TWI398262B - Immunogenic peptides of tumor associated antigen l6 and uses thereof in cancer therapy - Google Patents

Description

Immunopeptides of tumor-associated antigens and their use in cancer therapy

The present invention relates to an immunopeptide comprising a T cell epitope derived from a tumor associated antigen L6 (TAL6), and a nucleic acid encoding the same.

Related application

The present invention claims priority to U.S. Application Serial No. 12/846,092, filed on Jan. 29, 2010, the disclosure of which is hereby incorporated by reference.

Background of the invention

The tumor associated antigen L6 (TAL6) belongs to a member of the transmembrane-4 superfamily (TM4SF), a tumor marker antigen that is expressed on a variety of different cancer cells, such as lung cancer, breast cancer, colorectal cancer, and ovarian cancer cells. Previous studies have found that anti-TAL6 antibodies produce antibody-dependent cytotoxicity against cancer cells expressing TAL6 and inhibit tumor growth in nude mice, indicating that TAL6 is a target antigen that can be used in cancer immunotherapy.

A cancer vaccine based on the poisonous T lymphocyte (CTL) epitope is a viable anticancer drug. Such cancer vaccines contain a CTL-epitope derived from a tumor target antigen that elicits a toxic T cell response against a tumor cell expressing the target antigen, thereby destroying the tumor cell. For the development of cancer vaccines based on CTL epitopes, it is necessary to identify their CTL-epitope from tumor target antigens.

In one aspect, the invention features an isolated immunopeptide having up to 50 amino acids comprising an amino acid sequence derived from a T cell epitope of a tumor associated antigen L6 (TAL6), or encoding such an encoding The nucleic acid of an immunopeptide.

The term "isolated peptide" as used herein means a peptide which is substantially free of natural association molecules, i.e., the natural association molecule accounts for up to 20% by dry weight of the formulation containing the peptide. Purity can be measured by any suitable method, such as column chromatography, polypropylene gel electrophoresis, HPLC, and the like.

In one example, the immunopeptide comprises an HLA-A2-specific T-cell epitope, such as CIGHSLVGL (SEQ ID NO: 1), SLVGLALLC (SEQ ID NO: 2), ALLCIAANI (SEQ ID NO: 3), LLMLLPAFV (SEQ ID NO: 4), MLLPAFVFI (SEQ ID NO: 5), AMLSSVLAA (SEQ ID NO: 6), MLSSVLAAL (SEQ ID NO: 7), SVLAALIGI (SEQ ID NO: 8), GLAEGPLCL (SEQ ID NO: 9), HIVEWNVSL (SEQ ID NO: 10), SILLALGGI (SEQ ID NO: 11), ALGGIIFIL (SEQ ID NO: 12) or VINGVLGGI (SEQ ID NO: 13). In another example, the immunopeptide comprises an HLA-A24-specific T-cell epitope, such as CYGKCARCI (SEQ ID NO: 14), HSLVGLALL (SEQ ID NO: 15), LYFPNGETKY (SEQ ID NO: 16), KYASENHLS (SEQ ID NO: 17), RFVWFFSGI (SEQ ID NO: 18), FFSGIVGGGL (SEQ ID NO: 19), GYCVIVAAL (SEQ ID NO: 20), TFASTEGQYL (SEQ ID NO: 21), QYLLDTSTW (SEQ ID NO) :22) and EWNVSLFSI (SEQ ID NO: 23).

In addition to the TAL6-derived T-cell epitope, the immunopeptide of the invention may further comprise a T helper cell stimulating fragment, such as QYIKANSK FIGITE (SEQ ID NO: 24) or AKFVAAWTLK (SEQ ID NO: 25), and/or An endoplasmic reticulum target sequence (eg, MRYMILGLLALAAVCSA; SEQ ID NO: 26).

In another aspect, the invention features an immunological composition comprising any of the foregoing immunopeptides, a pharmaceutically acceptable carrier and, optionally, an adjuvant. The immunological composition can be used to treat cancer (eg, lung cancer, breast cancer, colon cancer, or ovarian cancer), or to enhance an immune response against TAL6.

