcttcag 721 tagaatgctt ttactaacgc taattacatg ttttgattat gaatgaacct aaaatagtgg 781 caatggcctt aacctaggcc tgtctttcct cagcctgaat gtgcttttga atggcacatt 841 tcacaccata cattcataat gcattagcgt tatggccatg atgttgtcat gagttttgta 901 tgggagaaaa aaaatcaatt tatcacccat ttattatttt ttccggttgt tcatgcaagc 961 ttattttcta ctaaaacagt tttggaatta ttaaaagcat tgctgatact taettcagat 1021 attatgtcta ggctctaaga atggtttcga catcctaaac agccatatga tttttaggaa 1081 tctgaacagt tcaaattgta ccctttaagg atgttttcaa aatgtaaaaa atatatatat 1141 atatatatat tccctaaaag aatattcctg tttattcttc tagggaagca aactgttcat 1201 gatgcttagg aagtcttttc agagaattta aaacagattg catattacca tcattgcttt 1261 aacattccac caattttact actagtaacc tgatatacac tgctttattt tttcctcttt 1321 ttttccctct attttccttt tgcctccccc tccctttgct ttgtaactca atagaggtgc 1381 cccaactcac tgacctaagc tttgttgata taaccgattc aagcatcggc ctgaggtgga 1441 ccccgctaaa ctcttccacc attattgggt accgcatcac agtagttgcg gcaggagaag 1501 gtatccctat ttttgaagat tttgtggact cctcagtagg atactacaca gtcacagggc 1561 tggagccggg cattgactat gatatcagcg ttatcactct cattaatggc ggcgagagtg 1621 cccctactac actgacacaa caaacgggtg aattttgaaa acttctgcgt ttgagacata 1681 gatggtgttg catgctgcca ccagttactc cggttaaata tggatgtttc atgggggaag 1741 tcagcaattg gccaaagatt cagataggtg gaattggggg gataaggaat caaatgcatc 1801 tgctaaactg attggagaaa aacacatgca atatcttcag tacactctca tttaaaccac 1861 aagtagatat aaagcctaga gaaatacaga tgtctgctct gttaaatata aaatagcaaa 1921 tgttcattca atttgaagac ctagaatttt tcttcttaaa taccaaacac gaataccaaa 1981 ttgcgtaagt accaattgat aagaatatat caccaaaatg taccatcatg ctcttccttc 2041 taccctttga taaactctac catgctcctt ctttgtagct aaaaacccat caaaatttag 2101 ggtagagtgg atgggcattg ttttgaggta ggagaaaagt aaacttggga ccattctagg 2161 ttttgttgct gtcactaggt aaagaaacac ctctttaacc acagtctggg gacaagcatg 2221 caacatttta aaggttctct gctgtgcatg ggaaaagaaa catgctgaga accaatttgc 2281 atgaacatgt tcacttgtaa gtagaattca ctgaatggaa ctgtagctct agatatctca 2341 catgggggga agtttaggac cctcttgtct ttttgtctgt gtgcatgtat ttctttgtaa 2401 agtactgcta tgtttctctt tgctgtgtgg caacttaagc ctcttcggcc tgggataaaa 2461 taatctgcag tggtattaat aatgtacata aagtcaacat atttgaaagt agattaaaat 2521 cttttttaaa tatatcaatg atggcaaaaa ggttaaaggg ggcctaacag tactgtgtgt 2581 agtgttttat ttttaacagt agtacactat aacttaaaat agacttagat tagactgttt 2641 gcatgattat gattctgttt cctttatgca tgaaatattg attttacctt tccagctact 2701 tcgttagctt taattttaaa atacattaac tgagtcttcc ttcttgttcg aaaccagctg 2761 ttcctcctcc cactgacctg cgattcacca acattggtcc agacaccatg cgtgtcacct 2821 ggg //
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Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5539084A (en) * | 1989-02-17 | 1996-07-23 | Coselco Mimotopes Pty. Ltd. | Method for the use and synthesis of peptides |
US5772995A (en) * | 1994-07-18 | 1998-06-30 | Sidney Kimmel Cancer Center | Compositions and methods for enhanced tumor cell immunity in vivo |
US5840839A (en) * | 1996-02-09 | 1998-11-24 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Alternative open reading frame DNA of a normal gene and a novel human cancer antigen encoded therein |
US6034218A (en) * | 1996-03-15 | 2000-03-07 | Corixa Corporation | Compounds and methods for immunotherapy and immunodiagnosis of prostate cancer |
CA2405363A1 (en) * | 2000-04-28 | 2001-11-08 | Ctl Immunotherapies Corp. | Epitope synchronization in antigen presenting cells |
US6342221B1 (en) * | 1999-04-28 | 2002-01-29 | Board Of Regents, The University Of Texas System | Antibody conjugate compositions for selectively inhibiting VEGF |
