Immunogenic peptides specific for BCMA and TACI antigens for cancer therapy

Abstract

The present disclosure relates to immunogenic peptides specific for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) and methods of use thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 553,669, filed September 1, 2017. The entire contents of the above are incorporated herein by reference.

[0003] Federally Sponsored Research Statement

[0004] This invention was made with government support under Grant Nos. P50-100007, P01-78378, and R01-50947 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to immunogenic peptides specific for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) and methods of use thereof. Background Art

[0006] Cancer is currently one of the diseases with the highest human mortality rate. According to statistics from the World Health Organization, the number of cancer cases and deaths worldwide reached 14 million and 8.2 million, respectively, in 2012. In the United States, cancer accounts for at least 25% of all deaths.

[0007] In recent years, new therapies for treating various types of cancer have been developed. Patients suffering from cancer are usually treated by using, for example, surgery, chemotherapy and / or immunotherapy. The prognosis of these patients is sometimes still unsatisfactory. Therefore, there is an urgent need for effective therapies and / or preventive programs for treating cancer. Summary of the invention

[0008] The present disclosure relates in part to immunogenic peptides specific for B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), T cells (e.g., CD8 + Cytotoxic T cells (CTL) and / or CD4 + Helper T cells) and peptide-encapsulated nanoparticles (e.g., polymeric nanocarriers or liposome nanoparticles) and methods of using the same.

[0009] In one aspect, the disclosure relates to a peptide comprising, consisting essentially of, or consisting of an amino acid sequence that is identical to or differs from the amino acid sequence set forth in any one of SEQ ID NOs: 13-17 by 1 to 6 amino acid residues. In some embodiments, the amino acid at position 1 of SEQ ID NOs: 13-17 is unchanged. In some embodiments, the amino acid at one or more of positions 1, 2, or 9 of SEQ ID NOs: 13-17 is substituted with another amino acid. For example, positions 1, 2, or 9 are substituted; positions 1 and 2 are substituted; positions 2 and 9 are substituted; positions 1 and 9 are substituted; or positions 1, 2, and 9 are substituted. In certain embodiments, the length of the peptide is 9 to 30 amino acids (i.e., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

[0010] In some embodiments, the amino acid sequence is SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0011] In some embodiments, the peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17, wherein the peptide comprises 1 to 15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N and / or C-terminus of the amino acid sequence.

[0012] In another aspect, the disclosure relates to a peptide comprising, consisting essentially of, or consisting of a first amino acid sequence and a second amino acid sequence heterologous to the first amino acid sequence, wherein the first amino acid sequence consists of an amino acid sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the amino acid at one or more of positions 1, 2, or 9 of SEQ ID NOs: 1-17 is substituted with another amino acid. For example, positions 1, 2, or 9 are substituted; positions 1 and 2 are substituted; positions 2 and 9 are substituted; positions 1 and 9 are substituted; or positions 1, 2, and 9 are replaced. In certain embodiments, the peptide is 9 to 30 amino acids in length (i.e., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

[0013] In another aspect, the disclosure relates to a peptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the amino acid at one or more of positions 3, 4, 5, 6, 7, or 8 of SEQ ID NOs: 1-17 is substituted with another amino acid. For example, position 3, 4, 5, 6, 7 or 8 is substituted; position 3 and 4 is substituted; position 3 and 5 is substituted; position 3 and 6 is substituted; position 3 and 7 is substituted; position 3 and 8 is substituted; position 4 and 5 is substituted; position 4 and 6 is substituted; position 4 and 7 is substituted; position 4 and 8 is substituted; position 5 and 6 is substituted; position 5 and 7 is substituted; position 5 and 8 is substituted; position 6 and 7 is substituted; position 6 and 8 is substituted; position 7 and 8 is substituted; or any combination of 3, 4, 5 or 6 different positions selected from the group of positions 3, 4, 5, 6, 7 and 8 is substituted.

