Compositions and methods for identifying tumor-specific neoantigens

Abstract

Methods are provided for identifying tumor-specific neoantigens that act alone or in combination with other tumor-associated peptides as pharmaceutically active ingredients in vaccine compositions that stimulate anti-tumor responses in immunotherapeutic peptides and immunotherapy, particularly cancer immunotherapy. The method includes identifying tumor-specific mutations in genes of subjects with cancer, wherein the mutant peptides bind to class I HLA proteins with higher affinity than the wild-type peptides and have an IC50 of less than 500 nm.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 334,866, filed May 14, 2010, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to the identification of tumor-specific neoantigens and the use of these neoantigens to generate cancer vaccines. [Background technology]

[0003] Background of the Invention Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., cytokines or TLR ligands) that cooperate to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Currently, nearly all vaccines contain either common tumor antigens or whole tumor cell preparations (Gilboa, 1999). Common tumor antigens are immunogenic proteins selectively expressed in tumors in many individuals and are typically delivered to patients as synthetic peptides or recombinant proteins (Boon et al., 2006). In contrast, whole tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations, or total mRNA (Parmiani et al., 2007). Because whole tumor cells are isolated from autologous patients, they express patient-specific tumor antigens in addition to common tumor antigens. Finally, there is a third class of tumor antigens that are rarely used in vaccines due to technical difficulties in identifying them (Sensi et al., 2006). This class consists of proteins with tumor-specific mutations that result in altered amino acid sequences. Such mutant proteins have the potential to (a) uniquely mark tumors (vs. non-tumor cells) for recognition and destruction by the immune system (Lennerz et al., 2005); and (b) circumvent central, and sometimes peripheral, T-cell tolerance and thus be recognized by more effective high-avidity T-cell receptors (Gotter et al., 2004).

[0004] Therefore, there is a need for methods to identify neoepitopes that are useful as tumor vaccines. Summary of the Invention

[0005] The present invention relates, in part, to the discovery of methods to identify peptides capable of eliciting tumor-specific T cell responses.

[0006] In one aspect, the present invention provides a method for identifying neoantigens by identifying tumor-specific mutations in expressed genes in subjects with cancer. In some aspects, when the mutation is a point mutation, the method further comprises identifying a mutant peptide having the mutation. Preferably, the mutant peptide binds to a class I HLA protein with higher affinity than the wild-type peptide and has an IC50 of less than 500 nm; in other aspects, when the mutation is a splice site mutation, a frameshift mutation, a read-through mutation, or a gene fusion mutation, the method further comprises identifying a mutant polypeptide encoded by the mutation. Preferably, the mutant polypeptide binds to a class I HLA protein.

[0007] Optionally, the method further comprises selecting a peptide or polypeptide that activates anti-tumor CD8 T cells.

[0008] The mutant peptide or polypeptide preferably binds to class I HLA proteins with higher affinity than the wild-type peptide, having an IC50 of less than 500 nM. Preferably, the peptide or polypeptide has an IC50 of less than 250 nM. More preferably, the peptide or polypeptide has an IC50 of less than 100 nM. Most preferably, the peptide or polypeptide has an IC50 of less than 50 nM.

[0009] The mutant peptide is about 8 to 10 amino acids in length, and in another aspect, about 8 to 50 amino acids in length. For example, the mutant peptide is greater than 10 amino acids in length, greater than 15 amino acids in length, greater than 20 amino acids in length, or greater than 30 amino acids in length. Preferably, the mutant peptide is about 24 to 40 amino acids in length.

[0010] In a further aspect, the present invention provides a method for inducing a tumor-specific immune response in a subject by administering one or more peptides or polypeptides identified according to the method of the present invention and an adjuvant.The adjuvant is, for example, a TLR-based adjuvant or a mineral oil-based adjuvant.In some embodiments, the peptide or polypeptide and the TLR-based adjuvant are emulsified with a mineral oil-based adjuvant.Optionally, the method further comprises administering an anti-immunosuppressant, such as an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody, or an IDO inhibitor.

[0011] In yet another aspect, the present invention provides a method for inducing a tumor-specific immune response in a subject by administering to the subject autologous dendritic cells or antigen-presenting cells pulsed with one or more of the peptides or polypeptides identified according to the methods of the present invention. Optionally, the method further comprises administering an adjuvant, such as a TLR-based adjuvant or a mineral oil-based adjuvant. In some embodiments, the peptide or polypeptide and the TLR-based adjuvant are emulsified with a mineral oil-based adjuvant. In some embodiments, the method further comprises administering an anti-immunosuppressant. Examples of anti-immunosuppressants include, for example, an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody, or an inhibitor of IDO.

[0012] In another aspect, the present invention provides a method for vaccinating or treating a subject against cancer by identifying multiple tumor-specific mutations in expressed genes in the subject, identifying mutant peptides or polypeptides bearing the identified tumor-specific mutations, selecting one or more of the identified mutant peptides or polypeptides that bind to a class I HLA protein, preferably with higher affinity than the wild-type peptide, and capable of activating anti-tumor CD8 T cells, and administering to the subject one or more selected peptides, polypeptides, or autologous dendritic cells or antigen-presenting cells pulsed with one or more identified peptides or polypeptides. The mutant peptides are approximately 8-10 amino acids in length, and in another aspect, approximately 8-50 amino acids in length. For example, the mutant peptides are greater than 10 amino acids, greater than 15 amino acids, greater than 20 amino acids, or greater than 30 amino acids in length. Preferably, the mutant peptides are approximately 24-40 amino acids in length.

[0013] Optionally, the method further comprises administering an adjuvant, such as a TLR-based adjuvant or a mineral oil-based adjuvant. In some embodiments, the peptide or polypeptide and the TLR-based adjuvant are emulsified with a mineral oil-based adjuvant. In some embodiments, the method further comprises administering an anti-immunosuppressant. Anti-immunosuppressants include, for example, anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CD25 antibodies, or inhibitors of IDO.

[0014] 23. The method of claim 22, wherein the subject has undergone a hematopoietic stem cell transplant.

[0015] The subject is a human, dog, cat, or horse. The cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, lymphoma such as B-cell lymphoma, or leukemia such as acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or T-lymphocytic leukemia.

[0016] Also included in the present invention is a pharmaceutical composition comprising a peptide or polypeptide identified according to the methods of the present invention and a pharmaceutically acceptable carrier.

[0017] For example, the present invention provides a method for producing SF3B1 fragments each of which is equal to or less than 50 amino acids in length, numbered based on wild-type SF3B1: leucine at amino acid position 625; histidine at amino acid position 626; glutamic acid at amino acid position 700; aspartic acid at amino acid position 742; or Arginine at amino acid position 903 The present invention provides a composition comprising at least two distinct SF3B1 peptides, wherein the composition comprises:

[0018] The present invention also provides compositions containing at least two distinct MYD88 peptides, each equal to or less than 50 amino acids in length and containing, when numbered based on wild-type MYD88, a threonine at amino acid position 232; a leucine at amino acid position 258; or a proline at amino acid position 265.

[0019] The present invention further provides compositions containing at least two distinct TP53 peptides, each equal to or less than 50 amino acids in length and containing, numbered based on wild-type TP53, an arginine at amino acid position 111; an arginine at amino acid position 215; a serine at amino acid position 238; a glutamine at amino acid position 248; a phenylalanine at amino acid position 255; a cysteine ​​at amino acid position 273; or an asparagine at amino acid position 281.

[0020] The present invention further provides compositions containing at least two distinct ATM peptides, each equal to or less than 50 amino acids in length and containing, numbered based on wild-type ATM, a phenylalanine at amino acid position 1252; an arginine at amino acid position 2038; a histidine at amino acid position 2522; or a cysteine ​​at amino acid position 2954.

[0021] Each is equal to or less than 50 amino acids in length, and, numbered based on wild-type ABL, includes: valine at amino acid position 244; valine at amino acid position 248; glutamic acid at amino acid position 250; alanine at amino acid position 250; histidine at amino acid position 252; arginine at amino acid position 252; phenylalanine at amino acid position 253; histidine at amino acid position 253; lysine at amino acid position 255; valine at amino acid position 255; glycine at amino acid position 276; isoleucine at amino acid position 315; and arginine at amino acid position 315. A composition comprising at least two distinct Abl peptides, each containing: paragine; leucine at amino acid position 317; threonine at amino acid position 343; threonine at amino acid position 351; glycine at amino acid position 355; valine at amino acid position 359; alanine at amino acid position 359; isoleucine at amino acid position 379; leucine at amino acid position 382; methionine at amino acid position 387; proline at amino acid position 396; arginine at amino acid position 396; tyrosine at amino acid position 417; or serine at amino acid position 486.

[0022] Further included in the present invention is a composition containing at least two distinct FBXW7 peptides, each of which is equal to or less than 50 amino acids in length and contains, when numbered based on wild-type FBXW7, a leucine at amino acid position 280; a histidine at amino acid position 465; a cysteine ​​at amino acid position 505; or a glutamic acid at amino acid position 597.

[0023] In a further aspect, the present invention provides a composition containing at least two distinct MAPK1 peptides, each of which is equal to or less than 50 amino acids in length and contains, when numbered based on wild-type MAPK1, an asparagine at amino acid position 162; a glycine at amino acid position 291; or a phenylalanine at amino acid position 316.

[0024] The present invention also provides a composition containing at least two distinct GNB1 peptides, each of which is equal to or less than 50 amino acids in length and contains a threonine at amino acid position 180, numbered based on wild-type GNB1.

[0025] The present invention also provides a method for treating a subject with an imatinib-resistant tumor, comprising administering to an HLA-A3-positive subject a composition of a Bcr-abl peptide of equal to or less than 50 amino acids in length, which contains a lysine at position 255, as numbered based on wild-type bcr-abl.

