WO1999029834A1 - Methods for preparation of vaccines against cancer - Google Patents
Methods for preparation of vaccines against cancer Download PDFInfo
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- WO1999029834A1 WO1999029834A1 PCT/US1998/026401 US9826401W WO9929834A1 WO 1999029834 A1 WO1999029834 A1 WO 1999029834A1 US 9826401 W US9826401 W US 9826401W WO 9929834 A1 WO9929834 A1 WO 9929834A1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/828—Cancer
Definitions
- hsp60, hsp70 and hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress.
- the present invention relates to methods for producing increased amounts of immunogenic material which can be used for prevention and treatment of cancer.
- the immunogenic compositions prepared by the methods of the invention comprise noncovalently associated molecular complexes of a heat shock protein (hsp) and an antigenic (or immunogenic) peptide.
- the complexes repared by the methods of the invention are intracellularly produced complexes comprising hsps from a selected recombinant host cell and antigenic peptides expressed from cDNAs of a cancer cell; the antigenic peptides of the complex are thus representative of antigenic peptides found in such cancer cell.
- the present invention provides methods for making a cDNA library from cancer cells, using the cDNA library to produce by recombinant DNA methods in host cells immunogenic hsp-peptide complexes that confer immunity to the cancer cells in an individual to which the complexes are administered.
- the methods of the invention comprise obtaining (e.g., isolating) cancer cells from one or more individuals, preparing RNA from the cancer cells, making cDNA from the RNA, introducing the cDNA into host cells, culturing the host cells so that the cancer-derived cDNAs are expressed, and purifying heat shock protein-peptide complexes from the host cells.
- the cDNA prepared from cancer cell RNA herein referred to as "cancer cDNA”
- the cDNAs are optionally inserted into a cloning vector for replication purposes prior to expression.
- host cells for expression of the cancer cDNAs can also be genetically engineered to coexpress recombinantly one or more hsp genes so that increased amounts of complexes comprising immunogenic peptides noncovalently associated with the hsps can be produced.
- Figure 1 Flow chart illustrating an exemplary method of the invention of producing hsp-peptide complexes. Optional steps are indicated by a dashed line.
- Immunogenic hsp-peptide complexes are produced naturally in cancer cells. As it is not always possible or feasible to obtain large number of cancer cells, the quantity of the hsp-peptide complexes obtainable from the cancer cells is sometimes very limited. It is therefore an object of the present invention to overcome the potential problem of having a restricted supply of starting cellular material by providing methods for making a cDNA library from cancer cells that is useful for producing large quantities of hsp-peptide complexes in recombinant host cells.
- preneoplastic cells may include antigenic cells that are infected with a cancer-causing infectious agent, such as a virus but which are not yet neoplastic; or antigenic cells that have been exposed to a mutagen or cancer-causing agent, such as, for example DNA- damaging agents, radiation, etc.
- a cancer-causing infectious agent such as a virus but which are not yet neoplastic
- Other cells that can be used to make the cDNA library are preneoplastic cells which are in transition from a normal to a neoplastic form as characterized by morphology, physiological or biochemical functions.
- the cancer cells and preneoplastic cells used in the methods of the invention are of mammalian origin.
- Mammals contemplated by this aspect of the invention include humans, companion animals (e.g., dogs and cats), livestock animals (e.g., sheep, cattle, goats, pigs and horses), laboratory animals (e.g., mice, rats and rabbits), and captive or free wild animals.
- companion animals e.g., dogs and cats
- livestock animals e.g., sheep, cattle, goats, pigs and horses
- laboratory animals e.g., mice, rats and rabbits
- the cancer cDNA libraries such as when made from total mRNA, contain at least one order of magnitude more cDNAs than the estimated number of mRNA species, e.g., a cancer cDNA library may contain 300,000 to 1,200,000 independent cDNA clones.
- a library of expression or replicable constructs comprising cancer cDNAs can be amplified in vitro (if desired) , aliquoted, and lyophilized or frozen as nucleic acid molecules for future use.
- the library can be thawed and directly introduced into host cells for production of hsp- peptide complexes.
- the library can be cloned and/or expanded by replication in a cloning vector in an intermediate cells, prior to introduction into suitable host cells for production of hsp-peptide complexes.
- the cancer cDNA library captures and preserves the antigenic genetic material that is actively expressed in the cancer cells.
- Expression constructs or expression vectors comprising cancer cDNAs can be introduced and maintained in the host cells by any methods known in the art.
- the cancer cDNAs of the cancer cells are transcribed, translated, and processed if appropriate, in the host cells or host organisms to produce the proteins or peptides of the cancer cells, some of which are antigenic and can induce an immune response when complexed with a stress protein.