In yet another aspect, the invention features an antibody that specifically binds to a TAL6 fragment having an amino acid sequence selected from the group consisting of CIGHSLVGL (SEQ ID NO: 1), SLVGLALLC (SEQ ID NO: 2), ALLCIAANI (SEQ ID NO: 3), LLLMLAFAV (SEQ ID NO: 4), MLLPAFVFI (SEQ ID NO: 5), AMLSSVLAA (SEQ ID NO: 6), MLSSVLAAL (SEQ ID NO: 7), SVLAALIGI (SEQ ID NO: 8), GLAEGPLCL (SEQ ID NO: 9), HIVEWNVSL (SEQ ID NO: 10), SILLALGGI (SEQ ID NO: 11), ALGGIIFIL (SEQ ID NO: 12), VINGVLGGI (SEQ ID NO: 13), CYGKCARCI ( SEQ ID NO: 14), HSLVGLALL (SEQ ID NO: 15), LYFPNGETKY (SEQ ID NO: 16), KYASENHLS (SEQ ID NO: 17), RFVWFFSGI (SEQ ID NO: 18), FFSGIVGGGL (SEQ ID NO: 19) ), GYCVIVAAL (SEQ ID NO: 20), TFASTEGQYL (SEQ ID NO: 21), QYLLDTSTW (SEQ ID NO: 22), and EWNVSLFSI (SEQ ID NO: 23).

The scope of the present invention also encompasses (1) a pharmaceutical composition for treating cancer or enhancing an immune response against TAL6, the pharmaceutical composition comprising any of the aforementioned immunopeptides, or a performance vector for producing the peptide, and (2) The use of the immunopeptide or the expression carrier for producing the same for the manufacture of the desired drug.

The details of one or more specific aspects of the invention are exemplified in the following embodiments. Other features and advantages of the present invention will be apparent from the following description and drawings.

The invention features an immunopeptide having up to 50 amino acid residues. The immunopeptide comprises a T-cell epitope (eg, a CTL epitope) derived from TAL6 and specific for a particular HLA allele, such as HLA-A2 or HLA-A24. A T cell epitope refers to a peptide that is capable of activating T cells and subsequently eliciting an immune response mediated by activated T-cells. A CTL epitope refers to a peptide capable of activating CTL (also known as Tc or killer T cells), which in turn stimulates a CTL response, ie, including abnormal cells (eg, by viral infection or tumor cells). The CTL epitope (representatively comprising an 8-11 amino acid residue) forms a complex with a specific class I MHC molecule (including heavy chain and β2 microglobulin) present on the surface of the antigen-presenting cell. When the complex binds to a T cell receptor of a T cell (e.g., CD8 T cell), it activates the T cell and then initiates a CTL response.

TAL6 is a known tumor target antigen that is expressed on a variety of different cancer cells. The amino acid sequence is shown below (SEQ ID NO: 30): Met Cys Tyr Gly Lys Cys Ala Arg Cys Ile Gly His Ser Leu Val Gly Leu Ala Leu Leu Cys Ile Ala Ala Asn Ile Leu Leu Tyr Phe Pro Asn Gly Glu Thr Lys Tyr Ala Ser Glu Asn His Leu Ser Arg Phe Val Trp Phe Phe Ser Gly Ile Val Gly Gly Gly Leu Leu Met Leu Leu Pro Ala Phe Val Phe Ile Gly Leu Glu Gln Asp Asp Cys Cys Gly Cys Cys Gly His Glu Asn Cys Gly Lys Arg Cys Ala Met Leu Ser Ser Val Leu Ala Ala Leu Ile Gly Ile Ala Gly Ser Gly Tyr Cys Val Ile Val Ala Ala Leu Gly Leu Ala Glu Gly Pro Leu Cys Leu Asp Ser Leu Gly Gln Trp Asn Tyr Thr Phe Ala Ser Thr Glu Gly Gln Tyr Leu Leu Asp Thr Ser Thr Trp Ser Glu Cys Thr Glu Pro Lys His Ile Val Glu Trp Asn Val Ser Leu Phe Ser Ile Leu Leu Ala Leu Gly Gly Ile Glu Phe Ile Leu Cys Leu Ile Gln Val Ile Asn Gly Val Leu Gly Gly Ile Cys Gly Phe Cys Cys Ser His Gln Gln Gln Tyr Asp Cys.