[0014] In some embodiments, the peptide binds to a major histocompatibility complex (MHC) molecule. In some embodiments, the peptide associated with the MHC molecule is recognized by an antigen-specific T cell receptor on a T cell. In some embodiments, the MHC molecule is a class I MHC molecule or a class II MHC molecule. In some embodiments, the class I MHC molecule is HLA-A (e.g., HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A30, HLA-A26, HLA-A68 or HLA-A11), HLA-B or HLA-C. In some embodiments, the MHC molecule is an HLA-A2 molecule or an HLA-A24 molecule.

[0015] The present disclosure also provides a composition comprising a peptide described herein and a second agent. In some embodiments, the second agent is selected from compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3 and anti-TIM3; (2) immune agonists, including anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS Compounds and / or HDAC inhibitors (e.g., ACY241), as single agents and / or in combination with dexamethasone; (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; (6) therapeutic agents that alter BCMA and TACI specific responses, including peptide-based vaccines, different types of vaccines (RNA vaccines, DNA vaccines), cell therapies, specific modulators, and / or specific inhibitors; and (7) therapeutic agents that have a broad coverage of methods for immune responses to diseases that are independent of BCMA and TACI targeted therapies, including biological and non-biological methods. In some embodiments, the second agent is an immunostimulatory agent (e.g., a cytokine or a T helper epitope). In some embodiments, the second agent is a T helper epitope. In some embodiments, the T helper epitope is a PADRE sequence or a universal tetanus toxoid T helper (TT Th) epitope. In some embodiments, the second agent is an adjuvant. The adjuvant can be selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand of a Toll receptor, QS21, RIBI, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT, and mutant Escherichia coli heat-labile toxin (MLT). In some embodiments, the second agent is a toll-like receptor 3 ligand (e.g., Poly ICLC), interferon alpha (IFNα), interferon gamma (IFNγ), granulocyte-macrophage colony stimulating factor (GM-CSF), anti-interleukin 6 (IL-6), IL-6 inhibitors, anti-OX40 antibodies, and anti-GITR antibodies. In some embodiments, the second agent is a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In some embodiments, the second agent is an immunomodulator including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241), as a single agent and / or in combination with dexamethasone.

[0016] On the one hand, the present disclosure also relates to a pharmaceutical composition comprising a peptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an agent selected from the group consisting of: a compound that enhances BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40, and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241), as a single agent and / or in combination with dexamethasone; (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a broad coverage of immune responses to diseases that are independent of BCMA and TACI targeted therapy methods. In some cases, the pharmaceutical composition comprises one or more of the following: an adjuvant (Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand for a Toll receptor, QS21, RIBI, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT, and mutant Escherichia coli heat-labile toxin (MLT)); an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody); a checkpoint inhibitor (e.g., an anti-LAG3 antibody); or an immunomodulator (e.g., lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241), as single agents and / or in combination with dexamethasone).

[0017] In some embodiments, the pharmaceutical composition further comprises a checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the pharmaceutical composition further comprises lenalidomide. In some embodiments, the pharmaceutical composition further comprises lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241), as a single agent and / or in combination with dexamethasone.

[0018] In some embodiments, the pharmaceutical composition further comprises T cells (e.g., CTLs) specific for BCMA. In some embodiments, the pharmaceutical composition further comprises T cells (e.g., CTLs) specific for TACI. CTLs are CTLs obtained by exposure to a peptide comprising one or more of SEQ ID NO: 13 or 14 or consisting of one or more of SEQ ID NO: 13 or 14. In other cases, CTLs are CTLs obtained by exposure to a peptide comprising any one or more of SEQ ID NO: 15-17 or consisting of any one of SEQ ID NO: 15-17. In some cases, CTLs are memory CD8 + In some cases, CTL are memory CD8 + CD45RO + In some cases, CTL are non-memory CD8 + In some cases, CTL is an effector CD8 + In some cases, CTL is activated CD8 + In some cases, the CTL is a tetramer-positive CD8 + In some cases, CTLs are CD8 + CTL. In certain instances, CTL are CD8 + In some cases, CTLs are CD8 T cells that produce cytokines and / or have upregulated expression and / or production of key cytolytic markers (e.g., CD107, granzymes, perforin). + In some cases, CTLs are CD8 + CTL.