- The- term "cancer cDNA host cells” will be used herein to refer to host cells containing cancer cDNA.
- peptides/proteins that complex with hsps confer specific immunity to a host against the cancer cell in which they are present (see PCT Publication WO 96/10411, dated April 11, 1996).
- the immunogenicity of such compositions can be tested by methods known in the art and described in Section 5.3.
- Noncovalent complexes of such cancer peptides with hsps can be used as a vaccine to treat or prevent the type of target cancer from which the antigenic cancer peptide originated.
- an immunogenic composition that is useful as a vaccine can be recovered or purified from a culture of recombinant host cells that are expressing cancer cDNAs and producing noncovalent complexes of cancer peptides and hsps.
- the recombinant host cells containing the cancer cDNA library can be pooled and/or aliquoted; or expanded to increase the number of clones containing the library; or archived by freezing down and storing under liquid nitrogen, so that batches of the recombinant host cells can be retrieved and used many times in the future.
- the antigenic peptides that are expressed in the cancer cells can be produced in large amounts in the recombinant host cells.
- the desirable immunogenic compositions useful for the treatment and prevention of cancer comprising noncovalent complexes of hsps of the host cells and antigenic proteins/peptides of the cancer cells, can be prepared or purified from a large-scale continuous or batch culture of cancer cDNA host cells.
- a cancer cDNA library can provide a consistent, reproducible and abundant source of the useful immunogenic composition.
- any cancer cells preferably human cancer cells, can be used in the present methods for making a cancer cDNA library.
- the cancer cells provide the RNAs which encode the proteins that are expressed in the cancer cells.
- Cancers which can be treated or prevented with immunogenic compositions prepared by methods of the invention include, but are not limited to, tumors such as sarcomas and carcinomas. Examples of cancers that are amenable to the methods of the invention are listed in Section 5.4.
- any tissues or cells isolated from a preneoplastic lesion, a cancer, including cancer that has metastasized to multiple remote sites can be used in the present method.
- cells found in abnormally growing tissue, circulating leukemic cells, metastatic lesions as well as solid tumor tissue can be used.
- cell lines derived from a preneoplastic lesion, cancer tissues or cancer cells can also be used, provided that the cells of the cell line have at least one or more antigenic determinants in common with antigens on the target cancer cells.
- Cancer tissues, cancer cells, cells infected with a cancer-causing agent, other preneoplastic cells, and cell lines of human origin are preferred.
- cancer cells are used that are excised from the patient to which ultimately the complexes are to be administered, although this need not be the case (e.g., the cancer cells can be from one or more different individuals) .
- Such immunogenic compositions are also capable of preventing the development of tumors and inhibiting the growth and progression of tumor cells.
- the immunogenic compositions can be used to induce an inflammatory reaction at the tumor site and ultimately cause a regression of the tumor burden in the cancer patients treated.
- the immunogenic compositions can also be administered autologously to the individual from whom the cancer t ssues were obtained, or to individuals at enhanced risk of cancer due to familial history or environmental risk factors.
- the antigenic peptides of the present invention are associated with hsps inside a cancer cell or a recombinant host cell expressing a cancer cDNA.
- Such antigenic peptide can be a fragment of an antigenic protein expressed in the cancer cell, such as for example, fragment of a tumor- specific antigen or tumor associated antigen.
- Such antigenic peptides are produced in cancer cDNA host cells that are expressing the cloned cancer cDNAs. However, it is not necessary to isolate or characterize or even know the identities of these antigens in advance of using the present methods.
- the cancer cDNAs that are expressed recombinantly in the host cells need not comprise full-length coding sequences although such is preferable.
- Heat shock proteins which are referred to interchangeably herein as stress proteins, useful in the treatment and prevention of cancer, can be selected from among any cellular protein that satisfies any one of the following criteria. It is a protein whose intracellular concentration increases when a cell is exposed to a stressful stimuli, it is capable of binding other proteins or peptides, and it is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH; or it is a protein showing at least 35% homology with any cellular protein having any of the above properties.
- ATP adenosine triphosphate
- hsp60, hsp70, hsp90 and protein disulfide isomerase families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress. Therefore it is contemplated that the definition of stress or heat shock protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75% to 85% amino acid identity with members of these families whose expression levels in a cell are enhanced in response to a stressful stimulus.
- Such recombinant hsps may also be fused to a heterologous polypeptide, such as an i munoglobulin constant region, which can facilitate purification of the noncovalent complex.