[0026] Further provided by the present invention is a method of treating a subject with an imatinib-resistant tumor, comprising administering to the subject one or more peptides containing a bcr-abl mutation that are equal to or less than 50 amino acids and bind to a class I HLA protein with an IC50 of less than 500 nm.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0028] [The present invention 1001] A method for identifying neoantigens, comprising the steps of: (a) identifying tumor-specific mutations in expressed genes in a subject with cancer; (b) If the mutation identified in step (a) is a point mutation: (i) identifying a mutant peptide having the mutation identified in step (a), wherein the mutant peptide binds to a class I HLA protein with a higher affinity than the wild-type peptide; and has an IC50 of less than 500 nm; (c) If the mutation identified in step (a) is a splice site mutation, a frameshift mutation, a readthrough mutation, or a gene fusion mutation: (i) identifying a mutant polypeptide encoded by the mutation identified in step (a), wherein the mutant polypeptide binds to a class I HLA protein. [The present invention 1002] 1001. The method of claim 1001, wherein said mutant peptide is about 8 to 10 amino acids in length. [The present invention 1003] 1001. The method of claim 1001, wherein said mutant peptide is greater than 10 amino acids in length. [The present invention 1004] 1004. The method of claim 1003, wherein said mutant peptide is greater than 15 amino acids in length. [The present invention 1005] 1005. The method of claim 1004, wherein said mutant peptide is greater than 20 amino acids in length. [The present invention 1006] 1005. The method of claim 10, wherein said mutant peptide is greater than 30 amino acids in length. [The present invention 1007] 1001. The method of claim 1001, wherein said mutant peptide is about 8 to 50 amino acids in length. [The present invention 1008] 1001. The method of claim 1001, wherein said mutant peptide is about 24 to 40 amino acids in length. [The present invention 1009] 1001. The method of claim 1001, wherein the tumor-specific mutations are identified by nucleic acid sequencing. [The present invention 1010] 1001. The method of claim 1001, further comprising the step of selecting a peptide identified in step (b) or a polypeptide of step (c), which activates anti-tumor CD8 T cells. [The present invention 1011] 1001. A method of inducing a tumor-specific immune response in a subject, comprising administering one or more peptides or polypeptides identified according to the present invention and an adjuvant. [The present invention 1012] 1011. The method of claim 10, wherein said adjuvant is a TLR-based adjuvant. [The present invention 1013] 1011. The method of claim 1011, wherein said peptide or polypeptide is emulsified with a mineral oil-based adjuvant. [The present invention 1014] 1011. The method of claim 1011, wherein said peptide or polypeptide and TLR-based adjuvant are emulsified with a mineral oil-based adjuvant. [The present invention 1015] The method of any one of claims 1 to 11, further comprising administering an anti-immunosuppressant. [The present invention 1016] 1015. The method of claim 10, wherein said anti-immunosuppressant is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody, or an inhibitor of IDO. [The present invention 1017] A method for inducing a tumor-specific immune response in a subject, comprising administering to the subject autologous dendritic cells or antigen-presenting cells pulsed with one or more of the peptides or polypeptides identified according to the present invention. [The present invention 1018] The method of claim 1017, further comprising administering an adjuvant. [The present invention 1019] The method of claim 1018, wherein the adjuvant is a TLR-based adjuvant. [The present invention 1020] The method of claim 1017, further comprising the step of administering an anti-immunosuppressant. [The present invention 1021] 1020. The method of claim 1020, wherein the anti-immunosuppressant is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody, or an inhibitor of IDO. [The present invention 1022] A method of vaccinating or treating a subject against cancer, comprising the steps of: (a) identifying a plurality of tumor-specific mutations in expressed genes of said subject, wherein the identified mutations are: (i) if the mutation is a point mutation, further identifying a mutant peptide having the point mutation; and / or (ii) if the mutation is a splice site mutation, a frameshift mutation, a read-through mutation, or a gene fusion mutation, further identifying the mutant polypeptide encoded by the mutation; (b) selecting one or more mutant peptides or polypeptides identified in step (a) that bind to class I HLA proteins; (c) selecting one or more mutant peptides or mutant polypeptides identified in step (b) that are capable of activating anti-tumor CD8 T cells; and (d) administering to the subject autologous dendritic cells or antigen-presenting cells pulsed with the one or more peptides or polypeptides selected in step (c). [The present invention 1023] The method of claim 1022, further comprising administering an adjuvant to said subject. [The present invention 1024] The method of claim 1023, wherein the adjuvant is a TLR-based adjuvant. [The present invention 1025] The method of claim 1022, further comprising the step of administering an anti-immunosuppressant. [The present invention 1026] 1025. The method of claim 1025, wherein said anti-immunosuppressant is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody, or an inhibitor of IDO. [The present invention 1027] 1023. The method of claim 1022, wherein said mutant peptide is about 8 to 10 amino acids in length. [The present invention 1028] 1023. The method of claim 1022, wherein the mutant peptide is about 8 to 50 amino acids in length. [The present invention 1029] 1023. The method of claim 1022, wherein the mutant peptide is about 24 to 40 amino acids in length. [The present invention 1030] The method of claim 1022, wherein the subject has undergone a hematopoietic stem cell transplant. [The present invention 1031] The method of claim 1022, wherein said subject is a human, dog, cat, or horse. [The present invention 1032] 1023. The method of claim 1022, wherein said cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, lymphoma, or leukemia. [The present invention 1033] 1033. The method of claim 1032, wherein said lymphoma is a B-cell lymphoma. [The present invention 1034] 1033. The method of claim 1032, wherein said leukemia is acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or T-lymphocytic leukemia. [This invention 1035] A pharmaceutical composition comprising a peptide identified according to the present invention and a pharmaceutically acceptable carrier. [The present invention 1036] A composition comprising at least two distinct peptides of: (a) each less than or equal to 50 amino acids in length, numbered based on wild-type SF3B1; (i) leucine at amino acid position 625; (ii) histidine at amino acid position 626; (iii) glutamic acid at amino acid position 700; (iv) aspartic acid at amino acid position 742; or (v) arginine at amino acid position 903 SF3B1 peptide, (b) each less than or equal to 50 amino acids in length, numbered based on wild-type MYD88; (i) threonine at amino acid position 232; (ii) a leucine at amino acid position 258; or (iii) Proline at amino acid position 265 containing the MYD88 peptide; (c) each less than or equal to 50 amino acids in length, numbered based on wild-type TP53; (i) arginine at amino acid position 111; (ii) arginine at amino acid position 215; (iii) serine at amino acid position 238; (iv) glutamine at amino acid position 248; (v) phenylalanine at amino acid position 255; (vi) a cysteine ​​at amino acid position 273, or (vii) asparagine at amino acid position 281 containing the TP53 peptide; (d) each less than or equal to 50 amino acids in length, numbered based on wild-type ATM; (i) phenylalanine at amino acid position 1252; (ii) arginine at amino acid position 2038; (iii) histidine at amino acid position 2522; or (iv) cysteine ​​at amino acid position 2954 containing the ATM peptide; (e) each less than or equal to 50 amino acids in length, numbered based on wild-type abl, (i) valine at amino acid position 244; (ii) valine at amino acid position 248; (iii) glutamic acid at amino acid position 250; (iv) alanine at amino acid position 250; (v) histidine at amino acid position 252; (vi) arginine at amino acid position 252; (vii) phenylalanine at amino acid position 253; (viii) histidine at amino acid position 253; (ix) lysine at amino acid position 255; (x) valine at amino acid position 255; (xi) glycine at amino acid position 276; (xii) isoleucine at amino acid position 315; (xiii) asparagine at amino acid position 315; (xiv) leucine at amino acid position 317; (xv) threonine at amino acid position 343; (xvi) threonine at amino acid position 351; (xvii) glycine at amino acid position 355; (xviii) valine at amino acid position 359; (xix) alanine at amino acid position 359; (xx) isoleucine at amino acid position 379; (xxi) leucine at amino acid position 382; (xxii) methionine at amino acid position 387; (xxiii) proline at amino acid position 396; (xxiv) arginine at amino acid position 396; (xxv) tyrosine at amino acid position 417; or (xxvi) Serine at amino acid position 486 Contains the ABL peptide; (f) each less than or equal to 50 amino acids in length, numbered based on wild-type FBXW7; (i) leucine at amino acid position 280; (ii) histidine at amino acid position 465; (iii) a cysteine ​​at amino acid position 505; or (iv) glutamic acid at amino acid position 597 containing the FBXW7 peptide; (g) each less than or equal to 50 amino acids in length, numbered based on wild-type MAPK1; (i) asparagine at amino acid position 162; (ii) glycine at amino acid position 291; or (iii) phenylalanine at amino acid position 316 a MAPK1 peptide containing (h) GNB1 peptides, each less than or equal to 50 amino acids in length and containing a threonine at amino acid position 180, numbered based on wild-type GNB1. [This invention 1037] The composition of the present invention 1036, further comprising an adjuvant. [The present invention 1038] A method for treating a subject having an imatinib-resistant tumor, comprising administering to an HLA-A3 positive subject a composition of a Bcr-abl peptide of equal to or less than 50 amino acids in length, which contains a lysine at position 255, as numbered based on wild-type bcr-abl. [This invention 1039] A method of treating a subject having an imatinib-resistant tumor, comprising administering to the subject one or more peptides containing a bcr-abl mutation, wherein the peptides are less than or equal to 50 amino acids and bind to class I HLA proteins with an IC50 of less than 500 nm. Other features and advantages of the present invention will become apparent from and be encompassed by the following detailed description and the appended claims. [Brief explanation of the drawings]

[0029] [Figure 1] We demonstrate the balance between specificity and autoimmune toxicity using three classes of antigens for tumor vaccines. Because the set of protein antigens expressed on tumor cells includes thousands of proteins that are also present in other cells of the body, whole tumor cells may be the least specific antigen preparation for tumor vaccines. Overexpressed tumor antigens are slightly more specific because they have been selected for their much stronger and more selective expression in tumors compared to other cells in the body. Nevertheless, it is not possible to test every cell in the body for expression of these antigens, and there is a substantial risk that other cells may express them. Finally, mutant proteins generate neoepitopes that are present only in tumor cells, providing the highest level of specificity. [Figure 2] This is a scheme for a personalized neoantigen vaccination strategy that can be applied to the treatment of any cancer. We also highlight the possibility of applying this strategy in two unique scenarios. In the first case, patients are vaccinated early after hematopoietic stem cell transplantation (HSCT) (e.g., as performed for CLL, CML, and other leukemias). The early post-HSCT period is a unique therapeutic setting because the immune system is competent for HSCT reconstitution and thus overcomes tumor- or treatment-induced host immune defects. Furthermore, in a lymphopenic environment, such as the early post-HSCT setting, abundant homeostatic cytokines can contribute to the rapid expansion of T cells. In the second case, patients are vaccinated early in the course of the disease, because immune competence may be more complete at an early stage of the disease, before it is impaired by chemotherapy exposure (e.g., for solid tumors or hematopoietic malignancies). Because the immune system is likely to be most active in these two specific situations, we propose that they are ideal settings for applying a tumor vaccination strategy. [Figure 3]We demonstrate a three-step strategy for identifying tumor neoepitopes: (1) use sequencing technology to detect genetic mutations present in a single patient's tumor but absent from their germline DNA; (2) use a prediction algorithm to predict whether mutant peptides have the potential to bind to an individual's HLA alleles; these predicted peptides can optionally be experimentally tested for binding to the appropriate HLA proteins. Furthermore, these genes must also be expressed on tumor cells. (3) generate T cells ex vivo and test whether they can recognize cells expressing the mutant proteins; alternatively, mass spectrometry can be used to detect peptides eluted from tumor cell surface HLA proteins. Our research to date has demonstrated that, for chronic lymphocytic leukemia, there are an average of 23 protein-altering mutations, 46 predicted binding mutant peptides, and 15–25 confirmed binding mutant peptides per patient. Of these, approximately 7–12 peptides are predicted to be expressed and processed in tumor cells (although this may vary depending on the tumor and patient). [Figure 4] Five mutation classes are shown to generate potential tumor neoepitopes. New tumor-specific epitopes can arise as a result of missense, splice-site, frameshift, or read-through point mutations (red asterisks), or from the fusion of two genes (or within the same gene). Specifically, splice-site, frameshift, and read-through mutations, and gene fusions, can each generate novel stretches of amino acids (magenta) that are normally not translated but become expressed and translated as a result of the mutation. Missense mutations result in peptides with a single amino acid change. [Figure 5]The frequency of each class of mutation in CLL patients is shown. Our study, applying next-generation sequencing to a series of seven CLL tumors, reveals that CLL cells harbor numerous mutations, providing a rich source of potential mutant peptides. We observe that the total number of non-silent gene alterations in CLL ranges from 17 to 155 per individual, the majority of which are somatically altered point mutations (missense). Four patient tumors also harbored splice site mutations; in three patients, novel gene fusions were identified by RNA sequencing. [Figure 6] Data from automated prediction of peptide binding (for peptides carrying specific missense mutations) to each of the patient's six HLA (MHC class I) alleles (step 2A of the strategy in Figure 3) are shown. Magenta = strong binders; green = moderate binders. [Figure 7] The method for confirming RNA expression of mutant genes is shown (step 2B of the strategy in Figure 3). (A) For CLL patient 7, we found that the majority of mutant genes, including predicted HLA-binding peptides, were expressed at the RNA level. (B) We also used RNA pyrosequencing to detect expressed RNA harboring specific mutations found in the DNA. (C) PCR-TOPO cloning of the breakpoint regions allowed us to verify the novel gene fusions seen by DNA sequencing (the fusion discovered for patient 2 is shown). [Figure 8]Methods and data for experimental demonstration of HLA-peptide binding are shown (step 2C of the strategy in Figure 3). (A) Scheme for experimental demonstration of peptide binding to specific HLA alleles. (B) Overview of mutant peptide candidates identified in patients 1 and 2. Shaded cells indicate ongoing analysis. (C) Data for patient 2 on predicted versus experimentally demonstrated binding affinity of peptides generated from genetic modifications (missense mutations or gene fusions). A prediction cutoff of IC50<120 nM (solid vertical line on the left) results in all peptides showing experimental binding to class I HLA. [Figure 9] Figure 1 shows the predicted differential binding of mutant peptides versus germline peptides (i.e., also referred to as parent, wild-type, or normal peptides) to HLA alleles. Twelve of the 25 predicted HLA-binding mutant peptides in Pt2 have more than twice the binding of the parent peptide (cutoff = red dotted line). This further increases the specificity of the mutant peptides. The mutant peptides are specific for two reasons: first, many of the T cell receptors that recognize the mutant peptides are likely to not detect the wild-type parent peptide; and second, some of the mutant peptides can bind to HLA with higher affinity than the parent peptide. Because the first property cannot be predicted computationally, the focus will be on predicting the second property and selecting for inclusion in the vaccine only those peptides that show higher HLA binding of the mutant peptides compared to the wild-type peptide. [Figure 10] Figure 3 shows T cell reactivity to individual CLL neoepitope candidates (step 3 of the strategy in Figure 3). We observed (using an Elispot assay) that T cells isolated from patient 1 after treatment were able to detect a specific mutant TLK2 peptide (peptide #7). [Figure 11]We show that BCR-ABL mutations generate many peptides predicted to bind to HLA-A and HLA-B alleles. By applying the NetMHC prediction algorithm (Nielsen et al. PLoS One. 2007, 2(8):e796), we predicted peptides generated from BCR-ABL mutations with binding potential to eight common HLA-A and HLA-B alleles. The most common BCR-ABL mutations are shown in order of decreasing frequency (from left to right), and the predicted IC50s of various class I MHC-binding peptides are illustrated. We predicted a total of 84 peptides that bind with good affinity, defined as an IC50 of less than 1000, across a wide range of HLA alleles. Of all predicted peptides, 24 / 84 (29%) were predicted to be strong binders, with an IC50 <50. 42 peptides (50%) were moderate binders, defined as an IC50 between 50 and 500. Eighteen peptides (21%) were weak binders, defined as IC50 between 500 and 1000. [Figure 12] We demonstrate that BCR-ABL peptides carrying the E255K mutation bind to HLA proteins and associate with specific polyfunctional T cells present in CML patients. (A) Experimentally obtained binding scores of E255K-B (and the parent peptide) to HLA A3 and supertype members. (B) In CD8+ T cells expanded from HLA A3+E255K+ patients after HSCT, we detected IFNγ secretion in response to the E255K-B (MUT) peptide and A3+-expressing APCs expressing the E255K minigene (MG). This response was abrogated in the presence of a class I blocking antibody (w6 / 32). (C) IFNγ-secreting cells were also tetramer+ for the mutant peptide and (D) were polyfunctional, secreting IP10, TNFα, and GM-CSF (based on Luminex assay). [Figure 13]We demonstrate that patient-derived T cell clones can recognize tumor-specific epitopes and kill cells displaying these epitopes. (A) Reactivity to the CML66 CD8+ T cell epitope (peptide 66-72C) is restricted to HLA B-4403. (B) CML66 mRNA can be efficiently nucleofected into CD40L-expanded B cells. (C) CML66-specific CD8+ T cells are cytotoxic to CD40L B cells expressing CML66 by RNA nucleofection or peptide pulsing, but not to control targets. [Figure 14A] Genes significantly mutated in CLL are shown. The 9 most significantly mutated genes in 64 CLL samples. All covered territories (base pairs) in N-64 sequenced samples. p- and q-values ​​were calculated by comparing the probability of seeing the observed mutation configuration with the calculated background mutation rate in the dataset. Red bars - genes previously unknown to be mutated in CLL; gray bars - genes previously reported to be mutated in CLL. [Figure 14B-01] Genes significantly mutated in CLL are shown. The type (missense, splice site, nonsense) and location (location and mutation in CLL samples are shown above the gene) of ATM, SF3B1, TP53, MYD88, FBXW7, DDX3X, MAPK1, and GNB1 mutations found in 64 CLLs compared with mutations previously reported in the literature or the COSMIC database (lines below the gene indicate the location of the mutation). [Figure 14B-02] FIG. 14B-02 is a diagram continuing from FIG. 14B-01. [Figure 15]SF3B1 is expressed in CLL samples (seventh column in the graph) and is more highly expressed than many control cells, including PBMC, M: monocytes, CC: cancer cell lines (including K562, Jurkat, IM9, MCF-7, Hela, Ovcar, RPMI, OTM, MCF-CAR, KM12BM, and MM1S). [Figure 16] We show that SF3B1 mutations generate peptides that are predicted to bind to patient-specific HLA alleles. For example, a peptide containing the common SF3B1 K700E mutation is predicted to bind strongly to HLA. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description of the Invention One of the key barriers to developing curative tumor-specific immunotherapy is the identification and selection of highly restricted tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations in malignant cells, represent the most tumor-specific antigen class. Neoantigens have rarely been used in vaccines due to technical challenges in their identification. Our approach to identifying tumor-specific neoepitopes involves three steps: (1) identification of DNA mutations using whole-genome or whole-exome (i.e., captured exons only) or RNA sequencing of tumor samples versus matched germline samples from each patient; (2) application of a validated peptide-MHC binding prediction algorithm to generate a set of candidate T cell epitopes based on non-silent mutations present in the tumor that can bind to the patient's HLA alleles; and (3) optionally, demonstration of antigen-specific T cells against the mutant peptide or demonstration that the candidate peptide binds to HLA proteins on the tumor surface.