[0019] In one aspect, the disclosure relates to nucleic acids encoding peptides as described herein. In some cases, the nucleic acid is RNA (e.g., mRNA). In other embodiments, the nucleic acid is DNA. In some cases, the RNA or DNA is encapsulated in a nanocarrier (e.g., polymeric such as PLGA, or liposomes). RNA and DNA may contain other regulatory sequences (e.g., start codons, stop codons, polyA tails).

[0020] In one aspect, the disclosure also relates to vectors comprising a nucleic acid encoding a peptide as described herein.In some embodiments, the nucleic acid sequence is operably linked to a promoter, regulatory element, or expression control sequence.

[0021] In another aspect, the disclosure also relates to a cultured cell comprising a vector as described herein. In some embodiments, the cell is a mammalian cell, a human cell, or an immune cell.

[0022] In another aspect, the disclosure provides a virus comprising a nucleic acid encoding a peptide as described herein. In some embodiments, the virus is a lentivirus, an adenovirus, an adeno-associated virus, a human foamy virus, a parvovirus, a myxoma virus, a Newcastle disease virus, a reovirus, a Seneca valley virus, a measles virus, a polio virus, a vaccinia virus, a herpes simplex virus, or a vesicular stomatitis virus.

[0023] In one aspect, the present disclosure relates to a combination of at least two different peptides, wherein at least two different peptides are selected from the group of peptides having the amino acid sequence shown in SEQ ID NO: 13-17. The combination may include peptides having 1-4 substitutions in one or more of SEQ ID NO: 13-17. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, position 1 is unchanged. In some cases, the length of the peptide is 9 to 30 amino acids. In some embodiments, the combination comprises at least 2, 3, 4 or all 5 peptides having the amino acid sequence shown in SEQ ID NO: 13-17. In some cases, the combination comprises two or more peptides shown in SEQ ID NO: 13-17, wherein the two or more peptides have 1 to 15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N and / or C-terminal of the amino acid sequence.

[0024] In one aspect, the present disclosure also relates to a pharmaceutical composition comprising a combination of peptides as described herein. In some embodiments, the pharmaceutical composition comprises an agent selected from the group consisting of compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241), as single agents and / or in combination with dexamethasone; (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a broad coverage of immune responses to diseases that are independent of BCMA and TACI targeted therapy methods. In some cases, the pharmaceutical composition comprises one or more of the following: an adjuvant (Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand for a Toll receptor, QS21, RIBI, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT, and mutant Escherichia coli heat-labile toxin (MLT)); an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody); a checkpoint inhibitor (e.g., an anti-LAG3 antibody); and / or lenalidomide, pomalidomide, a thalidomide analog, an IMiDS compound, and / or an HDAC inhibitor (e.g., ACY241), as a single agent and / or in combination with dexamethasone.

[0025] In some embodiments, the pharmaceutical composition further comprises an immunostimulant. In some cases, the immunostimulant can be an anti-OX40 antibody or an anti-GITR antibody. In some embodiments, the pharmaceutical composition further comprises a checkpoint inhibitor. In one case, the checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the pharmaceutical composition further comprises lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (eg, ACY241), as a single agent and / or or in combination with dexamethasone. On the one hand, the present disclosure also provides a composition comprising isolated dendritic cells, wherein the dendritic cells present peptide sequences on their surfaces, wherein the peptide sequences comprise BCMA antigens (SEQ ID NO: 18) and TACI antigens (SEQ ID NO: 19) One or both of at least one major histocompatibility complex (MHC) class I peptide epitope.

[0026] In some embodiments, the MHC class I peptide epitope is an HLA-A2 peptide epitope.