- the genetically engineered host cells may contain one or more copies of a nucleic acid sequence comprising a sequence that encodes a hsp, operably associated with regulatory region (s) that drive expression of the hsp nucleic acid sequence in the host cell. Any nucleic acid sequence encoding a hsp, including cDNA and genomic DNA, can be used. It is preferred that the recombinant hsp produced in the host cell or library cell is of the same species as the intended recipient of the immunogenic composition. Recombinant human hsp is most preferred. 5.1. Preparation of cancer cDNA Library
- Described herein are methods for the construction of a cancer cDNA library. Specifically described are the making of complementary DNA (cDNA) from cancer cell RNA, the insertion of cDNAs into an appropriate cloning vector, and the introduction of the cloned cDNAs into an appropriate host organism for propagation of the cancer cDNA library and/or for the production of hsp-peptide complexes.
- cDNA complementary DNA
- RNA isolated from cancer cells is further purified before conversion into complementary DNA 0 (cDNA) .
- cDNA complementary DNA 0
- mRNA messenger RNA
- poly- A poly (adenylic) acid
- Total RNA is 5 denatured to expose the poly-A tails.
- Poly-A+ RNA is then bound to oligo-dT cellulose, with the remainder of the RNA washing through. The poly-A+ RNA is eluted by removing salt from the solution.
- RNA enriched for poly-A+ RNA may be repeated to further enrich for messenger RNA.
- oligo-dT 0 matrices in different configurations may also be used, including but not limited to, simple gravity columns, paramagnetic particles, and spin columns.
- Substituted oligo-dT such as biotinylated oligo-dT, may also be used.
- the quantity and quality of RNA thus obtained may be determined 5 by methods such as formaldehyde agarose gel electrophoresis. The use of RNA enriched for poly-A+ RNA is most preferred.
- the first step in the making of cDNA involves the 5 oligonucleotide-primed synthesis of a first strand cDNA by a reverse transcriptase.
- a reverse transcriptase such as AMV reverse transcriptase, MMLV reverse transcriptase, or Superscript (Kotewicz et al . , 1988, Nucleic Acid Res. 16:265-277). Random hexamers may be used to prime first-strand synthesis from internal sites within the mRNA instead of oligo-dT primers resulting in shorter cDNAs which are enriched for the 5 • ends of long messenger RNAs.
- linkers or adaptors providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (Wu et al . , 1987, Methods in Enzymol 152:343-349). Cleavage with a restriction enzyme can be followed by- modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation.
- Such cDNAs having a desired site, such as EcoRI, at the 5' end and an Xhol site at the 3' end can be cloned unidirectionally into a vector such that the 5' end of the cDNAs are consistently positioned downstream from a promoter.
- cancer cDNAs or expression constructs comprising cancer cDNAs can be inserted into an expression vector for propagation and expression in host cells as described below.
- a potential drawback of a prokaryotic host-vector system is the inability to perform many of the post-translational processing of mammalian cells.
- an eukaryotic host- vector system is preferred, a mammalian host-vector system is more preferred, and a human host-vector system is the most preferred.
- the proteins of the cancer cells can be coordinately expressed with heat shock proteins or stress proteins of the recombinant host cell by exposure to the appropriate stress, such as high temperature.
- the efficiency of expression of the cancer cDNA in a host cell may be enhanced by the inclusion of appropriate transcription enhancer elements in the expression vector, such as those found in SV40 virus. Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ - actin (see Bittner et al . , 1987, Methods in Enzymol. 153:516- 544; Gorman, 1990, Curr. Op. in Biotechnol. 1:36-47).
- a number of viral-based expression systems may also be utilized with mammalian cells to make the cancer cDNA libraries.
- Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) (Hamer et al . , 1979, Cell 17:725), adenovirus (Van Doren et al . , 1984, Mol Cell Biol 4:1653), adeno-associated virus (McLaughlin et al . , 1988, J Virol 62:1963), and bovine papillomas ⁇ virus (Zinn et al . ,
- SV40 simian virus 40
- adenovirus Van Doren et al . , 1984, Mol Cell Biol 4:1653
- adeno-associated virus McLaughlin et al . , 1988, J Virol 62:1963
- bovine papillomas ⁇ virus Zainn et al .
- Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al . , 1990, DNA Prot Eng Tech 2:14-18); pDR2 and ⁇ DR2 (available from Clontech Laboratories) .
- the expression vector pDR2 carries the EBV origin which confers stable episo al maintenance to the vector when activated by EBNA-1. Extremely high transfection efficiencies up to 10 "1 can be obtained when pDR2 is transfected into cell lines which express EBNA-1.
- Host cells can be rendered proficient for high-efficiency transfections by first transfecting the host cells with an expression construct that produces EBNA-1.
- Retroviruses such as Moloney murine leukemia virus
- Moloney murine leukemia virus can be used since most of the viral gene sequence can be removed and replaced with cancer cDNA while the missing viral functions can be supplied in trans.