[0031] Thus, the present invention relates to methods for the identification and / or detection of T cell epitopes of antigens. In particular, the present invention provides methods for the identification and / or detection of tumor-specific neo-antigens useful in inducing tumor-specific immune responses in subjects.

[0032] Specifically, the present invention provides a method for vaccinating or treating a subject by identifying multiple tumor-specific mutations in the subject's genome. Mutant peptides and polypeptides that have the identified mutations and bind to class I HLA proteins are selected. Optionally, these peptides and polypeptides can bind to class I HLA with higher affinity than wild-type peptides and / or activate anti-tumor CD8 T cells. These peptides are administered to the subject. Alternatively, autologous antigen-presenting cells pulsed with the peptides are administered.

[0033] The importance of mutant antigens or neoepitopes in tumor immune control has been recognized in basic studies showing that: (a) mice and humans frequently mount T cell responses to mutant antigens (Parmiani et al., 2007; Sensi and Anichini, 2006); (b) mice can be protected from tumors by immunization with a single mutant peptide present in the tumor (Mandelboim et al., 1995); (c) natural or vaccine-mediated long-term melanoma survivors mount strong memory cytotoxic T cell (CTL) responses to mutant antigens (Huang et al., 2004; Lennerz et al., 2005; Zhou et al., 2005a); and (d) finally, follicular lymphoma patients exhibit molecular remission when immunized with patient-specific mutant immunoglobulin proteins present on their autologous tumor cells (Baskar et al., 2004). Furthermore, the CTL responses in these patients are directed against the mutated regions of the immunoglobulin proteins rather than the common regions. Furthermore, such mutant peptides have the potential to (a) uniquely mark tumors for recognition and destruction by the immune system, thus reducing the risk of autoimmunity; and (b) circumvent central and peripheral T cell tolerance, allowing antigens to be recognized by more effective high-avidity T cell receptors (Figure 1).

[0034] Identical mutations in specific genes are rarely found among tumors (even the most common driver mutations are low in frequency). Therefore, the method of the present invention will comprehensively identify patient-specific tumor mutations. Using highly parallel sequencing technology, HLA-peptide binding prediction tools, and biochemical assays, the method of the present invention allows for the following: (1) comprehensive identification of mutant peptides expressed in a patient's tumor and binding to HLA proteins present in the patient's tumor; (2) monitoring the cancer patient's innate immune response to these identified neoepitopes; and (3) determining whether cytotoxic T cells that recognize these peptides in the context of the patient's HLA proteins can selectively lyse autologous tumor cells ex vivo. This strategy addresses several fundamental questions regarding how a cancer patient's immune system interacts with tumor neoepitopes. These include: what fraction of tumor neoepitopes are detected by T cells, how many T cell precursors can respond to neoepitopes, how frequently neoepitope-specific memory and effector T cells are present in the circulation and in tumors, what avidity T cells have for these epitopes, and whether neoepitope-specific T cells are functional. The answers to these questions provide both justification and strategies for the use of tumor neoepitopes in human vaccines.

[0035] The human immune system can be divided into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against infection, quickly neutralizing most potential pathogens before they can cause significant infection. The adaptive immune system responds to molecular structures, called antigens, on invading organisms. There are two types of adaptive immune responses: humoral and cellular. In the humoral immune response, antibodies secreted into bodily fluids by B cells bind to antigens from pathogens and eliminate them through various mechanisms, such as complement-mediated lysis. In the cellular immune response, T cells, which can destroy other cells, are activated. For example, when disease-related proteins are present in cells, they are proteolytically fragmented into peptides within the cell. Specific cellular proteins then attach to the antigens or peptides thus formed and transport them to the cell surface, where they are presented to the body's molecular defense mechanisms, particularly T cells. Cytotoxic T cells recognize these antigens and kill cells that bear the antigens.

[0036] Molecules that transport and present peptides on cell surfaces are called major histocompatibility complex (MHC) proteins. MHC proteins are classified into class I and class II MHC proteins. The structures of the two MHC class proteins are very similar; however, they differ significantly in terms of function. MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. MHC class I proteins are generally loaded with antigens derived from endogenous proteins or intracellular pathogens and then presented to cytotoxic T lymphocytes (CTLs). Class II MHC proteins are present only on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. They primarily present peptides from external antigenic sources, i.e., processed peptides from outside the cell, to T helper (Th) cells. The majority of peptides bound by class I MHC proteins originate from cytoplasmic proteins produced within the healthy host organism itself and do not typically stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize such self-peptide-presenting MHC class I molecules are either eliminated in the thymus or eliminated or inactivated after release from the thymus, i.e., tolerized. MHC molecules can stimulate an immune response only when they present peptides to non-tolerized cytotoxic T lymphocytes. Cytotoxic T lymphocytes have both T cell receptors (TCRs) and CD8 molecules on their surface. T cell receptors can recognize and bind to peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0037] Before being presented on the cell surface, peptides are attached to MHC class I molecules in the endoplasmic reticulum through competitive affinity binding.Here, the affinity of each peptide is directly related to its amino acid sequence and the presence of specific binding motifs at defined positions in the amino acid sequence.If the sequence of such peptide is known, it is possible, for example, to use peptide vaccines to manipulate the immune system against diseased cells.

[0038] Using computer algorithms, it is possible to predict potential T cell epitopes, i.e., peptide sequences that bind to class I or class II MHC molecules in the form of peptide-presenting complexes and are then recognized by the T cell receptors of T lymphocytes in this form. Currently, two programs are specifically used: SYFPEITHI (Rammensee et al., Immunogenetics, 50 (1999), 213-219) and HLA_BIND (Parker et al., J. Immunol., 152 (1994), 163-175). The peptide sequences thus determined that may bind to class I MHC molecules must then be examined in vitro for their actual binding capacity.

[0039] The technical object of the present invention is to provide an improved method for identifying and screening potential T cell epitopes present on tumor cells that allows for the simultaneous and rapid investigation of large numbers of peptide sequences for their ability to bind to specific MHC molecules.

[0040] The technical objectives of the present invention are achieved by providing a method for detecting and / or identifying mutant antigens present in tumors but absent from normal tissues. The method uses massively parallel genome sequencing of the entire coding portion of a cancer patient's genome to identify specific mutant genes in tumors. To identify mutant peptides that bind more strongly to HLA than wild-type peptides and thus have the potential to confer tumor specificity, a well-established algorithm is used to predict peptides that bind to any of the six unique class I HLA alleles for each patient. A predicted IC50 is calculated for all 9- or 10-residue peptides with tumor-specific mutant residues versus those with germline residues. Typically, peptides with a predicted IC50 <50 nM are generally considered medium- to high-affinity binding peptides and will be selected for empirical affinity testing using biochemical assays of HLA binding. Finally, it will be determined whether the human immune system can mount an effective immune response against these mutant tumor antigens, thereby effectively killing tumors without killing normal cells.

[0041] definition "T cell epitope" should be understood as meaning a peptide sequence that, in the form of a peptide-presenting MHC molecule or MHC complex, is bound by an MHC molecule of class I or II and, in this form, is then recognized and bound by cytotoxic T lymphocytes or T helper cells, respectively.

[0042] The term "receptor" should be understood to mean a biological molecule or group of molecules that can bind to a ligand. A receptor can function to transmit information in a cell, cell formation, or organism. A receptor comprises at least one receptor unit, preferably two receptor units, and each receptor unit can consist of a protein molecule, specifically a glycoprotein molecule. The receptor has a structure that complements the structure of the ligand and can complex with the ligand as a binding partner. The information is transmitted specifically by a conformational change of the receptor after the ligand complexes on the surface of the cell. According to the present invention, a receptor should be understood to mean specifically MHC class I and II proteins that can form a receptor / ligand complex with a ligand, specifically a peptide or peptide fragment of an appropriate length.

[0043] The term "ligand" should be understood to mean a molecule that has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In accordance with the present invention, a ligand should be understood to mean, in particular, a peptide or peptide fragment of an appropriate length and containing an appropriate binding motif in its amino acid sequence so as to be capable of forming a complex with an MHC class I or MHC class II protein.

[0044] "Receptor / ligand complex" should also be understood to mean a "receptor / peptide complex" or a "receptor / peptide fragment complex", specifically a class I or class II MHC molecule presenting a peptide or peptide fragment.

[0045] The terms "major histocompatibility antigen (MHC) protein or molecule," "MHC molecule," "MHC protein," or "HLA protein" are specifically understood to mean proteins capable of binding peptides representing potential T cell epitopes resulting from proteolytic cleavage of protein antigens, transporting them to the cell surface, and presenting them to specific cells, specifically cytotoxic T lymphocytes or T helper cells. Major histocompatibility antigens in the genome contain gene regions whose gene products, expressed on the cell surface, bind and present endogenous and / or foreign antigens and are therefore important for regulating immunological processes. Major histocompatibility antigens are classified into two gene groups encoding different proteins: MHC class I molecules and MHC class II molecules. The two MHC class molecules are specialized for different antigenic origins. MHC class I molecules present endogenously synthesized antigens, such as viral proteins and tumor antigens. MHC class II molecules present protein antigens originating from exogenous sources, such as bacterial products. The cell biology and expression patterns of the two MHC classes are compatible with these different roles.

[0046] Class I MHC molecules consist of heavy and light chains and can bind peptides of approximately 8 to 11 amino acids, typically 9 or 10 amino acids, and present them to cytotoxic T lymphocytes if the peptides have the appropriate binding motif. The peptides bound by class I MHC molecules originate from endogenous protein antigens. The heavy chain of class I MHC molecules is preferably an HLA-A, HLA-B, or HLA-C monomer, and the light chain is β2-microglobulin.