[0027] In some embodiments, the MHC class I peptide epitope is an HLA-A24 peptide epitope.

[0028] In some embodiments, dendritic cells acquire the peptide sequence in vitro by exposure to a peptide comprising the peptide sequence.

[0029] In some embodiments, the peptide sequence is a synthetic peptide sequence. In some embodiments, the peptide sequence is a sequence shown in any one of SEQ ID NO: 1-12 and SEQ ID NO: 13-17. In some cases, the peptide sequence is a sequence shown in SEQ ID NO: 13-17 but with 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9. In a specific embodiment, the peptide sequence is SEQ ID NO: 13. In another embodiment, the peptide is SEQ ID NO: 13 but with 1 to 4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9. In another specific embodiment, the peptide sequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ ID NO: 16 but with 1 to 4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9.

[0030] In some embodiments, the composition comprises 10 5 Up to 10 8 Dendritic cells.

[0031] In some embodiments, the composition further comprises a peptide as shown in any one of SEQ ID NO: 1-12 and SEQ ID NO: 13-17. In a specific embodiment, the peptide sequence is SEQ ID NO: 13. In another specific embodiment, the peptide sequence is SEQ ID NO: 16. In some embodiments, the composition comprises an agent selected from the group consisting of: compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3 and anti-TIM3; (2) immune agonists, including anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors ( (e.g., ACY241), as a single agent and / or in combination with dexamethasone; (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; (6) therapeutic agents that alter BCMA and TACI specific responses, including peptide-based vaccines, different types of vaccines (RNA vaccines, DNA vaccines), cell therapies, specific modulators, and / or specific inhibitors; and (7) therapeutic agents that have a broad coverage of immune responses to diseases that are independent of BCMA and TACI targeted therapy methods (including biological and non-biological methods). In some embodiments, the composition further comprises an immune agonist (e.g., anti-OX40 antibody, anti-GITR antibody). In some embodiments, the composition further comprises a checkpoint inhibitor (e.g., anti-LAG3 antibody).

[0032] In one aspect, the disclosure relates to a method of inducing an immune response against BCMA and / or TACI expressing cells (e.g., cancer cells) in a human subject in need thereof, the method comprising administering to the human subject a peptide as described herein (e.g., a peptide comprising or consisting of SEQ ID NOs: 13-17) or a composition as described herein (e.g., a pharmaceutical composition). In another embodiment, the peptide is SEQ ID NO: 13, but has 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2, or 9. In another specific embodiment, the peptide sequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ ID NO: 16, but has 1 to 4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2, or 9.

[0033] In some embodiments, the subject has a cancer that expresses BCMA and / or TACI, and the immune response is directed against such cancer cells.

[0034] In some embodiments, the subject has non-cancerous cells that express BCMA and / or TACI, and the immune response is directed against such non-cancerous cells.

[0035] In some embodiments, the cancer is a hematological cancer (e.g., multiple myeloma). In some embodiments, the cancer cell is a plasma cell (e.g., cancerous plasma cell). In some embodiments, the human subject suffers from refractory multiple myeloma. In some embodiments, the human subject suffers from refractory multiple myeloma that relapsed after allogeneic transplantation.

[0036] In some embodiments, the cancer cells express BCMA, and the level of BCMA in the cancer cells is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% greater than in plasma cells of a healthy human subject.

[0037] In some embodiments, the cancer cells express TACI, and the level of TACI in the cancer cells is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% greater than in plasma cells of a healthy human subject.