US20030138432A1 (en) * | 1999-11-15 | 2003-07-24 | Drug Innovation & Design, Inc. | Selective cellular targeting: multifunctional delivery vehicles, multifunctional prodrugs, use as antineoplastic drugs |
US20030215425A1 (en) * | 2001-12-07 | 2003-11-20 | Simard John J. L. | Epitope synchronization in antigen presenting cells |
US6861234B1 (en) * | 2000-04-28 | 2005-03-01 | Mannkind Corporation | Method of epitope discovery |
US6897062B1 (en) * | 1999-04-09 | 2005-05-24 | Sloan-Kettering Institute For Cancer Research | DNA encoding the prostate-specific membrane antigen-like gene and uses thereof |
US6977074B2 (en) * | 1997-07-10 | 2005-12-20 | Mannkind Corporation | Method of inducing a CTL response |
US6994851B1 (en) * | 1997-07-10 | 2006-02-07 | Mannkind Corporation | Method of inducing a CTL response |
US7045605B2 (en) * | 2001-06-01 | 2006-05-16 | Cornell Research Foundation, Inc. | Modified antibodies to prostate-specific membrane antigen and uses thereof |
US7074400B1 (en) * | 1999-03-01 | 2006-07-11 | The Commonwealth Of Australia | Regulatory constructs comprising intron 3 of prostate specific membrane antigen gene |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9804121D0 (en) | 1998-02-26 | 1998-04-22 | Cancer Res Campaign Tech | Anti-angiogenic vaccines: materials and methods relating thereto |
WO1999045954A1 (en) * | 1998-03-13 | 1999-09-16 | Epimmune, Inc. | Hla-binding peptides and their uses |
IL125608A0 (en) * | 1998-07-30 | 1999-03-12 | Yeda Res & Dev | Tumor associated antigen peptides and use of same as anti-tumor vaccines |
-
2002
- 2002-03-07 EP EP10178936A patent/EP2295074A1/en not_active Withdrawn
- 2002-03-07 EP EP02715085A patent/EP1372736A4/en not_active Withdrawn
- 2002-03-07 US US10/094,699 patent/US7252824B2/en not_active Expired - Fee Related
- 2002-03-07 AU AU2002247304A patent/AU2002247304A1/en not_active Abandoned
- 2002-03-07 JP JP2002569085A patent/JP2005505242A/en active Pending
- 2002-03-07 CN CN02806006A patent/CN100589845C/en not_active Expired - Fee Related
- 2002-03-07 WO PCT/US2002/007204 patent/WO2002069907A2/en not_active Application Discontinuation
-
2005
- 2005-06-30 US US11/073,347 patent/US20050260234A1/en not_active Abandoned
-
2006
- 2006-05-29 HK HK06106248.1A patent/HK1083771A1/en not_active IP Right Cessation
-
2007
- 2007-07-02 US US11/772,811 patent/US20090208537A1/en not_active Abandoned
-
2011
- 2011-02-07 JP JP2011024307A patent/JP2011098980A/en active Pending
Patent Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5539084A (en) * | 1989-02-17 | 1996-07-23 | Coselco Mimotopes Pty. Ltd. | Method for the use and synthesis of peptides |
US5772995A (en) * | 1994-07-18 | 1998-06-30 | Sidney Kimmel Cancer Center | Compositions and methods for enhanced tumor cell immunity in vivo |
US5840839A (en) * | 1996-02-09 | 1998-11-24 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Alternative open reading frame DNA of a normal gene and a novel human cancer antigen encoded therein |
US6034218A (en) * | 1996-03-15 | 2000-03-07 | Corixa Corporation | Compounds and methods for immunotherapy and immunodiagnosis of prostate cancer |
US6994851B1 (en) * | 1997-07-10 | 2006-02-07 | Mannkind Corporation | Method of inducing a CTL response |
US6977074B2 (en) * | 1997-07-10 | 2005-12-20 | Mannkind Corporation | Method of inducing a CTL response |