- retroviruses can efficiently infect and transfer genes to a wide range of cell types including, for example, primary" hematopoietic cells.
- the host range for infection by a retroviral vector can be manipulated by the choice of envelope used for vector packaging.
- yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast) , Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts) .
- Autographa californica nuclear polyhydrosis virus (AcNPV) a baculovirus
- AcNPV Autographa californica nuclear polyhydrosis virus
- the cancer cDNA sequences may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) .
- AcNPV promoter for example the polyhedrin promoter
- Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, such as Chinese hamster ovary (CHO) cells, NIH/3T3, COS, HeLa, Daudi, 293, 293-EBNA, VERO, etc. (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990) .
- rodents such as Chinese hamster ovary (CHO) cells, NIH/3T3, COS, HeLa, Daudi, 293, 293-EBNA, VERO, etc.
- ⁇ DR2 uses the lox P mediated site-specific recombination to excise the expression vector pDR2 containing a cDNA insert from lambda clones which can recircularize to generate a plasmid.
- the plasmid pDR2 contains eukaryotic regulatory regions based on the Epstein- Barr virus and selection markers that allows direct introduction of the cDNA inserts as a library into permissive human host cells at high efficiency.
- Expression constructs containing cloned cancer cDNA can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al . , 1977, Cell 11:223-232), liposome-mediated transfection (Schaefer-Ridder et al . , 1982, Science 215:166- 168), electroporation (Wolff et al . , 1987, Proc Natl Acad Sci 84:3344), and microinjection (Cappechi, 1980, Cell 22:479- 488) .
- Cell lines that stably express hsp-peptide complexes may be engineered by using a vector that contains a selectable marker.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines.
- the recombinant host cells may be cultured under standard conditions of temperature, incubation time, optical density, and media composition.
- the recombinant host cells may be cultured under conditions emulating the nutritional and physiological requirements of the cancer cell from which the cancer cDNA was derived.
- conditions for maintenance and production of a cancer cDNA ] ibrary may be different from those for expression of antigenic proteins or peptides.
- Modified culture conditions and media may also be used to enhance production of hsp-peptide complexes.
- the host cells containing cancer cDNA may be exposed to heat or other environmental stress, or chemical stress prior to purification of the hsp-peptide complexes. Any techniques known in the art may be applied to establish the optimal conditions for producing hsp-peptide complexes.
- the protocols described hereinbelow may be used to recover and purify hsp-peptide complexes from any mammalian cells, for example, human cells, containing an expression construct comprising a cancer cDNA. 5.2.1. Preparation and Purification of Hsp90-peptide Complexes
- the cells may be lysed by mechanical shearing and 5 in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, ImM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
- the lysate is centrifuged at l,000g for 10 0 minutes to remove unbroken cells, nuclei and other cellular debris.
- the resulting supernatant is recentrifuged at 100,000g for 90 minutes, the supernatant harvested and then mixed with Con A SEPHAROSETM equilibrated with phosphate ' buffered saline (PBS) containing 2mM Ca 2+ and 2mM Mg 2+ .
- PBS phosphate ' buffered saline
- the supernatant is diluted with an equal volume of 2X lysis buffer prior to mixing with Con A SEPHAROSETM.
- the supernatant is then allowed to bind to the Con A SEPHAROSETM for 2-3 hours at 4°C.
- the material that fails to bind is harvested and dialyzed for _ 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, O.lmM EDTA, lOmM NaCl, ImM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q FPLCTM ion exchange 5 chromatography column equilibrated in 20mM Tris-Acetate pH 7.5, 20mM NaCl, O.lmM EDTA and 15mM 2-mercaptoethanol.
- the column is then developed with a 20mM to 500mM NaCl gradient and then eluted fractions fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized by immunoblotting using an appropriate anti- hsp70 antibody (such as from clone N27F3-4, from StressGen) .
- an appropriate anti- hsp70 antibody such as from clone N27F3-4, from StressGen
- the resulting precipitate is then harvested by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium sulfate.
- the washed precipitate is then solubilized and any residual ammonium sulfate removed by gel filtration on a SEPHADEXTM G25 column (Pharmacia).
- the hsp70 preparation thus obtained can be repurified through the Mono Q FPCL Column as described above.
- the hsp70-peptide complex can be purified to apparent homogeneity using this method.
- the supernatant is collected and the buffer is changed to buffer D (20mM Tris-acetate, 20mM NaCl, 15 mM B-mercaptoethanol, 3mM MgCl 2 , 0.5mM PMSF, pH7.5) by passing through a PD-10 column (SEPHADEXTM G-25, Pharmacia Biotech., Piscataway, NJ) .
- the sample is applied directly to an ADP-agarose column (Sigma Chemical Co. , St. Louis, MO) (5ml) equilibrated with buffer D.