[0038] In some embodiments, the method further comprises administering to a human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to a human subject a CTL specific for TACI. In some cases, the CTL is a CTL obtained by exposure to a peptide comprising one or more of SEQ ID NO: 13 or 14 or consisting of one or more of SEQ ID NO: 13 or 14. In some cases, the CTL is exposed to a peptide having 1-4 substitutions of SEQ ID NO: 13 or 14. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, the length of the peptide is 9 to 30 amino acids. In other cases, the CTL is a CTL obtained by exposure to a peptide comprising any one or more of SEQ ID NO: 15-17 or consisting of any one or more of SEQ ID NO: 15-17. In some cases, the CTL is exposed to a peptide having 1 to 4 substitutions of SEQ ID NO: 15, 16 or 17. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, the peptide is 9 to 30 amino acids in length. In some cases, the CTL is a memory CD8 + In some cases, CTL are memory CD8 + CD45RO + In some cases, CTL is an effector CD8 +In some cases, CTL is activated CD8 + In some cases, the CTL is a tetramer-positive CD8 + CTL. In some cases, CD8 + CTLs are CTLs that upregulate the expression of co-stimulatory molecules when stimulated with the peptides described herein. In some cases, CD8 + CTLs are CTLs that upregulate expression of checkpoint molecules when stimulated with the peptides described herein. In some cases, CTLs are CD8 + In some cases, CTLs are CD8 + CTL.

[0039] In some embodiments, the method further comprises administering an immune agonist to the human subject. In some embodiments, the immune agonist is anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40, and anti-GITR.

[0040] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to the human subject. In certain embodiments, the method further comprises administering lenalidomide to the human subject. In some embodiments, the method further comprises administering one or more of the following to the human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or an immunomodulator.

[0041] In some embodiments, the method further comprises determining whether an immune response against BCMA and / or a TACI-expressing cancer occurs in the human subject after administering the peptide or composition to the human subject.

[0042] In one aspect, the disclosure relates to a method of treating a human subject having cancer or a pre-malignant disease, the method comprising administering to the human subject a peptide as described herein or a composition as described herein.

[0043] In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is multiple myeloma, leukemia, lymphoma, or any B cell or plasma cell malignancy.

[0044] In some embodiments, the premalignant disease is monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma.

[0045] In some embodiments, the method further comprises detecting that one or more cancer cells in the human subject express or overexpress BCMA and / or TACI.

[0046] In some embodiments, the human subject has one or more cancer cells that overexpress BCMA and / or TACI, wherein the level of BCMA and / or TACI in the cancer cells is at least 20% greater than in normal cells (e.g., plasma cells in a healthy subject).

[0047] In some embodiments, the subject has one or more cancer cells that express MHC molecules.

[0048] In some embodiments, the method further comprises administering to a human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to a human subject a CTL specific for TACI. In some cases, the CTL is a CTL obtained by exposure to a peptide, the peptide comprising one or more of SEQ ID NO: 13 or 14 or consisting of one or more of SEQ ID NO: 13 or 14, in some cases, the CTL is exposed to SEQ ID NO: 13 or 14 but with 1-4 substituted peptides. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, the length of the peptide is 9 to 30 amino acids. In other cases, the CTL is a CTL obtained by exposure to a peptide, the peptide comprising any one or more of SEQ ID NO: 15-17 or consisting of any one or more of SEQ ID NO: 15-17. In some cases, the CTL is exposed to SEQ ID NO: 15, 16 or 17 but with 1 to 4 substituted peptides. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, the length of the peptide is 9 to 30 amino acids. In some cases, the CTL is a memory CD8+CTL. In some cases, the CTL is a memory CD8+CD45RO+CTL. In some cases, the CTL is an effector CD8+CTL. In some cases, the CTL is an activated CD8+CTL. In some cases, the CTL is a Tetramer positive CD8+CTL. In some cases, the CTL is a CD8+CTL with upregulated costimulatory molecule expression. In some cases, the CTL is a CD8+CTL with upregulated checkpoint molecule expression.

[0049] In some embodiments, the method includes administering to a human subject an agent selected from the group consisting of: compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a broad coverage of immune responses to diseases that are independent of BCMA and TACI targeted therapy methods. In some embodiments, the method also includes administering an immune agonist to a human subject. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0050] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to the human subject. In certain embodiments, the method further comprises administering lenalidomide to the human subject. In some embodiments, the method further comprises administering one or more of the following to the human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or an immunomodulator.