The T cell epitope derived from TAL6 can be identified by the following method. Peptides spanning the entire TAL6 amino acid sequence (e.g., containing 8-12 amino acids) can be prepared by conventional methods (e.g., chemical synthesis). The effect specific to a particular HLA allele is determined by analysis known in the art. In one example, an MHC-peptide complex formation assay is performed as follows to determine the Class I HLA specific effect of the TAL6 peptide. The heavy chain and β2 microglobulin encoded by a Class I HLA allele (eg, HLA-A2 or HLA-A24) are expressed and purified. They are then mixed with any of the foregoing peptides and any MHC-peptide complex formed thereby can be detected by, for example, ELISA. It is well known that heavy chains encoded by a particular class I HLA allele form a complex with β2 microglobulin only in the presence of a peptide specific for such an HLA allele. Thus, the formation of an MHC-peptide complex means that the peptide contains an epitope specific for the class I HLA allele.

After determining that a TAL6 peptide is specific for a Class I HLA allele, it can then be analyzed in vitro or in vivo to determine if it contains a T cell epitope (eg, a CTL epitope). The following is an example of an in vitro assay. Humans with a Class I HLA allele are identified by genotyping using methods known in the art. His or her peripheral blood monolayers and cells (PBMC) are collected and exposed to the peptide in the presence of self antigen presenting cells. If the synthetic peptide activates PBMC, it indicates that the epitope contained therein is a T cell epitope specific for the class I HLA allele. In another embodiment, the in vivo assay described below is used to determine whether the peptide includes a T cell epitope. A transgenic mouse that expresses a class I HLA allele is immunized with the peptide. Induction of an immune response (eg, secretion of a cytokine such as IFN-γ and IL-2, or induction of cytotoxicity) indicates that the peptide contains a T cell epitope specific for a Class I HLA allele.

The immunopeptide of the present invention may further comprise a T helper cell stimulating fragment, such as QYIKANSKFIGITE (tetanus toxoid 830-843; SEQ ID NO: 24) or AKFVAAWTLK (PADRE peptide; SEQ ID NO: 25). Alternatively or additionally, it may further comprise an endoplasmic reticulum target sequence. This target sequence facilitates entry of a polypeptide containing it into a Class I antigen display pathway, wherein the T-cell epitope of the polypeptide can form a complex with a Class I HLA molecule. Examples of target sequences include the peptides MRYMILGLLALAAVCSA (SEQ ID NO: 26) and RYMILGLLALAAVCSA (SEQ ID NO: 27), both derived from adenovirus E3 protein. Other target sequences include, but are not limited to, the peptide MRAAGIGILTVAAAAAG (SEQ ID NO: 28, see Minev et al. 2000, Eur J Immunol . 30(8): 2115-24), and the peptide MAGILGFVFTLAAAAAG (SEQ ID NO: 29, See Gueguen et al., 1994, J Exp Med . 1994, 180(5): 1989-94).

The immunopeptide of the present invention can be obtained by a conventional method such as chemical synthesis of a polypeptide or recombinant technique. To prepare a recombinant peptide, the nucleic acid encoding the same can be ligated to another nucleic acid encoding a fusion partner, such as glutathione-S-transferase (GST), 6x-His tag or M13 gene 3 protein. The resulting fusion nucleic acid is expressed in a suitable host and the fusion protein can be isolated by a method known in the art. The isolated fusion protein can be further processed (e.g., by enzymatic decomposition) to remove the fusion partner and obtain the recombinant immunopeptide of the present invention. If the immunopeptide contains a T-cell epitope derived from TAL6 (a cancer target antigen), it can be used to enhance the fight against cancer, such as lung cancer, colon cancer, breast cancer, ovarian cancer, gastric cancer, Kaposi's sarcoma and liver cancer. The immune response (eg, CTL reaction). This composition is effective in treating cancer when administered to an individual, preferably with an HLA-A2, HLA-A24 or equivalent HLA allele. An HLA allele equal to HLA-A2 or HLA-A24 is an allele that cross-reacts with a peptide specific for HLA-A2 or HLA-A24. Examples include, but are not limited to, HLA-A3 and HLA-A11.

In order to treat cancer or enhance the immune response against TAL6 in an individual in need thereof, any of the aforementioned immunopeptides, or an expression carrier capable of expressing the immunopeptide, may be mixed with a pharmaceutically acceptable carrier to form an immunogen. Sexual composition (eg vaccine).