US7074400B1 (en) * | 1999-03-01 | 2006-07-11 | The Commonwealth Of Australia | Regulatory constructs comprising intron 3 of prostate specific membrane antigen gene |
US6897062B1 (en) * | 1999-04-09 | 2005-05-24 | Sloan-Kettering Institute For Cancer Research | DNA encoding the prostate-specific membrane antigen-like gene and uses thereof |
US6342221B1 (en) * | 1999-04-28 | 2002-01-29 | Board Of Regents, The University Of Texas System | Antibody conjugate compositions for selectively inhibiting VEGF |
US7056509B2 (en) * | 1999-04-28 | 2006-06-06 | Board Of Regents The University Of Texas System | Antibody methods for selectively inhibiting VEGF |
US20030138432A1 (en) * | 1999-11-15 | 2003-07-24 | Drug Innovation & Design, Inc. | Selective cellular targeting: multifunctional delivery vehicles, multifunctional prodrugs, use as antineoplastic drugs |
US6861234B1 (en) * | 2000-04-28 | 2005-03-01 | Mannkind Corporation | Method of epitope discovery |
CA2405363A1 (en) * | 2000-04-28 | 2001-11-08 | Ctl Immunotherapies Corp. | Epitope synchronization in antigen presenting cells |
US7045605B2 (en) * | 2001-06-01 | 2006-05-16 | Cornell Research Foundation, Inc. | Modified antibodies to prostate-specific membrane antigen and uses thereof |
US20030215425A1 (en) * | 2001-12-07 | 2003-11-20 | Simard John J. L. | Epitope synchronization in antigen presenting cells |
Non-Patent Citations (12)
Title |
---|
Chai et al. (J. Virol. May 2007; 81 (10): 4912-8) * |
Chang et al. (Clin. Cancer Res. 1999 Oct; 5 (10): 2674-81) * |
Dumas et al. (Int. J. Cancer. 1999 Mar 15; 80 (6): 799-803) * |
Holzer et al. (PLoS One. 2013 Nov 14; 8 (11): e80292; pp. 1-15) * |
Kallergi et al. (Breast Cancer Res. 2009; 11 (6): R84; pp. 1-12) * |
Knight et al. (Prostate. 2009 Feb 1; 69 (2): 142-148) * |
Koh et al. (Virus Res. 2006 Dec; 122 (1-2): 35-44). * |
Kuratsukuri et al. (Eur. Urol. 2002 Jul; 42 (1): 67-73) * |
Liu et a. (Cancer Res. 1997 Sep 1; 57 (17): 3629-34) * |
Murphy et al. (Prostate. 1999; 39: 54-59) * |
Schroers et al. (Clin. Cancer Res. 2003 Aug 15; 9 (9): 3260-71) * |
Silver et al. (Clin. Cancer Res. 1997 Jan; 3 (1): 81-5) * |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8372393B2 (en) | 1997-07-10 | 2013-02-12 | Mannkind Corporation | Method of inducing a CTL response |
US20070004662A1 (en) * | 2005-06-17 | 2007-01-04 | Zhiyong Qiu | Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma |
US7999088B2 (en) | 2005-06-17 | 2011-08-16 | Mannkind Corporation | Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma |
US8674081B2 (en) | 2005-06-17 | 2014-03-18 | Mankind Corporation | Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma |
US20110189093A1 (en) * | 2008-04-14 | 2011-08-04 | Proscan Rx Pharma | Prostate specific membrane antigen antibodies and antigen binding fragments |
WO2011050344A2 (en) | 2009-10-23 | 2011-04-28 | Mannkind Corporation | Cancer immunotherapy and method of treatment |
Also Published As
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US7252824B2 (en) | 2007-08-07 |
EP2295074A1 (en) | 2011-03-16 |
AU2002247304A1 (en) | 2002-09-19 |
WO2002069907A3 (en) | 2003-02-13 |
JP2005505242A (en) | 2005-02-24 |
EP1372736A4 (en) | 2004-11-17 |
WO2002069907A8 (en) | 2004-02-12 |
CN1703247A (en) | 2005-11-30 |
WO2002069907A2 (en) | 2002-09-12 |
HK1083771A1 (en) | 2006-07-14 |
US20050260234A1 (en) | 2005-11-24 |
JP2011098980A (en) | 2011-05-19 |
CN100589845C (en) | 2010-02-17 |
US20030046714A1 (en) | 2003-03-06 |
EP1372736A2 (en) | 2004-01-02 |
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