[0047] Class II MHC molecules consist of α and β chains and can bind and present peptides of approximately 15 to 24 amino acids to T helper cells if the peptides have the appropriate binding motif. The peptides bound by class II MHC molecules generally result from extracellular or exogenous protein antigens. The α and β chains are specifically HLA-DR, HLA-DQ, and HLA-DP monomers.

[0048] "Vaccine" should be understood as meaning a composition for generating immunity for the prevention and / or treatment of disease. A vaccine is thus a pharmaceutical containing an antigen, intended for use in humans or animals to generate specific protection and protective substances by vaccination.

[0049] By "isolated" is meant that a polynucleotide or polypeptide, or a fragment, variant, or derivative thereof, has been essentially removed from other biological materials with which it is naturally associated, or is essentially free of other biological materials, for example, from a recombinant host cell that has been genetically engineered to express the polypeptide of the invention.

[0050] "Neoantigens" refers to a class of tumor antigens that arise from tumor-specific mutations in expressed proteins.

[0051] By "purified," it is meant that a polynucleotide or polypeptide, or a fragment, variant, or derivative thereof, is substantially free of other biological materials with which it is naturally associated, or is substantially free of other biological materials derived, for example, from a recombinant host cell genetically engineered to express the polypeptide of the invention. Thus, for example, a purified polypeptide of the invention is at least about 70-100% pure, i.e., the polypeptide is present in a composition comprising about 70-100% by weight of the total composition. In some embodiments, a purified polypeptide of the invention is about 75-99% pure by weight, about 80-99% pure by weight, about 90-99% pure by weight, or about 95-99% pure by weight.

[0052] Identification of tumor-specific mutations The present invention is based on the identification of certain mutations (e.g., variants or alleles) present in cancer cells. Specifically, these mutations are present in the genome of cancer cells of a subject with cancer, but are absent in normal tissue derived from that subject.

[0053] Genetic mutations in tumors may be useful for immunologically targeting tumors if they result in changes in the amino acid sequence of proteins exclusively in tumors. Useful mutations include: (1) nonsynonymous mutations that result in different amino acids in proteins; (2) read-through mutations that modify or delete stop codons, resulting in the translation of longer proteins with novel tumor-specific sequences at the C-terminus; (3) splice site mutations that result in the inclusion of introns in mature mRNA, thus resulting in unique tumor-specific protein sequences; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; (5) frameshift mutations or deletions that result in new open reading frames with novel tumor-specific protein sequences.

[0054] For example, peptides or mutant polypeptides with mutations arising from splice site mutations, frameshift mutations, read-through mutations, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or protein in tumor cells versus normal cells.

[0055] Peptides containing previously identified tumor-specific mutations are also within the scope of the present invention. Known tumor mutations can be found at http: / / www.sanger.ac.uk / cosmic.

[0056] A variety of methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this area have provided accurate, easy, and low-cost large-scale SNP genotyping. Recently, several new techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods require amplification of the target gene region, typically by PCR. Yet other newly developed methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling circle amplification may ultimately eliminate the need for PCR. Some of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. The method of the present invention is understood to include all available methods.

[0057] PCR-based detection methods can include simultaneous multiplex amplification of multiple markers.For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.Alternatively, it is possible to amplify different markers using primers that are differentially labeled and therefore can be differentially detected.Of course, hybridization-based detection methods allow for differential detection of multiple PCR products in a sample.Other techniques that allow for multiplex analysis of multiple markers are known in the art.

[0058] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. In one embodiment, single nucleotide polymorphisms can be detected by using specific exonuclease-resistant nucleotides, as disclosed, for example, in Mundy, CR (U.S. Pat. No. 4,656,127). According to this method, a primer complementary to the allelic sequence immediately 3' from the polymorphic site is hybridized to a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative present, that derivative will be incorporated into the end of the hybridized primer. Such incorporation renders the primer exonuclease-resistant, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative in the sample is known, the finding that the primer has become exonuclease-resistant reveals that the nucleotide present at the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.

[0059] In another embodiment of the present invention, a solution-based method is used to determine the identity of the nucleotide at a polymorphic site. Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO 91 / 02087). Similar to the method of Mundy in U.S. Pat. No. 4,656,127, a primer complementary to the allelic sequence immediately 3' to the polymorphic site is utilized. The method uses a labeled dideoxynucleotide derivative that will be incorporated onto the end of the primer if it is complementary to the nucleotide at the polymorphic site to determine the identity of the nucleotide at that site.

[0060] Another method, known as Genetic Bit Analysis or GBA®, is described by Goelet, P. et al. (PCT Application No. 92 / 15712). The Goelet, P. et al. method uses a mixture of labeled terminators and primers complementary to the sequence 3' to the polymorphic site. Thus, the labeled terminators incorporated are determined by and complementary to the nucleotide present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; PCT Application No. W091 / 02087), the Goelet, P. et al. method is preferably a heterogeneous assay in which either the primer or the target molecule is immobilized on a solid phase.

[0061] Recently, several primer-guided nucleotide incorporation methods have been described for assaying polymorphic sites in DNA. TIFF0007801134000001.tif38169. These methods differ from GBA® in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at polymorphic sites. In such formats, signal is proportional to the number of incorporated deoxynucleotides, so that polymorphisms occurring in a run of identical nucleotides can result in a signal proportional to the length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0062] Numerous initiatives are currently underway to obtain sequence information directly and in parallel from millions of individual DNA or RNA molecules. Real-time single-molecule sequencing-by-synthesis (SBS) techniques rely on the detection of fluorescent nucleotides as they are incorporated into nascent strands of DNA complementary to the template being sequenced. In one method, 30-50 base-long oligonucleotides are covalently attached at their 5' ends to a coverslip. These attached strands serve two functions. First, if the template is configured with a capture tail complementary to the surface-bound oligonucleotide, they serve as capture sites for the target template strand. They also act as primers for template-directed primer extension, which forms the basis of sequence readout. The capture primers serve as immobilized sites for sequencing using multiple cycles of synthesis, detection, and chemical cleavage of the dye linker to remove the dye. Each cycle consists of the addition of a polymerase / labeled nucleotide mixture, rinsing, imaging, and dye cleavage. In another method, the polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, and each nucleotide is color-coded with an acceptor fluorescent moiety attached to the gamma phosphate. The system detects the interaction between the fluorescently tagged polymerase and the fluorescently modified nucleotide as the nucleotide is incorporated into the nascent strand. Other SBS techniques also exist.

[0063] Preferably, any suitable SBS platform can be used to identify mutations. As mentioned above, four major SBS platforms are currently available: Roche / 454 Life Sciences' Genome Sequencers, Illumina / Solexa's 1G Analyzer, Applied BioSystems' SOLiD system, and Helicos Biosciences' Heliscope system. SBS platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies. Each of these platforms can be used in the methods of the present invention. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize nucleic acids on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. Nucleic acids can also be attached to a support by hybridizing the capture sequence to a complementary sequence covalently attached to the support. A capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to a sequence attached to the support, which can double as a universal primer.

[0064] Instead of a capture sequence, a member of a coupling pair (such as, for example, an antibody / antigen pair, a receptor / ligand pair, or an avidin-biotin pair, e.g., as described in U.S. Patent Application Publication No. 2006 / 0252077) may be linked to each fragment for capture onto a surface coated with the respective second member of the coupling pair.

[0065] After capture, the sequence can be analyzed by single-molecule detection / sequencing, including, for example, template-dependent SBS, as described in the Examples and U.S. Patent No. 7,283,337. In SBS, the surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated at the 3' end of the growing chain. This can be done in real time or in a step-and-repeat mode. For real-time analysis, each nucleotide can incorporate a different optical label, and multiple lasers can be used to stimulate the incorporated nucleotides.

[0066] Any cell type or tissue can be used to obtain the nucleic acid sample for use in the diagnosis described herein.In a preferred embodiment, DNA sample or RNA sample is obtained from tumor or body fluid, for example, blood obtained by known technique (for example, venipuncture) or saliva.Alternatively, nucleic acid test can be carried out on dry sample (for example, hair or skin).

[0067] Alternatively, protein mass spectrometry may be used to identify or demonstrate the presence of mutant peptides bound to MHC proteins on tumor cells. Peptides can be acid-eluted from tumor cells or from HLA molecules immunoprecipitated from tumors and then identified using mass spectrometry.

[0068] Neoantigenic peptides The present invention further includes isolated peptides containing tumor-specific mutations identified by the methods of the present invention, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods of the present invention. These peptides and polypeptides are referred to herein as "neo-antigenic peptides" or "neo-antigenic polypeptides." The term "peptide" typically refers to a series of residues, typically L-amino acids, connected to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acids, and is therefore used interchangeably herein with "mutant peptide" and "neo-antigenic peptide." Similarly, the term "polypeptide" typically refers to a series of residues, typically L-amino acids, connected to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acids, and is therefore used interchangeably herein with "mutant polypeptide" and "neo-antigenic polypeptide." Polypeptides or peptides can be of various lengths, can be in neutral (uncharged) form or in salt form, and can be free of or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein.

[0069] In certain embodiments, the size of the at least one neo-antigenic peptide molecule can include, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In a specific embodiment, the neo-antigenic peptide molecule is less than or equal to 50 amino acids.

[0070] In some embodiments, specific neo-antigenic peptides and polypeptides of the invention are 13 residues in length or less, generally about 8 to about 11 residues, specifically 9 or 10 residues, for MHC class I; and 15 to 24 residues for MHC class II.

[0071] Longer peptides may be designed in several ways. In one case, when HLA-binding peptides are predicted or known, the longer peptides may consist of either: (1) individual binding peptides with 2-5 amino acid extensions toward the N- and C-termini of each corresponding gene product; or (2) concatenation of some or all of the binding peptides with their respective extension sequences. In another case, when sequencing reveals a long (>10 residue) neoepitope sequence present in the tumor (e.g., due to frameshift, readthrough, or intron inclusion resulting in a novel peptide sequence), the longer peptide may consist of (3) the entire novel tumor-specific stretch of amino acids, thus avoiding the need for computer prediction or in vitro testing of peptide binding to HLA proteins. In either case, the use of longer peptides may allow for endogenous processing by the patient's cells, resulting in more effective antigen presentation and induction of T cell responses.

[0072] Neo-antigenic peptides and neo-antigenic polypeptides bind to HLA proteins. In some aspects, neo-antigenic peptides and neo-antigenic polypeptides bind to HLA proteins with higher affinity than wild-type peptides. The neo-antigenic peptides or neo-antigenic polypeptides have an IC50 of at least 5000 nM, at least 500 nM, at least 250 nM, at least 200 nM, at least 150 nM, at least 100 nM, at least 50 nM, or less.

[0073] The neoantigenic peptides and polypeptides do not induce an autoimmune response and / or induce immune tolerance when administered to a subject.

[0074] The present invention also provides compositions comprising at least two or more neo-antigenic peptides. In some embodiments, the composition contains at least two distinct peptides. Preferably, the at least two distinct peptides are derived from the same polypeptide. Distinct polypeptides refer to peptides that differ in length, amino acid sequence, or both. The peptides are derived from any polypeptide known to contain tumor-specific mutations or discovered by the methods of the present invention. Suitable polypeptides from which neo-antigenic peptides can be derived can be found, for example, in the COSMIC database (http: / / www.sanger.ac.uk / cosmic). COSMIC maintains comprehensive information on somatic mutations in human cancers. The peptides contain tumor-specific mutations. In some aspects, the tumor-specific mutations are driver mutations for a particular cancer type. In some aspects, the peptides are derived from an SF3B1 polypeptide, a MYD88 polypeptide, a TP53 polypeptide, an ATM polypeptide, an Abl polypeptide, an FBXW7 polypeptide, a DDX3X polypeptide, a MAPK1 polypeptide, or a GNB1 polypeptide.

[0075] An SF3B1 peptide refers to a peptide containing a portion of the SF3B1 polypeptide. Preferably, the SF3B1 peptide contains, when numbered based on wild-type SF3B1, a leucine at amino acid position 625, a histidine at amino acid position 626, a glutamic acid at amino acid position 700, an aspartic acid at amino acid position 742, or an arginine at amino acid position 903. Wild-type SF3B1 is shown in Table A (SEQ ID NO: 1).

[0076] (Table A) Wild type SF3B1 (SEQ ID NO:1) TIFF0007801134000002.tif133136

[0077] MYD88 peptide refers to a peptide containing a portion of the MYD88 polypeptide. Preferably, the MYD88 peptide contains, when numbered based on wild-type MYD88, a threonine at amino acid position 232, a leucine at amino acid position 258, or a proline at amino acid position 265. Wild-type MYD88 is shown in Table B (SEQ ID NO:2).