Immunopeptides may require chemical modification first because they may not have a significant long half-life. Chemically modified peptides or peptide analogs include any of the functional chemical equivalents of the peptide that are characterized in vivo or in vitro for the stability and/or efficacy of the practice of the present invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. Peptide analogs can be modified by, including but not limited to, side chains, incorporation of unnatural amino acids and/or derivatives thereof during peptide synthesis, and procedures using crosslinkers, with other peptides Or a method of forcing configuration limitation on or the like. Examples of side chain modifications include modification of an amine group, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ; benzimidination with methyl acetimimine; and with acetic anhydride B Deuteration; methylation of cyanate esters; trinitrobenzylation of amines with 2,4,6-trinitrobenzenesulfonic acid (TNBS); amines and succinic anhydrides and alkylation of action tetrahydrophthalic anhydride; and lysine with pyridoxal-5'-phosphate ester group of subsequent acts to NaBH ₄ reduction of pyridoxine. The thiol group of arginine can be modified by forming a heterocyclic condensation product with a reagent such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group can be modified by activation of carbodiimide formed by o-mercaptoisourea followed by derivatization (for example) in the corresponding guanamine. The sulfhydryl group can be carboxymethylated with, for example, iodoacetic acid or iodine with decylamine; oxidized with formic acid to cystine; forms a mixed disulfide with other thiol compounds; with maleic imine, maleic anhydride Or other substituted quinone imine reactions; use of 4-chloromercury benzoate, 4-chloromercuric acid, phenylmercuric chloride, 2-chloromercury-4-nitrophenol and other mercury-based compounds to form mercury radicals Derivatives; modification with cyanate esters at a basic pH for amine mercaptolation. The tryptophan residue can be oxidized, for example, with N-bromosuccinylamine, or its alkylation of an anthracene ring with a brominated 2-hydroxy-5-nitrobenzyl or sulfonyl halide. Modification. The tyrosine residue can be modified by the formation of a 3-nitrotyrosine derivative by digestion with tetranitromethane. The imidazole ring of the histidine residue can be modified by alkylation with an iodoacetic acid derivative or with N-carbon ethoxylation of diethyl dicarbonate. Examples of incorporation of non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, using n-leucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenyl Valeric acid, 6-aminohexanoic acid, t-butylglycine, n-proline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methyl D-isomer of heptanoic acid, 2-thienylalanine and/or amino acid.

The foregoing composition can be prepared by a conventional method. It comprises an immunopeptide/expression carrier of the invention, a pharmaceutically acceptable carrier such as a phosphate buffered saline solution, a bicarbonate solution and/or an adjuvant. When the immunopeptide does not include the T helper cell stimulating fragment as described above, such a fragment may be included in the composition to enhance the immune response. The choice of vector is based on the form and route of administration and standard pharmaceutical practice procedures. Suitable pharmaceutical carriers and diluents, as well as the pharmaceutically necessary ingredients for their use, are described in Remington's Pharmaceutical Sciences. If desired, adjuvants such as cholera toxin, E. coli thermolabic endotoxin (LT), liposomes, immunostimulating complex (ISCOM) or immunostimulatory sequence oligodeoxynucleotides (ISS-ODN) may also be included. In the composition of the present invention. The composition may also include a polymer that facilitates delivery in vivo. See Audran R. et al., Vaccine 21: 1250-5, 2003; and Denis-Mize et al. Cell Immunol ., 225: 12-20, 2003. In one example, the immunopeptide is a component of a multivalent vaccine composition against cancer. The multivalent composition contains at least one of the aforementioned immunopeptides, and at least one protective antigen isolated from influenza virus, parainfluenza virus 3, Streptococcus pneumoniae, B. sphaericus, S. aureus or respiratory syncytial virus With or without adjuvant. In another example, the immunopeptide is formulated as a virion that contains a functional viral outer membrane glycoprotein, such as influenza virus hemagglutinin (HA).

Methods for preparing vaccines are generally known in the art, as exemplified by U.S. Patent Nos. 4,601,903; 4,599,231; 4,599,230 and 4,596,792.

The vaccine can be prepared as an injectable, in the form of a liquid solution or emulsion. The immunopeptides of the invention can be combined with physiologically acceptable and compatible excipients. Excipients can include water, saline, dextrose, glycerol, ethanol, and combinations thereof. The vaccine may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents or adjuvants for enhancing the effectiveness of the vaccine. Methods for achieving the adjuvant efficacy of the vaccine include the use of a reagent such as aluminum hydroxide or aluminum phosphate (alumina), typically a solution of 0.05 to 0.1 percent dissolved in phosphate buffered saline. The vaccine can be administered parenterally, by subcutaneous or intramuscular injection. Alternatively, other forms of administration may be desired including suppositories and oral formulations. For suppositories, an adhesive and a carrier such as a polyalkylene glycol or a triglyceride may be included. Oral formulations may include excipients that are normally used, such as pharmaceutical grade saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of the immunopeptides described herein.