- the column is washed with buffer D containing 0.5M NaCl and then buffer D alone until no more protein could be detected by the Bradford protein assay (BioRad, Richmond, California) .
- IX Lysis buffer consisting of 5mM sodium phosphate buffer (pH 7) , 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and
- the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
- the cells may be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, ImM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
- the eluted fractions are fractionated by SDS-PAGE and fractions containing the hsp90-peptide complexes are identified by immunoblotting using an anti-hsp90 antibody, such as 3G3 (Affinity Bioreagents) .
- An apparently homogenous preparation of hsp90-peptide complexes can be obtained using this procedure.
- 150-200 ⁇ g of hsp90-peptide complex can be purified from lg of cells or tissue.
- a pellet of recombinant host cells is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and ImM PMSF and the cells allowed to swell on ice 20 minutes.
- the cell pellet is then homogenized in a Dounce homogenizer (the appropriate clearance of the homogenizer will vary according to each cells type) on ice until >95% cells are lysed.
- the lysate is centrifuged at 1,000 g for 10 minutes to remove unbroken cells, nuclei and other debris.
- the supernatant from this centrifugation step then is recentrifuged at 100,000 g for 90 minutes.
- the gp96-peptide complex can be purified either from the pellet after centrifugation at 100,000 g or from the supernatant.
- the supernatant When purified from the supernatant, the supernatant is diluted with equal volume of 2X lysis buffer and the supernatant mixed for 2-3 hours at 4°C with Con A SEPHAROSETM equilibrated with PBS containing 2mM Ca 2+ and 2mM Mg 2+ . Then, the slurry is packed into a column and washed with IX lysis buffer until the OD 280 drops to baseline. Then, the column is washed with 1/3 column bed volume of 10% ⁇ -methyl mannoside ( ⁇ -MM) dissolved in PBS containing 2mM Ca 2+ and 2mM Mg 2+ , the column sealed with a piece of parafilm, and incubated at 37°C for 15 minutes.
- ⁇ -MM ⁇ -methyl mannoside
- the column is cooled to room temperature and the parafilm removed from the bottom of the column.
- Five column volumes of the ⁇ -MM buffer are applied to the column and the eluate analyzed by SDS-PAGE. Typically the resulting material is about 60-95% pure, however this depends upon the cell type and the tissue-to-lysis buffer ratio used.
- the sample is applied to a Mono Q FPLCTM ion exchange chromatography column (Pharmacia) equilibrated with a buffer containing 5mM sodium phosphate, pH 7.
- the proteins then are eluted from the column with a 0-1M NaCl gradient and the gp96 fraction elutes between 400mM and 550mM NaCl.
- the procedure may be modified by two additional steps, used either alone or in combination, to consistently produce apparently homogeneous gp96-peptide complexes.
- One optional step involves an ammonium sulfate precipitation prior to the Con A puri ication step and the other optional step involves DEAE-SEPHAROSETM purification after the Con A purification step but before the Mono Q FPLCTM ion exchange chromatography step.
- the supernatant resulting from the 100,000g centrifugation step is brought to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate.
- the ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water.
- the solution is stirred from about 1/2 to 12 hours at 4°C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor).
- the supernatant resulting from this step is removed, brought to 70% ammonium sulfate saturation by the addition of ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor).
- the gp96 containing fractions eluted from the Con A column are pooled and the buffer exchanged for 5mM sodium phosphate buffer, pH 7, 300mM NaCl by dialysis, or preferably by buffer exchange on a SEPHADE5CTM G25 column.
- the solution is mixed with DEAE-SEPHAROSETM previously equilibrated with 5mM sodium phosphate buffer, pH 7, 300mM NaCl.
- the protein solution and the beads are mixed gently for 1 hour and poured into a column. Then, the column is washed with 5mM sodium phosphate buffer, pH 7, 300mM NaCl, until the absorbance at 280nM drops to baseline.
- the pellet When the gp96 fraction is isolated from the 100,000g pellet, the pellet is suspended in 5 volumes of PBS containing either 1% sodium deoxycholate or 1% oxtyl glucopyranoside (but without the Mg 2+ and Ca 2+ ) and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg 2+ and Ca 2+ ) to remove the detergent. The dialysate is centrifuged at 100,000g for 90 minutes, the supernatant harvested, and calcium and magnesium are added to the supernatant to give final concentrations of 2mM, respectively. Then the sample is purified by either the unmodified or the modified method for isolating gp96-peptide complex from the 100,000g supernatant, see above.
- the gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10 to 20 ⁇ g of gp96 can be isolated from lg cells or tissue.
- a pellet of recombinant host cells is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and ImM PMSF and the cells allowed to swell on ice 20 minutes.