[0078] (Table B) Wild type MYD88 (SEQ ID NO:2) TIFF0007801134000003.tif37136

[0079] A TP53 peptide refers to a peptide containing a portion of a TP53 polypeptide. Preferably, the TP53 peptide contains, numbered based on wild-type TP53, an arginine at amino acid position 111; an arginine at amino acid position 215; a serine at amino acid position 238; a glutamine at amino acid position 248; a phenylalanine at amino acid position 255; a cysteine ​​at amino acid position 273, or an asparagine at amino acid position 281. Wild-type TP53 is shown in Table C (SEQ ID NO:3).

[0080] (Table C) Wild type TP53 (SEQ ID NO:3) TIFF0007801134000004.tif41134

[0081] The ATM peptide refers to a peptide containing a portion of the SF3B1 polypeptide. Preferably, the ATM peptide contains, based on wild-type ATM, phenylalanine at amino acid position 1252, arginine at amino acid position 2038, histidine at amino acid position 2522, or cysteine ​​at amino acid position 2954.

[0082] Wild-type ATM is shown in Table D (SEQ ID NO:4).

[0083] (Table D) Wild type ATM (SEQ ID NO:4) TIFF0007801134000005.tif36136TIFF0007801134000006.tif231134TIFF0007801134000007.tif35134

[0084] The term "Abl peptide" refers to a peptide containing a portion of an Abl polypeptide. Preferably, the Bcr-abl peptide, when numbered based on wild-type Abl, contains the following amino acids: valine at amino acid position 244; valine at amino acid position 248; glutamic acid at amino acid position 250; alanine at amino acid position 250; histidine at amino acid position 252; arginine at amino acid position 252; phenylalanine at amino acid position 253; histidine at amino acid position 253; lysine at amino acid position 255; valine at amino acid position 255; glycine at amino acid position 276; isoleucine at amino acid position 315. and / or a serine at amino acid position 486. Wild-type AbI is shown in Table E (SEQ ID NO:5).

[0085] (Table E) Wild type Abl (SEQ ID NO:5) TIFF0007801134000008.tif70157

[0086] FBXW7 peptide refers to a peptide containing a portion of the FBXW7 polypeptide. Preferably, the FBXW7 peptide contains, based on wild-type FBXW7, a leucine at amino acid position 280, a histidine at amino acid position 465, a cysteine ​​at amino acid position 505, or a glutamic acid at amino acid position 597. Wild-type FBXW7 is shown in Table F (SEQ ID NO: 6).

[0087] (Table F) Wild type FBXW7 (SEQ ID NO:6) TIFF0007801134000009.tif75134

[0088] A DDX3X peptide refers to a peptide containing a portion of a DDX3X polypeptide. A DDX3X peptide is a peptide that is the result of a missense mutation at amino acid position 24, a splice site at amino acid position 342, or a frameshift at amino acid position 410, based on wild-type DDX3X. Wild-type DDX3X is shown in Table G (SEQ ID NO:7).

[0089] (Table F) Wild type DDX3X (SEQ ID NO:7) TIFF0007801134000010.tif70134

[0090] A MAPK1 peptide refers to a peptide containing a portion of a MAPK1 polypeptide. Preferably, the MAPK1 peptide contains, based on wild-type MAPK1, an asparagine at amino acid position 162, a glycine at amino acid position 291, or a phenylalanine at amino acid position 316. Wild-type MAPK1 is shown in Table H (SEQ ID NO:8).

[0091] (Table F) Wild type MAPK1 (SEQ ID NO:8) TIFF0007801134000011.tif41134

[0092] A GNB1 peptide refers to a peptide containing a portion of the GNB1 polypeptide. Preferably, the GNB1 peptide contains a threonine at amino acid position 180, based on wild-type GNB1. Wild-type GNB1 is shown in Table I (SEQ ID NO:9).

[0093] (Table I) Wild type GNB1 (SEQ ID NO:9) TIFF0007801134000012.tif36134

[0094] Neoantigenic peptides and polypeptides with desired activity may be modified, if necessary, to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide in binding to desired MHC molecules and activating appropriate T cells. For example, neoantigenic peptides and polypeptides can undergo various changes, such as conservative or non-conservative substitutions, which may provide certain advantages in use, such as improved MHC binding. Conservative substitutions refer to the exchange of an amino acid residue with another amino acid that is biologically and / or chemically similar, e.g., the exchange of hydrophobic residues with one another, or the exchange of polar residues with another. Substitutions include Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and combinations such as Phe and Tyr. The effects of single amino acid substitutions may also be explored using D-amino acids. Such modifications can be made using well-known peptide synthesis methods, such as those described in, for example, Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0095] Neoantigenic peptides and polypeptides may be modified, for example, by adding or deleting amino acids to extend or reduce the amino acid sequence of the compound. Peptides, polypeptides, or analogs may also be modified by altering the order or composition of certain residues, although it is readily recognized that certain amino acid residues essential for biological activity, such as those at critical contact sites or conserved residues, generally cannot be altered without adverse effects on biological activity. Non-essential amino acids are not necessarily limited to those naturally occurring in proteins, such as L-α amino acids or their D-isomers, but also include unnatural amino acids such as β-γ-δ-amino acids, and many derivatives of L-α amino acids.

[0096] Typically, a series of peptides with single amino acid substitutions are used to determine the effects on binding of electrostatic charge, hydrophobicity, etc. For example, to reveal patterns of differential sensitivity to various MHC molecules and T cell receptors, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide. Additionally, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be used. Substitutions may be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the spacing required between essential contact points and certain functional attributes (e.g., hydrophobicity vs. hydrophilicity) desired. Increased binding affinity for MHC molecules or T cell receptors compared to that of the parent peptide may also be achieved by such substitutions. In any case, such substitutions should utilize amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference that could disrupt binding.

[0097] Amino acid substitutions are typically single residue substitutions. Substitutions, deletions, insertions, or any combination thereof may be combined to obtain the final peptide. Substitution variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place. When it is desired to finely modulate the characteristics of a peptide, such substitutions are generally made according to the following table. TIFF0007801134000013.tif128137

[0098] Substantial alterations in function (e.g., affinity for MHC molecules or T cell receptors) can be made by selecting substitutions that are less conservative than those in the table above, i.e., by selecting residues that differ more significantly in terms of (a) the effect on maintaining the structure of the peptide backbone in the area of ​​the substitution, e.g., sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. In general, the substitutions expected to most significantly alter peptide properties will be (a) the substitution of a hydrophilic residue, such as seryl, for a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) the substitution of a residue with a positively charged side chain, such as lysyl, arginyl, or histidyl, for a negatively charged residue, such as glutamyl or aspartyl; or (c) the substitution of a residue with a bulky side chain, such as phenylalanine, for one without a side chain, such as glycine.

[0099] Peptides and polypeptides may contain two or more isosteres of a neoantigenic peptide or polypeptide. An isostere, as defined herein, is a sequence of two or more residues that can be substituted for a second sequence because the conformation of the first sequence is compatible with the binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, alpha carbon, amide carbonyl, complete replacement, extension, deletion, or backbone crosslinking of the amide bond. See generally Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0100] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids is particularly useful for increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in a variety of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). The half-life of the peptides of the present invention is conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows: Pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0101] Peptides and polypeptides may be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a T helper cell response. Particularly preferred immunogenic peptide / T helper conjugates are linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that the optional spacer need not be composed of identical residues and may therefore be a hetero- or homo-oligomer. If present, the spacer will generally be at least one or two residues, more typically three to six residues. Alternatively, the peptide may be linked to the T helper peptide without a spacer.

[0102] The neoantigenic peptide can be linked to a T helper peptide either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the neoantigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398, and 378-389.

[0103] Proteins or peptides can be produced by any technique known to those skilled in the art, including expressing proteins, polypeptides, or peptides through standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotides corresponding to various genes, as well as protein, polypeptide, and peptide sequences, have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0104] In a further aspect, the present invention provides nucleic acids (e.g., polynucleotides) encoding the neo-antigenic peptides of the present invention. The polynucleotides can be, for example, single- and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or native or stabilized polynucleotides, e.g., polynucleotides with phosphorothioate backbones, or combinations thereof, and may or may not contain introns, so long as they encode the peptide. Of course, only peptides containing naturally occurring amino acid residues joined by naturally occurring peptide bonds can be encoded by the polynucleotide. A further aspect of the present invention provides expression vectors capable of expressing the polypeptides of the present invention. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0105] Vaccine Composition The present invention relates to immunogenic compositions, e.g., vaccine compositions, capable of generating specific T cell responses, comprising mutant peptides and mutant polypeptides corresponding to tumor-specific neoantigens identified by the methods described herein.

[0106] Those skilled in the art will be able to select preferred peptides, polypeptides, or combinations thereof, for example, by testing in vitro T cell generation and their efficiency and total abundance, proliferation, affinity, and expansion of certain T cells to certain peptides, and T cell functionality, e.g., by analyzing IFN-γ production or tumor killing by T cells. Typically, the most efficient peptides are then combined as a vaccine.

[0107] A suitable vaccine will preferably contain between 1 and 20 different peptides, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different peptides, even more preferably 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, and most preferably 12, 13, or 14 different peptides.

[0108] In one embodiment of the present invention, different peptides and / or polypeptides are selected so that a single vaccine composition comprises peptides and / or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecules. Preferably, a single vaccine composition comprises peptides and / or polypeptides capable of associating with the most frequently occurring MHC class I molecules. Thus, a vaccine composition according to the present invention comprises different fragments capable of associating with at least two, more preferably at least three, and even more preferably at least four preferred MHC class I molecules.

[0109] The vaccine composition may generate a specific cytotoxic T cell response and / or a specific helper T cell response.

[0110] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The peptides and / or polypeptides in the composition may be associated with a carrier, such as, for example, a protein or an antigen-presenting cell, such as, for example, a dendritic cell (DC), which can present the peptide to T cells.

[0111] An adjuvant is any substance that, when incorporated into a vaccine composition, increases or otherwise modifies the immune response to a mutant peptide. A carrier is a scaffold, such as a polypeptide or polysaccharide, to which a neo-antigenic peptide can associate. Optionally, the adjuvant is covalently or non-covalently conjugated to the peptide or polypeptide of the present invention.

[0112] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised against the antigen, and an increase in T cell activity is typically manifested in increased cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also modify the immune response, for example, by changing a primarily humoral or Th response to a primarily cellular or Th response.

[0113] Suitable adjuvants include 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA Adjuvants include, but are not limited to, 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel™ vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts, and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are preferred. Several immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines may also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNFα), promoting dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety), and functioning as immunoadjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0114] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or inactivated viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates, in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, even in the absence of CD4 T cell help, leading to enhanced activation of TH1 cells and the generation of potent cytotoxic T lymphocytes (CTLs). The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly needed to induce strong responses when the antigen is relatively weak. In some experiments, they have also accelerated immune responses, enabling antigen doses to be reduced by approximately two orders of magnitude while still achieving antibody responses comparable to full-dose vaccines without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, pp. 471-484). U.S. Patent No. 6,406,705 B1 describes the use of CpG oligonucleotides in combination with non-nucleic acid adjuvants and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical compositions of the present invention. Other TLR binding molecules, such as RNA that binds to TLR7, TLR8, and / or TLR9, may also be used.

[0115] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and also include, but are not limited to, small molecules and antibodies with immunological activity, such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0116] Vaccine compositions of the present invention may contain multiple different adjuvants. Additionally, the present invention encompasses therapeutic compositions containing any adjuvant substance, including any of the above or a combination thereof. It is contemplated that the peptide or polypeptide and the adjuvant may be administered separately, in any appropriate order.

[0117] The carrier may be present independently of the adjuvant. The function of the carrier may be, for example, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life, particularly to increase the molecular weight of the variant. Furthermore, the carrier may assist in presenting the peptide to T cells. The carrier may be any suitable carrier known to those skilled in the art, such as a protein or antigen-presenting cell. The carrier protein may be, but is not limited to, a serum protein such as keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, an immunoglobulin, or a hormone such as insulin, or palmitic acid. For human immunization, the carrier must be a physiologically acceptable carrier that is tolerable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers in one embodiment of the present invention. Alternatively, the carrier may be dextran, such as Sepharose.

[0118] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than the complete foreign antigen itself. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible only when a trimeric complex of peptide antigen, MHC molecule, and APC is present. Therefore, not only can peptides be used to activate CTLs, but also, when APCs bearing the respective MHC molecules are added, they can enhance the immune response. Therefore, in some embodiments, the vaccine composition of the present invention further contains at least one type of antigen-presenting cell.

[0119] Antigen-presenting cells (or stimulator cells) typically have MHC class I or II molecules on their surface and, in one embodiment, are themselves substantially incapable of loading MHC class I or II molecules with a selected antigen. As described in more detail below, MHC class I or II molecules can readily be loaded with a selected antigen in vitro.

[0120] Preferably, the antigen-presenting cells are dendritic cells. Suitably, the dendritic cells are autologous dendritic cells pulsed with neoantigenic peptides. The peptides may be any suitable peptide that confers an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides derived from tumor-associated antigens is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.