The aforementioned immunological composition may also be a dendritic cell-based vaccine containing dendritic cells of any of the immunopeptides described herein.

Methods for preparing dendritic cell-based vaccines are well known in the art. See Slingluff et al.Clin Cancer Res . 12 : 2342s-2345s, 2006; Buchsel et al.Clin J Oncol Nurs . 10 :629-40,2006; and Yamauchi et al.Expert Opin Biol Ther . 7 :645-9,2007.

An effective amount of the composition is administered to an individual (e.g., a human) via a conventional route for treating cancer or enhancing an immune response against TAL6-expressing cells. Exemplary routes of administration include, but are not limited to, oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or via an implanted container. The term "parenteral" is used herein to include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intra-abdominal, intrathecal, intralesional, and intracranial injection or immersion techniques. The term "effective amount" as used herein, refers to the amount of each active agent required to produce a therapeutic effect in an individual, either alone or in combination with one or more other active agents. The effective amount (as would be appreciated by those skilled in the art) will vary with the route of administration, the use of excipients, and the use of other active agents. The amount to be administered will depend on the individual being treated, including, for example, the individual's immune system synthesizing antibodies, and the ability to produce a cell-mediated immune response if desired. The amount of active ingredient required to be administered is determined by the judgment of the physician. However, a suitable dosage range can be readily determined by those skilled in the art and can be a polypeptide of the invention in the microgram range. The appropriate course of treatment for initial and additional doses may also vary, but may include initial administration followed by subsequent administration. The dose of the vaccine may also depend on the route of administration and will vary depending on the size of the host.

A cancer patient can be identified and administered to the aforementioned immunological composition. The dosage of the composition will depend, for example, on the particular immunopeptide, whether or not an adjuvant is co-administered with the immunopeptide, the type of co-administered adjuvant, the form and frequency of administration, as determined by those skilled in the art. If necessary, it can be decided by the skilled person to repeat the administration. For example, after the dose is initiated, three additional doses can be administered at weekly intervals. Additional injections may be given 4 to 8 weeks after the first immunization, and a second addition may be given between 8 and 12 weeks, using the same formulation. Serum or T cells can be collected from an individual for testing the immune response elicited by the immunological compositions described herein. Methods for analyzing cytotoxic T cells against a certain protein are well known in the art. Additional additions can be given if needed. The immune program can be optimized to maximize the anti-cancer immune response by varying the amount of immunopeptide, composition dose, and frequency of administration.

The immunopeptides of the invention can also be used to produce antibodies in animals (for the purpose of producing antibodies) or humans (for the purpose of treating diseases). Methods for making single and multiple antibodies and fragments thereof in animals are known in the art. See, for example, Harlow and Lane, (1988) Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact immunoglobulin molecules, antigen-binding fragments thereof (eg, Fab, F(ab') ₂ , Fv), and genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, and single chains. Antibodies and dAbs (Functional Domain Antibodies; Ward et al. (1989) Nature , 341 , 544). These antibodies can be used to detect cancers that exhibit TAL6, or for cancer treatment.

Typically, to make an antibody against a peptide, the peptide can be coupled to a carrier protein (e.g., KLH), mixed with an adjuvant, and injected into a host animal.

The antibody produced in the animal can then be purified by peptide affinity chromatography. Host animals commonly used include rabbits, mice, guinea pigs and rats. A variety of adjuvants that can be used to increase the immunological response are dependent on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, CpG, surfactants such as lysolecithin, pro-poly Pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin and dinitrophenol. Human adjuvants that can be used include BCG (Bacillus Calmette-Guerin) and Corynebacterium.

Multiple strains of antibodies (heterologous antibody molecule populations) are present in the serum of the immunized individual. Monoclonal antibodies (homologous antibody populations against the polypeptides of the invention) can be prepared using standard fusion knob techniques (see, for example, Kohler et al. (1975) Nature 256 , 495; Kohler et al. (1976) Eur. J. Immunol. 6 , 511; Kohler et al. (1976) Eur J Immunol 6 , 292; and et al. (1981) Monoclonal antibodies and T cell fusion tumors, Elsevier, NY). In particular, monoclonal antibodies can be made by any technique for making antibody molecules by continuous culture of cell lines, as described, for example, in Kohler et al. (1975) Ndture 256, 495 and U.S. Patent 4,376,110; Human B-cell fusion tumor technology. (Kosbor et al. (1983) Immunol Today 4 , 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026); and EBV-fused tumor technology (Cole et al. (1983) monoclonal antibody and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The fusion tumor producing the monoclonal antibody of the present invention can be cultured in vitro or in vivo. The ability to produce high-potency monoclonal antibodies in vivo can be a particularly useful manufacturing method.