- the cell pellet is then homogenized in a Dounce homogenizer (the appropriate clearance of the homogenizer will vary according to each cells type) on ice until >95% cells are lysed.
- the lysate is centrifuged at 1,000 g for 10 minutes to remove unbroken cells, nuclei and other debris.
- the supernatant from this centrifugation step then is recentrifuged at 100,000 g for 90 minutes.
- the supernatant is brought to a final concentration of 80% ammonium sulfate by the addition of ammonium sulfate.
- the ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water. The solution is stirred from about 1/2 to 12 hours at 4°C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor).
- the resulting pellet is harvested and dissolved in PBS containing 2mM Ca 2+ and 2mM Mg 2+ (2X lysis buffer) .
- the solution is mixed for 2-3 hours at 4°C with Con A SEPHAROSETM equilibrated with PBS containing 2mM Ca 2+ and 2mM Mg 2+ and the slurry is packed into a column and washed with IX lysis buffer until the OD 280 drops to baseline.
- the flow-through is collected and passed through a PD-10 column (Pharmacia) to change the buffer to 0.025M sodium citrate (pH 5.1).
- the solution is loaded onto a CM-Sephadex 50 (Pharmacia) cation-exchange chromatographic column, and the flow-through is collected.
- the purified stress protein-peptide complexes can be assayed for immunogenicity using the mixed lymphocyte target culture assay (MLTC) well _ known in the art.
- MLTC mixed lymphocyte target culture assay
- mice are injected subcutaneously with the candidate stress protein-peptide complexes.
- Other mice are injected with either other stress _ protein-peptide complexes from normal, non-recombinant cells or whole infected cells which act as positive controls for the assay.
- the mice are injected twice, 7-10 days apart.
- Ten days after the last immunization the spleens are removed and the lymphocytes released.
- the released lymphocytes may 0 be restimulated subsequently in vitro by the addition of dead cells that expressed the complex of interest.
- 8xl0 6 immune spleen cells may be stimulated with 4xl0 4 mitomycin C treated or ⁇ -irradiated (5- 10,000 rads) infected cells (or cells transfected with an 5 appropriate gene, as the case may be) in 3ml RPMI medium containing 10% fetal calf serum. In certain cases 33% secondary mixed lymphocyte culture supernatant may be included in the culture medium as a source of T cell growth factors (See, Glasebrook et al . , 1980, J. Exp. Med. 151:876). To test the primary cytotoxic T cell response after immunization, spleen cells may be cultured without 5 stimulation. In some experiments spleen cells of the immunized mice may also be restimulated with antigenically distinct cells, to determine the specificity of the cytotoxic T cell response.
- the target cells are prelabelled by incubating lxlO 6 target cells in culture medium containing 200 mCi 51 Cr/ml for one hour at 37 °C. The cells are washed three times following labeling. Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to
- the percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided by the total detergent released cpm minus spontaneously released cpm.
- Preparations for mucosal administrations are suitable in various formulations as described below.
- the route of administration can be varied during a course of treatment.
- Preferred dosages, routes of administration and therapeutic regimens are described herein, and in PCT International patent applications published as WO 96/10411 and WO 97/10001, incorporated by reference herein in their entireties.
- the complex may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
- an appropriate buffer for example, phosphate buffered saline or other physiologically compatible solutions.
- the resulting complex may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
- the noncovalent complexes and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
- the complexes may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the complexes may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the complexes may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the noncovalent complexes.
- the pack may for example comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of the noncovalent hsp- peptide complexes in pharmaceutically acceptable form.
- the hsp-peptide complexes in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
- preferred methods of treatment or prevention of cancer comprise isolating RNA molecules from cancer cells obtained from one or more individual, preferably the individual in need of treatment, and making cDNA molecules from the RNA molecules isolated therefrom.
- the cancer cDNAs are manipulated by methods described above in section 5.i, such that the cDNA molecules, in the form of an expression construct, or intrachromosomally integrated, are suitable for expression of the cDNA molecules in one or more preselected host cells.
- the recombinant host cells containing the cDNA molecules are cultured under conditions such that peptides encoded by the cDNA molecules are expressed by the recombinant host cells.
- the invention provides a method of treating or preventing cancer in an individual in whom treatment or prevention of cancer is desired comprising the steps of : (a) culturing host cells containing cDNA molecules made from RNA molecules of cancer tissue obtained from one or more human individuals, under conditions such that proteins encoded by the cDNA molecules are expressed by the host cells;
- the present invention provides a method of treating an individual having cancer comprising the following steps:
- Cancers that can be treated or prevented by using noncovalent hsp-peptide complexes prepared by the methods of the present invention include, but not limited to human sarcomas and carcinomas, e.g. , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma , osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma , lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma , colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcino a, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcino
- the cancer is metastatic.