[0121] Thus, in one embodiment of the invention, a vaccine composition containing at least one antigen-presenting cell is pulsed or loaded with one or more peptides of the invention. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded with peptides ex vivo and injected back into the patient.

[0122] Alternatively, the antigen-presenting cells comprise an expression construct encoding a peptide of the invention. The polynucleotide may be any suitable polynucleotide, but preferably is capable of transducing dendritic cells, thereby resulting in presentation of the peptide and induction of immunity.

[0123] treatment method The present invention further provides methods for inducing a tumor-specific immune response in a subject, vaccinating against a tumor, treating cancer in a subject, or alleviating symptoms thereof, by administering to the subject a neo-antigenic peptide or neo-antigenic vaccine composition of the present invention.

[0124] The subject is diagnosed with cancer or is at risk of developing cancer. The subject has imatinib-resistant tumor. The subject is human, dog, cat, horse, or any animal for which tumor-specific immune response is desired. The tumor is any solid tumor such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs, and blood tumors such as lymphoma and leukemia, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-lymphocytic leukemia, and B-cell lymphoma.

[0125] The peptides or compositions of the invention are administered in an amount sufficient to induce a CTL response.

[0126] In specific embodiments, the present invention provides a method for treating imatinib-resistant tumors by administering one or more neoantigenic peptides containing bcr-abl mutations to a subject. In some embodiments, the subject is HLA-A3. Bcr-abl mutations include, for example, T315I, E255K, M351T, Y253H, Q252H, F317L, F359V, G250E, Y253F, E355G, E255V, M244V, L248V, G250A, Q252R, D276G, T315N, M343T, F359A, V379I, F382L, L387M, H396P, H396R, S417Y, and F486S.

[0127] The neoantigenic peptide, neoantigenic polypeptide, or neoantigenic vaccine compositions of the present invention can be administered alone or in combination with other therapeutic agents, such as chemotherapeutic agents, radiation, or immunotherapy. Any appropriate therapeutic treatment for the particular cancer can be administered. Examples of chemotherapeutic agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, and ifosfamide. , interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. A preferred chemotherapeutic agent that can be combined with anti-CTLA-4 for the treatment of prostate cancer is paclitaxel (Taxol®).

[0128] Furthermore, the subject may be further administered an anti-immunosuppressant / immunostimulatory agent. For example, the subject may be further administered an anti-CTLA antibody, or anti-PD-1, or anti-PD-L1. Blocking CTLA-4 or PD-L1 with an antibody can enhance the immune response against cancer cells in patients. In particular, CTLA-4 blockade has been shown to be effective when used in conjunction with a vaccination protocol.

[0129] The optimal amount and optimal dosing regimen of each peptide to be included in the vaccine composition can be determined by one of skill in the art without undue experimentation. For example, the peptide or its variant can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 1 to 500 mg, 50 μg to 1.5 mg, or preferably 125 μg to 500 μg of peptide or DNA can be given, depending on the respective peptide or DNA. Doses in this range have been used successfully in previous studies (Brunsvig PF, et al., Cancer Immunol Immunother. 2006; 55(12):1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administering vaccine compositions are known to those skilled in the art.

[0130] Pharmaceutical compositions of the present invention can be formulated so that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, and / or patient-specific. For example, the precise selection of peptides can be guided by the expression pattern of the parent protein in a given tissue to avoid side effects. Selection can depend on the specific type of cancer, the state of the disease, the initial treatment regimen, the patient's immune status, and, of course, the patient's HLA haplotype. Furthermore, vaccines of the present invention can contain components that are tailored to the individual needs of a particular patient. Examples include the expression of relevant neoantigens in a particular patient, varying the amount of peptides depending on the individual's allergies or other unwanted side effects of treatment, and adjusting for secondary treatments after an initial round or scheme of treatment.

[0131] In order to use the composition as a vaccine against cancer, peptides whose endogenous parent protein is highly expressed in normal tissues will be avoided or present in low amounts in the compositions of the present invention. On the other hand, if a patient's tumor is known to highly express a certain protein, each pharmaceutical composition for treating that cancer may be present in high amounts and / or may include multiple peptides specific for that particular protein or pathway of that protein.

[0132] Pharmaceutical compositions containing the peptides of the present invention can be administered to individuals already suffering from cancer. In therapeutic applications, the compositions are administered to patients in an amount sufficient to induce an effective CTL response against tumor antigens and to cure or at least partially arrest symptoms and / or complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Effective amounts for this use will depend, for example, on the composition of the peptide, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician, but generally, for a 70 kg patient, an initial immunization (therapeutic or prophylactic administration) will range from about 1.0 μg to about 50,000 μg of peptide, followed by a booster schedule of about 1.0 μg to about 10,000 μg of peptide over several weeks to several months, depending on the patient's response and condition, as determined by measuring specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present invention may generally be utilized in severe disease states, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized. In such cases, given the minimization of foreign substances and the relatively non-toxic nature of the peptides, it may be possible and desirable for the treating physician to administer substantial overdose of these peptide compositions.

[0133] For therapeutic use, administration should begin at the time of tumor detection or surgical removal, followed by booster doses until at least symptoms are substantially alleviated and for some period thereafter.

[0134] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are suitable for parenteral, topical, nasal, oral, or local administration. Preferably, pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. To induce a localized immune response against tumors, compositions can also be administered at the site of surgical resection. The present invention provides parenteral compositions comprising a solution of peptides, where the vaccine composition is dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solution can be packaged for immediate use or lyophilized, and the lyophilized preparation can be combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0135] The concentration of the peptide of the present invention in a pharmaceutical formulation may vary widely, i.e., from less than about 0.1% by weight, generally at least about 2% by weight, up to 20-50% by weight, and will be selected primarily depending on the liquid volume, viscosity, etc., depending on the particular mode of administration selected.

[0136] The peptides of the present invention may be administered via liposomes, which target the peptide to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for increasing the half-life of peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar materials, and the like. In these preparations, the peptide to be delivered is incorporated as part of the liposome, either alone or in combination with a molecule that binds to a receptor prevalent in lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Liposomes loaded with the desired peptide of the present invention are thus directed to the site of lymphoid cells, where they can then deliver the selected therapeutic / immunogenic peptide composition. Liposomes for use in the present invention are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by considerations such as liposome size, acid lability and stability of the liposomes in the bloodstream. See, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. 4,235,871 No., no. 4,501,728 No., no. 4,837,028 No., and No. 5,019,369 A variety of methods are available for the preparation of liposomes, such as those described in US Pat. No. 6,239,999.

[0137] Ligands incorporated into liposomes for targeting to immune cells include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. Liposomal suspensions containing peptides can be administered intravenously, topically, locally, etc., at doses that vary depending, inter alia, on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0138] For solid compositions, conventional or nanoparticulate non-toxic solid carriers may be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, pharmaceutically acceptable non-toxic compositions are formed by incorporating any of the commonly employed excipients, such as the above-listed carriers, with the active ingredient, i.e., one or more peptides of the present invention, generally in a concentration of 10-95%, more preferably 25-75%.

[0139] For aerosol administration, the immunogenic peptide is preferably supplied in finely divided form along with a surfactant and propellant. Typical proportions of peptide are 0.01-20% by weight, preferably 1-10% by weight. The surfactant must, of course, be nontoxic and preferably soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6-22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid, with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed glycerides or natural glycerides, may also be utilized. The surfactant may comprise 0.1-20% by weight, preferably 0.25-5% by weight, of the composition. The remainder of the composition is usually the propellant. A carrier may be included, as desired, such as lecithin for intranasal delivery.

[0140] For therapeutic or immunization purposes, nucleic acids encoding the peptides of the invention, and optionally one or more of the peptides described herein, may be administered to a patient. A number of methods are conveniently used to deliver nucleic acids to a patient. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and U.S. Pat. 5,580,859 No. and No. 5,589,466Nucleic acids are described, for example, in U.S. Pat. 5,204,253 The DNA may be administered using ballistic delivery, as described in the article "Dysfunctional DNA Delivery," or by administering particles composed solely of DNA. Alternatively, the DNA may be attached to particles, such as gold particles.

[0141] Nucleic acids can also be delivered complexed with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988); Rose, U.S. Pat. 5,279,833 No. WO 91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0142] The peptides and polypeptides of the present invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences encoding the peptides of the present invention. When introduced into an acutely or chronically infected host, or into an uninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, thereby eliciting a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. 4,722,848 No. 6,239,663. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as Salmonella typhi vectors, will become apparent to those skilled in the art from the description herein.

[0143] A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding multiple epitopes. To generate DNA sequences (minigenes) encoding selected CTL epitopes for expression in human cells, the amino acid sequences of the epitopes are reverse-translated. A human codon usage table is used to guide codon selection for each amino acid. The DNA sequences encoding these epitopes are directly adjacent to each other to create a continuous polypeptide sequence. Additional elements may be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse-translated and included in the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Furthermore, MHC presentation of CTL epitopes can be improved by including synthetic flanking sequences (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitopes.

[0144] The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then be cloned into the desired expression vector.

[0145] To ensure expression in the target cells, standard regulatory sequences well known to those skilled in the art are included in the vector. Several vector elements are required: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin resistance or kanamycin resistance). Many promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. Other suitable promoter sequences are described in U.S. Pat.5,580,859 No. and No. 5,589,466 Please refer to the issue.

[0146] Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences may also be considered to increase minigene expression. It has recently been proposed that immune stimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. These sequences can be included outside the minigene coding sequence in the vector if found to enhance immunogenicity.

[0147] In some embodiments, a bicistronic expression vector can be used to allow production of a minigene-encoded epitope and a second protein included to enhance or reduce immunogenicity. Examples of proteins or polypeptides that can beneficially enhance immune responses when coexpressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), or costimulatory molecules. Helper (HTL) epitopes can be conjugated to intracellular targeting signals and expressed separately from CTL epitopes. This would allow the HTL epitopes to be directed to different cellular compartments than the CTL epitopes. If required, this can facilitate more efficient entry of the HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically reducing the immune response by coexpressing immunosuppressive molecules (e.g., TGF-β) can be beneficial in certain diseases.

[0148] Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene and all other elements contained in the vector are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as master and working cell banks.

[0149] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA with sterile phosphate-buffered saline (PBS). A variety of methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. Additionally, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective, interactive, non-condensing (PINC) compounds can be complexed with purified plasmid DNA to affect variables such as stability, intramuscular distribution, or transport to specific organs or cell types.

[0150] Target cell sensitization can be used as a functional assay for expression and MHC class I presentation of the minigene-encoded CTL epitope. Plasmid DNA is introduced into a suitable mammalian cell line as a target for a standard CTL chromium release assay. The transfection method used will depend on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow for direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) may also be cotransfected to allow enrichment of transfected cells using fluorescence-activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. Cell lysis, detected by 51Cr release, indicates the occurrence of MHC presentation of the minigene-encoded CTL epitope.

[0151] In vivo immunogenicity is a second approach for functional testing of minigene-DNA formulations. Transgenic mice expressing the appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are formulation-dependent (e.g., IM for DNA in PBS, IP for DNA complexed with lipids). 21 days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromium-51-labeled target cells using standard techniques. Lysis of target cells primed by MHC loading of peptides corresponding to minigene-encoded epitopes indicates that the DNA vaccine is functional for in vivo induction of CTLs.

[0152] Peptides may be used to induce CTLs ex vivo. The resulting CTLs can be used to treat chronic tumors in patients who do not respond to other conventional types of therapy or who would not respond to peptide vaccine therapeutic approaches. Ex vivo CTL responses against specific tumor antigens are induced by incubating the patient's CTL precursor cells (CTLp) with a source of antigen-presenting cells (APCs) and the appropriate peptide in tissue culture. After an appropriate incubation period (typically 1-4 weeks), during which the CTLp are activated and mature and expand into effector CTLs, the cells are infused back into the patient, where they will destroy specific target cells (i.e., tumor cells). To optimize in vitro conditions for the generation of specific cytotoxic T cells, stimulator cell cultures are maintained in an appropriate serum-free medium.

[0153] Prior to incubation of the stimulator cells with the cells to be activated, e.g., precursor CD8+ cells, a sufficient amount of antigenic peptide is added to the stimulator cell culture to load human class I molecules and express them on the surface of the stimulator cells. In the present invention, a sufficient amount of peptide is an amount that will allow approximately 200, preferably 200 or more, peptide-loaded human class I MHC molecules to be expressed on the surface of each stimulator cell. Preferably, the stimulator cells are incubated with >2 μg / ml of peptide. For example, the stimulator cells are incubated with >3, 4, 5, 10, 15 μg / ml or more of peptide.