Further, a technique developed to produce a "chimeric antibody" can be used. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. Nature 312, 604; and Takeda et al. (1984) Nature 314: 452. A chimeric antibody is one in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody, and a human immunoglobulin constant region. Alternatively, phage libraries of single chain Fv antibodies can be made using the techniques described for the manufacture of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692). The single-stranded anti-system is formed by bridging the heavy and light chain fragments of the Fv region via an amino acid. Moreover, antibody fragments can be produced by known techniques. For example, such fragments include, but are not limited to, F(ab') ₂ fragments which can be produced by pepsin decomposition of antibody molecules, and can be bridged by reduction of the disulfide of the F(ab') ₂ fragment. The resulting Fab fragment.

Antibodies can also be humanized by methods known in the art. For example, monoclonal antibodies having the desired binding activity can be humanized by commercial methods (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully human antibodies, such as those found in transgenic animals, are also characteristic of the invention (see, for example, Green et al. (1994) Nature Genetics 7 , 13; and U.S. Patent Nos. 5,545,806 and 5,569,825).

The invention also features a unicellular nucleic acid encoding an immunopeptide of the invention, comprising a vector which enables the expression of the immunopeptide. Such a nucleic acid can be used (as a DNA vaccine) for immunization by administering the nucleic acid directly to a subject via a living vector, such as a Salmonella, BCG, adenovirus, poxvirus, vaccinia virus or non-viral vector. Nucleic acid-based immunological methods are well known in the art.

The following specific examples are merely illustrative and are not intended to limit the remainder of the disclosure in any way. Without further elaboration, it is believed that those skilled in the art can use the invention to the fullest extent thereof. All publications cited herein are incorporated by reference in their entirety. Moreover, any mechanism proposed below does not limit the scope of the invention in any way.

Example 1: Identification of HLA-A2-specific T cell epitopes derived from TAL6 protein

Sixteen peptides (all derived from TAL6) were synthesized by chemical synthesis and are listed in Table 1 below. These peptides were subjected to MHC-peptide complex formation assays (described in U.S. Patent Application Serial No. 12/235,872) to identify those specific to HLA-A2 from among them.

Briefly, His-Tag-fused human HLA-A2 heavy chain (His-HLA-A2) and His-Tag-fused human β2-microglobulin (His-β2) were expressed in Escherichia coli according to the following procedure. , to obtain a His-Tag fusion protein. The expression vector encoding the fusion protein was introduced into E. coli BL2 (DE3). The resulting transformant was grown at 37 °C. When its O.D. value reached 0.5, 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added to the E. coli culture. After further culturing at 37 ° C for 3-4 hours, E. coli cells were harvested by centrifugation at 6000 rpm for 20 minutes. The cell pellet was suspended in Buffer A containing 8 M urea, 20 mM HEPES (pH 8.0) and 50 mM NaCl, and then subjected to ultrasonic oscillation. The ultrasonically oscillated cells were again centrifuged, and the resulting supernatant was collected and applied to a Ni-NTA-Sepharose column to purify His-HLA-A2 or His-β2.

His-A2, His-β2 and various peptides (test peptides) or positive control peptides listed in Table 1 above, together with 100 mM Tris-HCl (pH 8), 400 mM L-arginine, 2 The cells were incubated at 4 ° C for 72 hours in a refolding buffer of mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione. The method for determining the formation analysis of the MHC-peptide complex is described below.