- the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e.g., chemotherapy radiation) prior to administration of the hsp-peptide molecule complexes of the invention.
- the cancer is a tumor .
- the effect of immunotherapy with hsp-peptide complexes on progression of neoplastic diseases can be monitored by any methods known to one skilled in the art, including but not limited to measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g.> carcinoembryonic (CEA) antigens; d) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; e) changes in levels of putative biomarkers of risk for a particular cancer in individuals at high risk, and f) changes in the morphology of tumors using a sonogram. Other techniques that can also be used include scintigraphy and endoscopy.
- the cultures are tested for cytotoxity in a 4 hour 51 Cr-release assay.
- the spontaneous 51 Cr-release of the targets should reach a level less tha'n 20%.
- a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of 12.5% (Heike et al . , 1994, J. Immunotherapy 15:165-174) .
- cytokine release is detected by antibodies which are specific for a particular cytokine, such as interleukin-2 , tumor necrosis fact.or ⁇ or interferon-7 (for example, see Scheibenbogen et al . , 1997, Int. J. Cancer, 71:932-936).
- the assay is carried out in a microtitre plate which has been pre-coated with an antibody specific for a cytokine of interest which captures the cytokine secreted by T cells.
- the cytotoxic T cells are removed and replaced with a second labelled antibody that recognizes a different epitope on the cytokine. After extensive washing to remove unbound antibody, an enzyme substrate which produces a colored reaction product is added to the plate. The number of cytokine-producing cells is counted under a microscope.
- This method has the advantages of short assay time, and sensitivity without the need of a large number of cytotoxic T cells.
- the immune response in subjects after immuninization can be assessed by an IFN- ⁇ ELISPOT assay kit (Mabtech, Sweden) as follows.
- Blood samples (20 ml) drawn from patients are heparinized, and peripheral blood mononuclear cells are separated from the blood samples by Ficoll (Pharmacia) gradient centrifugation.
- the separated blood cells may be stored in aliquots of 5xl0 6 cells at -130°C.
- CD8+ T cells are isolated from the blood cells by standard techniques, such as the use of magnetic beads (Dynal) .
- HA-Multiscreen plates (Millipore) are coated with 100 ⁇ l of mouse-anti-human IFN-7 antibody (10 ⁇ g/ml) .
- CD8+ T cells are plated at a concentration of 8xl0 4 cells/well.
- Tumor cells (5xl0 4 cells/Veil) or tumor cell membranes (5xl0 7 cell equivalents/well) are added and incubated for 20 hours at 37 °C. After the incubation, the cells are removed by extensive washing with PBS/ 0.05% Tween 20, and 100 ⁇ l of biotinylated capture-antibody against human IFN- ⁇ are added at a concentration of 2 ⁇ g/ml. Spot development is performed by standard techniques and spots are counted using a stereomicroscope at a 40-fold magnification. Each spot corresponds to one CD8+ T cell secreting IFN-7.
- the preventive effect of immunotherapy using hsp- peptide complexes may also be estimated by determining levels of a putative biomarker for risk of a specific cancer.
- serum prostate-specific antigen PSA
- CEA serum prostate-specific antigen
- CEA is measured by methods known in the art
- 16- ⁇ -hydroxylation of estradiol is measured by the procedure described by Schneider et al . , 1982, Proc. Natl. Acad. Sci. USA 79:3047-3051.
- EXAMPLE TREATMENT OF HEPATOCELLULAR CARCINOMA
- a liver biopsy sample (approximately 10 6 -10 7 cells, or 200 mg to 1 g of tissue) obtained from a patient with hepatocellular carcinoma is lysed gently in the presence of guanidinium isothiocyanate and phenol/chloroform. After centrifugation of the cell lysate, 1-2 mg of total RNA is extracted from about 1 g of tissue, and precipitated in isopropanol.
- Total poly-A + RNA is isolated from total RNA by column chromatography using commercially available prepacked oligo-dT cellulose spin columns (Clontech Laboratories, CA) . Approximately 50 ⁇ g of poly-A + RNA is yielded from 1 mg of total RNA.
- a commercial cDNA synthesis kit is used to make cDNA molecules from the poly-A + RNA with the approriate termini which is cloned unidirectionally into the BamHI/Xbal sites of the vector ⁇ DR2 (Clontech Laboratories, CA) .
- This cloning vector contains an embedded version of the Epstein- Barr virus shuttle vector pDR2 which provides the elements for stable gene expression in permissive human cell lines.
- the 5* end of the cDNAs are inserted at the BamHI site which is downstream of the promoter of the Rous sarcoma virus long terminal repeat (RSV LTR) for expression in human cells.