[0154] The resting or precursor CD8+ cells are then incubated in culture with appropriate stimulator cells for a period sufficient to activate the CD8+ cells. Preferably, the CD8+ cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8+ (effector) cells to stimulator cells may vary from individual to individual and may depend on variables such as the adaptability of the individual's lymphocytes to the culture conditions and the nature and severity of the disease state or other condition for which the treatment modalities described herein are used. Preferably, however, the lymphocyte:stimulator cell ratio is within the range of about 30:1 to 300:1. The effector / stimulator cell culture may be maintained for as long as necessary to stimulate a therapeutically usable or effective number of CD8+ cells.

[0155] In vitro CTL induction requires specific recognition of peptides bound to allele-specific MHC class I molecules on APCs. The number of specific MHC / peptide complexes per APC is important for CTL stimulation, particularly in primary immune responses. While a small number of peptide / MHC complexes per cell is sufficient to sensitize cells to CTL lysis or stimulate secondary CTL responses, a significantly higher number of MHC / peptide complexes is required for successful activation of CTL precursors (pCTLs) in primary responses. Peptide loading of empty major histocompatibility complex molecules on cells allows for the induction of primary cytotoxic T lymphocyte responses.

[0156] Because mutant cell lines for all human MHC alleles do not exist, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APCs, and then load the resulting empty MHC molecules with the immunogenic peptide of interest.The use of non-transformed (non-tumorigenic) uninfected cells, preferably the patient's autologous cells, as APCs is desirable for designing CTL induction protocols for the development of ex vivo CTL therapy.The present application discloses a method for stripping endogenous MHC-associated peptides from the surface of APCs, and then loading the desired peptide.

[0157] Stable MHC class I molecules are trimeric complexes formed from the following components: (1) a peptide, typically 8–10 residues long; (2) a transmembrane polymorphic protein heavy chain that harbors peptide-binding sites in the α1 and α2 domains; and (3) a noncovalently associated nonpolymorphic light chain, β2-microglobulin. Removal of the bound peptide and / or dissociation of β2-microglobulin from the complex renders the MHC class I molecule nonfunctional, unstable, and subject to rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to MHC class I molecules on APCs without causing degradation before exogenous peptides are added.

[0158] Two possible methods for removing bound peptides from MHC class I molecules include lowering the culture temperature from 37°C to 26°C overnight to destabilize β2-microglobulin, and stripping endogenous peptides from cells using mild acid treatment. These methods release previously bound peptides into the extracellular environment, allowing new exogenous peptides to bind to empty class I molecules. The low-temperature incubation method allows exogenous peptides to efficiently bind to MHC complexes, but requires overnight incubation at 26°C, which may slow the metabolic rate of the cells. Cells that do not actively synthesize MHC molecules (e.g., resting PBMCs) are also unlikely to produce large amounts of empty surface MHC molecules using the low-temperature method.

[0159] Strong acid stripping involves peptide extraction with trifluoroacetic acid (pH 2) or acid denaturation of immunoaffinity-purified class I-peptide complexes. Because it is important to remove endogenous peptides while preserving the viability and optimal metabolic state of APCs, which are critical for antigen presentation, these methods are not feasible for CTL induction. Weakly acidic solutions with a pH of 3, such as glycine buffer or citrate-phosphate buffer, have been used to identify endogenous peptides and identify tumor-associated T cell epitopes. This treatment is particularly effective in that only MHC class I molecules are destabilized (and associated peptides are released), while other surface antigens, including MHC class II molecules, remain intact. Most importantly, treatment of cells with weakly acidic solutions does not affect cell viability or metabolic state. Weak acid treatment is rapid, as stripping of endogenous peptides occurs within 2 minutes at 4°C, and APCs are ready to perform their functions after being loaded with the appropriate peptides. This technique is utilized herein to generate peptide-specific APCs for the generation of primary antigen-specific CTLs. The resulting APCs are efficient in inducing peptide-specific CD8+ CTLs.

[0160] Activated CD8+ cells can be effectively separated from stimulator cells using one of a variety of known methods. For example, a monoclonal antibody (or a segment thereof) specific for stimulator cells, a peptide loaded onto the stimulator cells, or CD8+ cells can be used to bind to an appropriate complementary ligand. The antibody-tagged molecule can then be extracted from the stimulator-effector cell mixture via appropriate means, for example, via well-known immunoprecipitation or immunoassay methods.

[0161] The effective cytotoxic amount of activated CD8+ cells may vary between in vitro and in vivo use, and may also vary depending on the amount and type of cells that are the ultimate target of these killer cells. The amount may also vary depending on the patient's condition and should be determined by the practitioner after considering all appropriate factors. However, preferably, the amount is about 1 x 10 6 ~Approx. 1×10 12 , more preferably about 1 × 10 8 ~Approx. 1×10 11 , more preferably about 1 × 10 9 ~Approx. 1×10 10 activated CD8+ cells are utilized for adult humans, compared to approximately 5×10 in mice. 6 ~5×10 7 cells are used.

[0162] Preferably, as described above, activated CD8+ cells are harvested from cell culture prior to administration of the CD8+ cells to the individual being treated. However, it is important to note that, unlike other existing and proposed treatment modalities, the method of the present invention uses a non-tumorigenic cell culture system. Thus, if complete separation of stimulator cells from activated CD8+ cells is not achieved, there are no inherent risks known to be associated with administering small numbers of stimulator cells, but administration of mammalian tumor-promoting cells can be very harmful.

[0163] Methods for reintroducing cellular components are known in the art and are described in US Pat. No. 6,233,599 to Honsik et al. 4,844,893No. and Rosenberg U.S. Pat. 4,690,915 For example, administration via intravenous infusion of activated CD8+ cells is suitable.

[0164] The invention is further described in the following examples, which do not limit the scope of the invention described in the appended claims. [Example]

[0165] Example 1: Strategy for identifying neoepitopes for vaccination Our approach to identifying tumor-specific neoepitopes involves three steps: (1) Identifying DNA mutations using whole-genome or whole-exome (i.e., captured exons only) sequencing of tumor samples versus matched germline samples from each patient. Our preliminary studies have demonstrated that CLL cells contain many distinct genetic alterations that can alter amino acid sequences and generate potential novel T cell epitopes. (2) Applying a highly validated peptide-MHC binding prediction algorithm to generate a set of candidate T cell epitopes based on non-silent mutations present in tumors. We will confirm the expression of mutant genes as RNA in CLL samples and then confirm the peptide-HLA binding predictions using an experimental approach to quantify binding of candidate peptides to HLA alleles. (3) Generating antigen-specific T cells against the mutant peptides.

[0166] Example 2: Sequencing of tumor and normal genomes to identify mutated genes in tumors of patients with chronic lymphocytic leukemia (Step 1) To detect tumor-specific mutations (not present in normal tissues), samples were collected from each patient's tumor and normal tissue. For leukemia, tumors were purified using magnetic bead isolation or fluorescence-activated cell sorting with tumor cell-specific antibodies. For example, tumor cells from patients with chronic lymphocytic leukemia (CLL) express the surface markers CD5 and CD19. Skin fibroblasts were used as normal tissue controls. DNA or RNA for sequencing was purified from isolated tumor cells or normal tissue cells. For melanoma, ovarian tumors, and other solid tumors (which may contain non-tumor cell contamination), DNA and RNA were isolated from relatively homogenous short-term cultures of tumor cells or laser-captured tumors. PBMCs were used as normal control cells. For all samples, PBMCs were cryopreserved until needed for the expansion of mutant peptide-specific T cells. Finally, short-term cultures of tumor cells were also cryopreserved for later use as targets for expanded T cells. Isolated genomic DNA or RNA was tested for nucleic acid integrity and purity before sequencing.

[0167] For each DNA sample, either the entire genomic DNA was sheared and sequenced, or coding exons were captured with complementary oligonucleotides using hybrid selection and then sequenced (Gnirke et al., Nat Biotechnol. 2009, 27(2):182-9). DNA and RNA libraries were generated and sequenced using Illumina next-generation sequencing instruments.

[0168] Sequencing of 64 patients with chronic lymphocytic leukemia (CLL) revealed an average of 23 non-silent mutations in tumors that altered protein amino acid sequences relative to the germline DNA sequence (Figure 3). These non-silent mutations fell into five distinct classes with the potential to generate neoepitopes: missense, splice site, frameshift (indels, insertions, and deletions), readthrough, and gene fusion (Figure 4). The frequency of these mutations varied among individual patients (Figure 5). While all of these mutations provide potential neoepitopes for immunization, frameshift, readthrough, and splice site mutations (e.g., involving retained introns) generate longer stretches of novel peptides, missense mutations result in short peptides with single amino acid changes, and finally, gene fusions generate hybrid peptides with novel junction sequences.

[0169] Example 3: Identification of HLA-binding peptides derived from expressed proteins carrying tumor-specific mutations (Step 2) The next question was whether the mutant genes could generate peptides that could be presented by the patient's MHC / HLA proteins. First, using several algorithms, we predicted HLA-binding peptides with IC50 scores <500 nM for 30 of the 10 missense mutations in patient 1, 53 missense mutations in patient 2, 1 indel, and 137 of the two gene fusions. An example of one missense mutation in a patient with six specific HLA alleles is shown, along with two predicted binding peptides from 54 combinations of a 9-residue peptide and an HLA allele (Figure 6). To confirm that these genes were expressed in tumors, we measured RNA levels for the mutant genes (using several approaches depending on the mutation class (Figure 7)) and found that 98% of the mutant genes with HLA-binding peptides were expressed.

[0170] The HLA-binding ability of all predicted peptides for which RNA expression was demonstrated was then experimentally verified by performing competitive binding assays of the test peptides against reference peptides known to bind to HLA alleles (Sidney et al. Curr Protoc Immunol. 2001, Chapter 18: Unit 18.3) (Figure 8A). Of the subset subjected to experimental confirmation of HLA binding, 8 of 17 (47%) predicted peptides from missense mutations in Pt1 were confirmed to have high binding affinity for HLA alleles (IC). 50 <500) (Figure 8B). For Pt2, 25 of the 49 predicted peptides were experimentally confirmed as HLA-binding (Figure 8B). These results support the predicted IC 50 All peptides with affinity <150 nM experimentally demonstrated HLA binding, suggesting that a cutoff of <500 nM would yield 40–50% true binding peptides at that time (Figure 8C). Notably, 12 of the 25 confirmed mutant peptides in Pt2 had binding affinity >2-fold better than the germline peptide (Figure 9). While such peptides are preferred for incorporation into tumor vaccines because they reduce the probability of T cell cross-reactivity with the germline peptide, even peptides that do not exhibit differential binding may provide tumor-specific responses due to differential recognition of mutant versus germline peptides by T cell receptors.

[0171] Example 4: CD8+ T cell responses to mutant peptides identified by sequencing of CLL patient samples (Step 3) Based on predicted or experimentally confirmed HLA-binding mutant peptides, it became possible to determine whether T cells recognizing these tumor-specific mutant peptides could be generated. Therefore, we synthesized peptides with binding scores below 1000 nM derived from genes whose expression was demonstrated in tumor cells. To generate T cells with the desired specificity, we stimulated sequenced patient T cells with autologous APCs (dendritic cells and autologous B cells expanded with CD40L) pulsed with peptides (using individual peptides or peptide pools) weekly in the presence of IL-2 and IL-7. After 3–4 stimulations, the expanded CD8+ cells were tested by ELISpot for evidence of reactivity to the peptides based on IFNγ secretion. Among 17 candidate peptides from patient 1, IFNγ secretion was detected in T cells against autologous DCs pulsed with a mutant peptide derived from the TLK2 gene (Figure 10).

[0172] Example 5: Mutant BCR-ABL gene can bind to patient MHC / HLA proteins and induce mutant peptide-specific CD8+ T cells The present inventors conducted a more thorough study of T cell responses to tumor-specific mutant peptides in patients with another type of leukemia, chronic myeloid leukemia (CML). CML is defined by the expression of a tumor-specific translocation that is the product of the BCR-ABL gene fusion. BCR-ABL mutations occur in CML patients who develop drug resistance to state-of-the-art pharmacological treatment with imatinib mesylate, which targets BCR-ABL. These mutations, when bound to MHC proteins, may generate neoepitopes that can be recognized by T cells derived from the host or transplanted normal donors; these T cells are likely to be minimally tolerated.

[0173] We considered the 20 most common mutations evolved in patients with resistance to imatinib and predicted the binding of 9- and 10-residue peptides surrounding each mutation. Using either the prediction algorithms NetMHC (Nielsen et al. PLoS One. 2007, 2(8):e796) or IEDB (Vita R et al. Nucleic Acids Res. 2010, 38:D854-62), we predicted the binding of 84 peptides derived from the 20 common mutations to one or more of the eight common HLA alleles (IC). 50 <1000). Many peptides were derived from the three most common mutations. 24 of the 84 peptides were strong binders (IC 50 <50)(Fig. 11 ), 42 peptides were intermediate binders (50 <IC 50 <500, 18 peptides were weak binders (500 <IC 50 <1000).