The ELISA plate was covered with anti-HLA antibody W6/32 (HB-95; ATCC.) (50 μL, concentration 5 μg/ml in 100 mM carbonate buffer, pH 9.6 at 4 °C overnight). The plate was incubated with 250 μl/well of 5% w/v skimmed milk powder in PBS for 2 hours at room temperature, and then washed three times with 300 μl/well of 0.05% Tween-20 (Sigma) in PBS. The complex was diluted in PBS containing 1% BSA and then added to the antibody-coated flat disk. The plates were incubated for 2 hours at room temperature to allow the complex to bind to the anti-HLA antibody. After incubation, the plate was washed twice, then 100 μl/well of horseradish peroxidase (HRP)-labeled rabbit anti-human β2-microglobulin antibody (DAKO, Japan; diluted 1:2500 in 1%) BSA in PBS solution). The plates were incubated for 2 hours at room temperature, washed six times with PBS/0.05% Tween 20, and then HRP-conjugated anti-rabbit antibody (1:2000) was added. After incubation for one hour at room temperature, add 3,3'-5,5'-tetramethylbenzidine (TMB, Sigma) to a flat plate, incubate for 30 minutes, and use an ELISA reader at 450 nM. The chromaticity thus developed in each well is read. The intensity of the color indicates the MHC-peptide formation rate of the peptide. Based on the MHC-peptide formation rate of each peptide relative to the MHC-peptide formation rate of the positive control peptide, the relative binding activity was determined according to the following formula: relative binding activity = (MHC-peptide formation rate of the test peptide - blank control peptide) MHC-peptide formation rate) / (MHC-peptide formation rate of positive control peptide - MHC-peptide formation rate of blank control peptide). The relative binding activities of the respective test peptides are shown in Table 1 above. A peptide having a relative binding activity of greater than 50% is a T cell epitope specific for HLA-A2.

Example 2: HLA-A2 transgenic mice immunized with HLA-A2-specific T cell epitopes derived from TAL6

One milligram of each of the peptides (test peptides) identified as HLA-A2-specific T cell epitopes in Example 1 above, and 1 mg of PADRE peptide (AKFVAAWTLKAAA; SEQ ID NO: 25) (both soluble) In 0.5 ml PBS), mix with 0.5 mL of incomplete Freund's adjuvant (IFA). An EBV-derived HLA-A2-specific epitope (ie, GLCTLVAML (GLC; SEQ ID NO: 35)) was used as a positive control, and an EBV-derived HLA-A24-specific epitope (ie, TYGPVFMCL (TYG; SEQ ID NO: 37)) was used as a negative control group. 100 μl of the mixture (containing 50 μg of the test peptide) was subcutaneously injected into the base of the tail of the HLA-A2 transgenic mouse, and twice in total every 7 days. Seven days after the second injection, spleen cells were collected from the treated mice and cultured in a medium containing 10 μg/mL of the test peptide. The supernatant of the cell culture was subjected to ELISpot analysis to detect the total amount of IFN-γ contained therein, which was proportional to the activity of the test peptide to stimulate T cells. As shown in Figure 1, some of the test peptides (e.g., peptides 2 and 5) induce T cells to secrete IFN-[gamma], indicating that they are HLA-A2-specific T cell epitopes.

Example 3: Identification of HLA-A24-specific T cell epitopes derived from TAL6

The TAL6 peptides listed in Table 2 below were subjected to MHC-peptide complex formation analysis according to the procedure described in Example 1 above to identify those who can form MHC complexes with human HLA-A24 and β2-microglobulin. . The results obtained by this are shown in Table 2 below. A peptide having a relative binding activity of greater than 50%, which is confirmed to be a TAL6 T cell epitope specific for HLA-A24.

Example 4: HLA-A24 transgenic mice immunized with HLA-A24-specific T cell epitopes derived from TAL6

HLA-A24 transgenic mice were immunized according to the procedure described in Example 2 above using various peptides (test peptides) identified as HLA-A24-specific T-cell epitopes in Example 3 above. . The EBV peptides GLC and TYG were used as negative and positive control groups, respectively. As shown in Figure 2, some of the test peptides (e.g., peptides 27, 29, and 30) induce T cells to secrete IFN-[gamma], indicating that they are HLA-A24-specific T cell epitopes.

Example 5: HLA-A2 transgenic mouse to produce TAL6 expression vector can also induce T cell response specific to TAL6 peptide

The plastid pEK/TAL6 was constructed by the conventional recombinant technique using the expression vector pCIneo (Promega, Madison, WI, USA), which was designed to express an endoplasmic reticulum target sequence, H-2Kb-specific sequence. SIINFEKL (SEQ ID NO: 46; derived from ovalbumin), HLA-A2-specific sequence GILGFVFTL (SEQ ID NO: 47; M protein derived from influenza virus) and TAL6 fusion protein.