[0051] In some embodiments, the method further comprises administering chemotherapy or radiation therapy to the human subject.

[0052] In one aspect, the disclosure relates to a method of generating and / or expanding BCMA-specific cytotoxic T cells, the method comprising contacting one or more cytotoxic T cells with one or more antigen presenting cells pulsed with a peptide comprising an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14. In some cases, the CTLs are exposed to a peptide of SEQ ID NO: 13 or 14 but with 1-4 substitutions. In some cases, the substitutions are at one or more of positions 1, 2, or 9. In some cases, the peptide is 9 to 30 amino acids in length.

[0053] In some embodiments, cytotoxic T cells are memory cytotoxic T cells. In some embodiments, cytotoxic T cells are effector cytotoxic T cells. In some cases, CD8+CTL is a CTL that raises the expression of costimulatory molecules when stimulated with a peptide as described herein. In some cases, CD8+CTL is a CTL that raises the expression of checkpoint molecules when stimulated with a peptide as described herein. In some cases, CTL is a CD8+CTL that produces cytokines and / or has key cytolytic markers (e.g., CD107, granzymes, perforin) expressed and / or produced. In some cases, CTL is a CD8+CTL with anti-tumor or other target activity.

[0054] In some embodiments, the antigen presenting cell is a dendritic cell (DC). In a specific embodiment, the peptide comprises SEQ ID NO: 13 or consists of SEQ ID NO: 13. On the other hand, the present disclosure relates to a method for generating TACI-specific cytotoxic T cells, the method comprising contacting one or more cytotoxic T cells with one or more antigen presenting cells pulsed with a peptide comprising an amino acid sequence selected from SEQ ID NO: 15-17. In some cases, CTLs are exposed to peptides of SEQ ID NO: 15, 16 or 17 but with 1-4 substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9. In some cases, the length of the peptide is 9 to 30 amino acids.

[0055] In a specific embodiment, the peptide comprises or consists of SEQ ID NO:16.

[0056] In one aspect, the disclosure also relates to a method of killing a target cell, the method comprising contacting the target cell with one or more BCMA-specific cytotoxic T cells, wherein the target cell expresses or overexpresses BCMA. In some embodiments, the target cell expresses HLA-A.

[0057] In some embodiments, the method further comprises contacting one or more BCMA-specific cytotoxic T cells with an agent selected from the group consisting of compounds that enhance BCMA- and TACI-specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241), as single agents and / or in combination with dexamethasone; (4) adjuvants; (5) therapeutic agents that increase BCMA- and TACI-specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a method of broadly covering immune responses against disease that is independent of BCMA- and TACI-targeted therapies.

[0058] In some embodiments, the method further comprises contacting the one or more BCMA-specific cytotoxic T cells with an immune agonist.

[0059] In some embodiments, the method comprises further administering a peptide comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 13 or 14. In some cases, the peptide may have 1 to 15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids added to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO.: 13 or 14.

[0060] In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0061] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to a human subject. In certain embodiments, the method further comprises administering lenalidomide to a human subject. In some embodiments, the method further comprises administering lenalidomide to a subject. In certain embodiments, the method further comprises administering one or more of the following to a human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0062] In another aspect, the disclosure relates to a method of killing a target cell, the method comprising contacting the target cell with one or more TACI-specific cytotoxic T cells, wherein the target cell expresses or overexpresses TACI. In some embodiments, the target cell expresses HLA-A.

[0063] In some embodiments, the method comprises further administering a peptide comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 15 to 17. In some cases, the peptide may have 1 to 15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids added to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NOs.: 15-17.

[0064] In some embodiments, the method further comprises contacting one or more TACI-specific cytotoxic T cells with an agent selected from the group consisting of: compounds that enhance BCMA and TACI-specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI-specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a method of broadly covering immune responses against diseases that are independent of BCMA and TACI targeted therapies.