[0174] The present inventors have demonstrated that the IC 50 The mutant peptide (E255K-B) generated by the E255K mutation is predicted to bind at a frequency of 1.5 kJ / s. 255-263 ) (KVYEGVWKK) (SEQ ID NO: 10). Using a competitive MHC binding assay (FIG. 8A), we found that E255K-B has a high binding affinity (IC 50 = 17 nM) was experimentally confirmed. The HLA binding of the mutant peptide was approximately 10 times stronger than that of the parent (wild-type) peptide (Figure 1). 12 A) E255K-B is a member of the HLA-A family of other A3 supertypes. * 1101 and HLA-A * We next generated T cell lines against E255K-B from a normal HLA-A3+ donor and two E255K+ / HLA-A3+ CML patients, each of which showed greater specificity for the mutant than for the parent peptide (Figure 1). 12 B.12 C). E255K-B appears to be endogenously processed and presented, since T cells reactive with E255K-B also responded to HLA-A3+ APCs transfected with a minigene encompassing the 227 base pairs surrounding the E255K mutation. Finally, E255K reactivity in one patient first occurred after definitive allogeneic HSCT (Figure 1). 12 D). These studies demonstrate that leukemia-driven genetic alterations can provide novel immunogenic, tumor-specific antigen targets that are associated with in vivo clinical responses. Thus, our approach to identifying immunogenic T cell epitopes of mutant BCR-ABL illustrates an effective strategy for applying bioinformatics tools to discover T cell epitopes derived from mutant genes.

[0175] Example 6: Patient T cell clones that recognize tumor epitopes can selectively kill cells presenting mutant epitopes Confirmation of T cell target specificity is best addressed by characterizing individual T cell clones. Thus, mutant peptide-specific T cell clones are typically isolated by limiting dilution of reactive T cell lines, and then standard chromium release assays are used to screen for T cell clones that exhibit differential killing of autologous APCs pulsed with the mutant peptide versus those pulsed with the germline peptide. A standard dilution series is used for each peptide to determine the concentration of peptide required for 50% killing. If the ratio of wild-type to mutant peptides required for 50% killing is greater than 10-fold, we conclude that there is differential recognition of these peptides by T cells, as previously seen for mutant tumor antigens. We performed this procedure with the CML tumor antigen CML66. To determine whether CML66 peptide-specific T cells recognize processed and presented epitopes, CML66 peptide-reactive T cells were incubated with autologous APCs transduced to express the entire CML66 protein. CML66 was expressed by nucleofection of either plasmid DNA or in vitro transcribed RNA (in DCs, CD40L-expanded B cells, or K562 cells with engineered HLA molecules). 13 As shown in A, stimulated T cells were specific for a CML66-derived peptide epitope (peptide 66-72C) bound to HLA-B4403. When CD40L-expanded B cells were nucleofected with CML66 mRNA, the complete CML66 protein was efficiently expressed (Fig. 13 B), we found that we could use these cells (or cells pulsed with the peptide) as targets in a standard chromium release assay and that T cells effectively lyse these target cells (Figure 1). 13 C) Comparable assays involving lysis of patient-matched tumor cells are performed on each of the mutant peptide-specific T cell lines generated from each cancer patient (e.g., using the T cell lines described in Examples 6 and 7).

[0176] Example 7: Mutant tumor drivers as potential tumor antigens Among 1188 non-silent mutations in 64 patients, we identified eight recurrent mutations, including SF3B1 (16% of CLL patients), TP53 (12.5%), MYD88 (9%), ATM (9%), FBXW7 (6%), MAPK1 (5%), GNB1 (3%), and M6PR (3%) (Figure 14A~14C ). These mutations (especially the most frequent ones: SF3B1, TP53, MYD88, and ATM) are predicted to be driver mutations essential for tumor development or progression. These driver genes represent promising tumor-specific antigens for inclusion in vaccines.

[0177] SF3B1 is the most frequently mutated gene in CLL, is mutated at a conserved site, and is highly expressed in CLL patients (Figure 1). 15 ), which has not been previously described. The most common SF3B1 mutation is K700E (40% of SF3B1 mutations); genotyping of an additional 89 unrelated CLL patients revealed tumors in six more patients harboring this mutation. Applying a peptide-HLA binding algorithm to SF3B1 mutations predicted binding of mutant peptides to the most common HLA-A2 alleles (Figure 1). 16 If a peptide carrying the most common mutation in CLL (SF3B1 K700E) binds to the most common class I HLA allele (HLA-A2), this peptide would be an excellent candidate for inclusion in a CLL vaccine for many CLL patients.

[0178] References TIFF0007801134000014.tif180165TIFF0007801134000015.tif215165TIFF0007801134000016.tif231166TIFF0007801134000017.tif223165TIFF0007801134000018.tif223166TIFF0007801134000019.tif223166TIFF0007801134000020.tif230165TIFF0007801134000021.tif231165TIFF0007801134000022.tif165165

Claims

1. 1. A pharmaceutical composition for use in treating cancer in a subject patient in need thereof, the pharmaceutical composition comprising: (i), (ii), (iii) or (iv) of the following: (i) one or more polypeptides comprising neo-antigenic sequences of at least two peptide sequences; (ii) one or more polynucleotides encoding neo-antigen sequences of the at least two peptide sequences; (iii) antigen-presenting cells pulsed with (i) or containing an expression construct comprising (ii); or (iv) cytotoxic T cells (CTLs) that specifically bind to a complex of (1) a protein encoded by an HLA allele of said one subject patient and (2) a neo-antigen sequence of said at least two peptide sequences, said CTLs being stimulated ex vivo by (iii). Contains The at least two peptide sequences are synthesized by the following steps (a) to (d): (a) comparing the nucleic acid sequence of (a) with the nucleic acid sequence of (b) below: (A) A nucleic acid sequence obtained by whole genome or whole exome sequencing of cancer cells derived from said single subject patient. (i) a nucleic acid sequence obtained by whole genome or whole exome nucleic acid sequencing of a normal cell derived from said single subject; (b) identifying a plurality of cancer-specific nucleic acid sequences that are unique to cancer cells of said single subject patient based on (a), wherein the identified plurality of cancer-specific nucleic acid sequences encode two or more different peptide sequences of two or more different proteins expressed by the cancer cells; each of the two or more different peptide sequences of the two or more different proteins comprises a cancer-specific amino acid mutation that is not present in normal cells derived from the single subject patient; (c) calculating the binding affinity of a peptide sequence encoded by the genome of a cancer cell of said subject patient and containing the cancer-specific amino acid mutation identified in (b) to a protein encoded by the HLA allele of said subject patient by HLA peptide binding analysis using a program implemented on a computer system; and (d) IC<150 nM 50 selecting at least two peptide sequences calculated in (c) to have binding affinity to proteins encoded by HLA alleles of said one subject patient; A pharmaceutical composition selected by a method comprising:

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains one or more polypeptides comprising neoantigen sequences of at least two peptide sequences selected in (d).

3. The pharmaceutical composition of claim 2, wherein the one or more polypeptides comprising the neoantigen sequences of at least two peptide sequences selected in (d) are expressed polypeptides or synthetic polypeptides.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition contains one or more polynucleotides encoding neoantigen sequences of at least two peptide sequences selected in (d).

5. The pharmaceutical composition of claim 1 , further comprising an adjuvant.

6. 6. The pharmaceutical composition of claim 5, wherein the adjuvant comprises polyI:polyC.

7. 3. The pharmaceutical composition of claim 2, wherein each of said at least two peptide sequences is present in the pharmaceutical composition in an amount of 50 μg to 1.5 mg.

8. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is capable of inducing a tumor-specific T cell response.

9. The pharmaceutical composition of claim 1 , further comprising an immunostimulant or an anti-immunosuppressant.

10. 10. The pharmaceutical composition of claim 9, wherein the immunostimulatory or anti-immunosuppressant is selected from the group consisting of an anti-CTLA-4 agent, an anti-PD1 agent, an anti-PD-L1 agent, an anti-CD25 agent, or an indoleamine-(2,3)-dioxygenase (IDO) inhibitor.

11. 2. The pharmaceutical composition of claim 1, (i) each peptide sequence of the one or more polypeptides comprising neo-antigenic sequences of at least two peptide sequences selected in (d) has a length of 8 to 50 amino acids; or (ii) each peptide sequence encoded by the one or more polynucleotides encoding the neo-antigen sequences of at least two peptide sequences selected in (d) has a length of 8 to 50 amino acids; Pharmaceutical compositions.

12. 12. The pharmaceutical composition of claim 11, (i) each peptide sequence of the one or more polypeptides comprising neo-antigenic sequences of at least two peptide sequences selected in (d) is greater than 15 amino acids in length; or (ii) each peptide sequence encoded by the one or more polynucleotides encoding the neo-antigenic sequences of at least two peptide sequences selected in (d) is greater than 15 amino acids in length; Pharmaceutical compositions.

13. 2. The pharmaceutical composition of claim 1, (i) a first neoantigen sequence of the at least two peptide sequences selected in (d) is predicted to bind to a protein encoded by a first HLA allele of the one subject patient; and (ii) the first neo-antigen sequence or a second neo-antigen sequence of the at least two peptide sequences selected in (d) is predicted to bind to a protein encoded by a second HLA allele of the single subject patient that is different from the first HLA allele; Pharmaceutical compositions.

14. The protein encoded by the HLA allele of said one subject patient is an MHC class I protein, and each of the at least two peptide sequences selected in (d) is 8 to 12 amino acids in length. The pharmaceutical composition according to claim 1.

15. 2. The pharmaceutical composition of claim 1, wherein the at least two peptide sequences selected in (d) comprise a maximum of 20 peptide sequences.

16. 2. The pharmaceutical composition of claim 1, wherein the at least two peptide sequences selected in (d) comprise a minimum of four peptide sequences.

17. The pharmaceutical composition according to claim 1, wherein the cancer-specific amino acid mutation, which is not present in normal cells derived from the single subject patient, is encoded by a frameshift mutation.

18. The step of selecting comprises selecting an IC of less than 100 nM. 50 The pharmaceutical composition of claim 1, comprising selecting at least one peptide sequence calculated in (c) to have binding affinity to a protein encoded by an HLA allele of said one subject patient.

19. The pharmaceutical composition of claim 1, wherein the identifying step comprises identifying two or more different proteins expressed by cancer cells of the single subject patient by measuring the levels of RNA encoding the two or more different proteins in the cancer cells of the single subject patient.

20. The pharmaceutical composition according to claim 1, wherein the cancer cells of the subject patient are cancer cells of a solid cancer.

21. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains antigen-presenting cells pulsed with one or more polypeptides comprising neo-antigen sequences of at least two peptide sequences selected in (d), or comprising an expression construct comprising one or more polynucleotides encoding neo-antigen sequences of at least two peptide sequences selected in (d).

22. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains CTLs that specifically bind to a complex of (1) a protein encoded by an HLA allele of the subject patient and (2) a neo-antigen sequence of at least two peptide sequences selected in (d), and the CTLs are CTLs stimulated ex vivo by antigen-presenting cells pulsed with (i) or antigen-presenting cells containing an expression construct comprising (ii).

23. 1. Use of a pharmaceutical composition prepared by a method comprising the following steps (a) to (e) in the manufacture of a medicament for treating cancer in a subject patient in need thereof: (a) comparing the nucleic acid sequence of (a) with the nucleic acid sequence of (b) below: (A) A nucleic acid sequence obtained by whole genome or whole exome sequencing of cancer cells derived from said single subject patient. (i) a nucleic acid sequence obtained by whole genome or whole exome nucleic acid sequencing of a normal cell derived from said single subject; (b) identifying a plurality of cancer-specific nucleic acid sequences that are unique to cancer cells of said single subject patient based on (a), wherein the identified plurality of cancer-specific nucleic acid sequences encode two or more different peptide sequences of two or more different proteins expressed by the cancer cells; each of the two or more different peptide sequences of the two or more different proteins comprises a cancer-specific amino acid mutation that is not present in normal cells derived from the single subject patient; (c) calculating the binding affinity of a peptide sequence encoded by the genome of a cancer cell of said one subject patient and containing the cancer-specific amino acid mutation identified in (b) to a protein encoded by the HLA allele of said one subject patient by HLA peptide binding analysis using a program implemented on a computer system; (d) IC<150 nM 50 selecting at least two peptide sequences calculated in (c) to have binding affinity to proteins encoded by HLA alleles of said one subject patient; (e) A step of manufacturing the pharmaceutical composition, wherein the pharmaceutical composition is a pharmaceutical composition containing: (i) one or more polypeptides comprising neo-antigen sequences of at least two peptide sequences selected in (d); (ii) one or more polynucleotides encoding neo-antigen sequences of at least two peptide sequences selected in (d); (iii) antigen-presenting cells pulsed with (i) or comprising an expression construct comprising (ii); or (iv) CTLs that specifically bind to a complex of (1) a protein encoded by an HLA allele of the single subject patient and (2) neo-antigen sequences of at least two peptide sequences selected in (d), wherein the CTLs are stimulated ex vivo by antigen-presenting cells pulsed with (i) or comprising an expression construct comprising (ii).

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