Mice were immunized 2-3 times over 3 weeks via intramuscular injection with pEK/TAL6 or pCIneo. Seven days after the last injection, the immunized mice were sacrificed, their spleen cells were collected and stimulated with various test TAL6 peptides. The presence of IFN-γ secreting cells activated by the test TAL6 peptide was determined by IFN-γ ELISpot. As shown in Figure 3, spleen cells from mice immunized with pEK/TAL6 reacted with peptides 2 and 5 listed in Table 1 above. This result shows that DNA vaccines expressing TAL6 can induce T cell responses specific to certain TAL6 peptides.

Example 6: Immunization with TAL6 peptide inhibits tumor growth in HLA-A2 transgenic mice

Peptide 2 or peptide 5 (mixed with IFA) listed in Table 1 above was administered subcutaneously to HLA-A2 transgenic mice (50 μg/mouse) or wild-type C57BL/6 mice for two weeks. Give it twice. After twenty-one mice were each implanted subcutaneously immunized at 2 x 10 ⁵ tumor cells EL4 / TAL6 / HLA-A2 (stable expression of HLA-A2 and TAL6 EL4 cells), to the immunized in Tumor growth was induced in mice. Tumor size in treated mice was detected every 2-3 days. Tumor volume was calculated using the following formula: tumor volume = length x width x width/2.

Immunization with peptide 5 significantly inhibited tumor growth in HLA-A2 transgenic mice, but did not inhibit tumor growth in wild-type mice. See Figure 4. Immunization with peptide 2 also reduced tumor growth in HLA-A2 transgenic mice, but to a lesser extent. These results show that immunization with HLA-A2-specific immunopeptide derived from TAL6 is effective in inhibiting tumor growth in HLA-A2 carriers.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

<110> National Institute of Health

<120> Immunopeptides of tumor-associated antigens and their use in cancer therapy

<130> 38301-014001

<150> 61/233,229

<151> 2009-08-12

<160> 47

<170> PatentIn version 3.5

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Figure 1 is a graph showing the induction of IFN-γ production by spleen cells derived from HLA-A2 transgenic mice immunized with several immunopeptides derived from TAL6. "NP" means that no peptide is present.

Figure 2 is a graph showing the induction of IFN-γ production by spleen cells derived from HLA-A24 transgenic mice immunized with several immunopeptides derived from TAL6. "N" refers to the negative control group, while "N" refers to the positive control group.

Figure 3 is a graph showing the induction of peptide-specific T-cell responses in mice immunized with transplasty pEK/TAL6 DNA. IFN-γ ELISpot analysis was performed to detect cells secreting IFN-γ. "N" refers to the negative control group, while "N" refers to the positive control group.

Figure 4 is a graph showing inhibition of tumor growth in immunized mice by immunopeptides derived from TAL6. Tumor volume was calculated using the following formula: tumor volume = length x width x width/2. Parallel group (a): HLA-A2 transgenic mice. Parallel group (b): wild type mice.

Claims (9)

An isolated immunopeptide comprising a T cell epitope specific for HLA-A2 derived from a tumor associated antigen L6 (TAL6), wherein the T cell epitope is by SLVGLALLC (SEQ ID NO: 2) or The amino acid sequence of LLMLLPAFV (SEQ ID NO: 4) consists of. The isolated immunopeptide according to claim 1 of the patent application further comprises an endoplasmic reticulum target fragment. The isolated immunopeptide according to claim 1 of the patent application further comprises a T helper cell stimulating fragment. The isolated immunopeptide according to item 3 of the patent application, wherein the T helper cell stimulating fragment has an amino acid sequence of QYIKANSKFIGITE (SEQ ID NO: 24) or AKFVAAWTLK (SEQ ID NO: 25). An isolated immunopeptide according to the first aspect of the patent application, which consists of the amino acid sequence of SLVGLALLC (SEQ ID NO: 2). An isolated immunopeptide according to the first aspect of the patent application, which consists of the amino acid sequence of LLMLLPAFV (SEQ ID NO: 4). An immunological composition comprising the immunopeptide according to item 1 of the scope of the patent application, and a pharmaceutically acceptable carrier. An immunological composition according to item 7 of the patent application, which further comprises an adjuvant. An antibody that specifically binds to a TAL6 fragment of an amino acid sequence having SLVGLALLC (SEQ ID NO: 2) or LLMLLPAFV (SEQ ID NO: 4).