- the cloned cDNAs are packaged in vitro using commercially available E.
- the cloned cDNAs are converted into pDR2 plasmids carrying the expression constructs.
- the expression plasmids are purified from E . coli cells, and transfected into 293-EBNA cells (Invitrogen, CA) , a human cell line constitutively expressing the neomycin resistance gene and the Epstein-Barr virus nuclear antigen which confers stable episomal maintenance of the expression plasmid. About 10-20 copies of the plasmid is maintained per cell and expression of the cloned cDNAs are driven by the RSV LTR promoter.
- the transfected human cells are cultured in batches or continuously to allow recombinant production of the cloned cDNAs.
- Heat shock protein-peptide complexes are purifed from the recombinant cells as described in Section 5.2.
- hsp-antigen complexes prepared as described above Treatment with hsp-antigen complexes prepared as described above is started any time after surgery. However, if the patient has received chemotherapy, hsp-antigen complexes are usually administered after an interval of four weeks or more so as to allow the immune system to recover. The immunocompetence of the patient is tested by procedures described in sections 5.9 above.
- the therapeutic regimen includes weekly injections of the hsp-antigen complex, dissolved in saline or other physiologically compatible solution. ' ⁇ ⁇
- the dosage used for hsp70 or gp96 is in the range of 10 to 600 micrograms, with the preferred dosage for a human patient being 10 to 100 micrograms.
- the dosage used for hsp90 is in the range of 50 to 5,000 micrograms, with the preferred dosage for a human patient being 50 to 200 micrograms, e.g., 100 micrograms.
Abstract
Description
Claims
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CA002314005A CA2314005A1 (en) | 1997-12-11 | 1998-12-11 | Methods for preparation of vaccines against cancer |
AU19106/99A AU728036B2 (en) | 1997-12-11 | 1998-12-11 | Methods for preparation of vaccines against cancer |
JP2000524407A JP2001526025A (en) | 1997-12-11 | 1998-12-11 | Method for preparing vaccine against cancer |
EP98963869A EP1037965A1 (en) | 1997-12-11 | 1998-12-11 | Methods for preparation of vaccines against cancer |
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US08/988,878 US5948646A (en) | 1997-12-11 | 1997-12-11 | Methods for preparation of vaccines against cancer comprising heat shock protein-peptide complexes |
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WO2000033861A2 (en) * | 1998-12-04 | 2000-06-15 | Pharmaproducts Uk Limited | Pharmaceutical compositions containing protein-disulfide isomerases |
WO2001014411A1 (en) * | 1999-08-19 | 2001-03-01 | Minister For Agriculture, Minister For Land And Water Conservation For And On Behalf Of The State Of New South Wales | Recombinant subunit vaccine |
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US7666581B2 (en) | 2001-08-20 | 2010-02-23 | University Of Connecticut Health Center | Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease |
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US7595192B2 (en) | 2002-08-16 | 2009-09-29 | Glycotype Gmbh | Process for the production of temperature-induced tumor cell lysates for use as immunogenic compounds |
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US10280230B2 (en) | 2006-09-10 | 2019-05-07 | Glycotope Gmbh | Use of human cells of myeloid leukemia origin for expression of antibodies |
US9494587B2 (en) | 2006-11-10 | 2016-11-15 | Glycotope Gmbh | Microorganisms or fractions thereof capable of activating cellular immunity against carbohydrates |
US8592165B2 (en) | 2006-11-10 | 2013-11-26 | Glycotope Gmbh | Carbohydrate specific cellular immunity inducing microorganisms and fractions thereof |
US9700610B2 (en) | 2011-08-22 | 2017-07-11 | Glycotope Gmbh | Microorganisms carrying a tumor antigen |
WO2013037714A1 (en) * | 2011-09-12 | 2013-03-21 | The Provost, Fellows, Foundation Scholars, And The Other Members Of Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin | BIOMARKERS FOR BREAST TUMOURS FROM Hsp70-ASSOCIATED PEPTIDES |
US11872289B2 (en) | 2018-05-18 | 2024-01-16 | Daiichi Sankyo Co., Ltd. | Anti-MUC1 antibody-drug conjugate |
Also Published As
Publication number | Publication date |
---|---|
EP1037965A1 (en) | 2000-09-27 |
JP2001526025A (en) | 2001-12-18 |
US5948646A (en) | 1999-09-07 |
US6406700B1 (en) | 2002-06-18 |
AU1910699A (en) | 1999-06-28 |
US6410027B1 (en) | 2002-06-25 |
CA2314005A1 (en) | 1999-06-17 |
AU728036B2 (en) | 2001-01-04 |
US6410026B1 (en) | 2002-06-25 |
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