[0065] In some embodiments, the method further comprises contacting one or more TACI-specific cytotoxic T cells with an immune agonist.

[0066] In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist.

[0067] In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to a human subject. In certain embodiments, the method further comprises administering lenalidomide to a human subject. In some embodiments, the method further comprises administering one or more of the following to a human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody) or lenalidomide.

[0068] In one aspect, the disclosure relates to a method of treating a human subject having a disease or cancer expressing BCMA or TACI, the method comprising administering to the human subject a plurality of BCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells.

[0069] In some embodiments, the method further comprises administering to a human subject a peptide comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 13-17. In some cases, the peptide has a sequence as set forth in one of SEQ ID NOs: 13-17, except that it has 1-4 substitutions. In some cases, the substitutions may be at one or more of positions 1, 2, or 9. In some cases, the peptide is 9 to 30 amino acids in length.

[0070] In some embodiments, the method further comprises administering to the human subject an agent selected from the group consisting of: compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a method of broadly covering immune responses against diseases that are independent of BCMA and TACI targeted therapies.

[0071] In some embodiments, the method further comprises administering an immune agonist to the subject. In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0072] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to a human subject. In certain embodiments, the method further comprises administering lenalidomide to a human subject. In some embodiments, the method further comprises administering lenalidomide to a subject. In some embodiments, the method further comprises administering one or more of the following to a human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0073] In some embodiments, the cytotoxic T cells are derived from cells of a human subject. In some embodiments, the cytotoxic T cells are derived from induced pluripotent stem cells.

[0074] In some embodiments, the cancer expresses BCMA and / or TACI. In some embodiments, the human subject has multiple myeloma.

[0075] In one aspect, the present disclosure relates to a method comprising:

[0076] (a) obtaining bone marrow-derived mononuclear cells from a subject;

[0077] (b) culturing mononuclear cells in vitro under conditions where the mononuclear cells adhere to a culture container;

[0078] (c) selecting adherent mononuclear cells;

[0079] (d) culturing the adherent mononuclear cells in the presence of one or more cytokines under conditions whereby the cells differentiate into antigen presenting cells; and

[0080] (e) contacting an antigen presenting cell with a peptide as described herein (eg, SEQ ID NOs: 13-17), thereby generating an antigen presenting cell that presents the peptide on a major histocompatibility complex (MHC) molecule.

[0081] In some embodiments, the major histocompatibility complex molecule is an MHC class I molecule.

[0082] In some embodiments, the one or more cytokines include granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4).

[0083] In some embodiments, the one or more cytokines comprise tumor necrosis factor-α (TNF-α).

[0084] In some embodiments, bone marrow-derived cells are obtained from a subject diagnosed with multiple myeloma.

[0085] In one aspect, the present invention relates to a method for identifying a T cell antigen receptor sequence for BCMA, the method comprising

[0086] (a) generating and / or expanding BCMA-specific cytotoxic T cells by the method described herein;

[0087] (b) Determination of the T cell antigen receptor sequence of BCMA in BCMA-specific cytotoxic T cells.

[0088] In another aspect, the present disclosure relates to a method for treating a human subject having a disease or cancer that expresses BCMA, the method comprising: administering to the human subject a composition comprising a chimeric antigen receptor T cell (CAR-T cell), wherein the CAR-T cell expresses a chimeric antigen receptor, wherein the chimeric antigen receptor binds BCMA.

[0089] In some embodiments, the cancer expresses BCMA.In some embodiments, the human subject has multiple myeloma.

[0090] In some embodiments, the method further comprises administering to a human subject a peptide comprising or consisting of a sequence as set forth in any one of SEQ ID NO.: 13 or 14. In some cases, the peptide has a sequence as set forth in SEQ ID NO: 13 or 14, except that it has 1-4 amino acid substitutions. In certain cases, the substitution is at one or more of positions 1, 2, or 9. In some cases, the peptide is 9 to 30 amino acids in length. In some embodiments, the method further comprises administering to the human subject an agent selected from the group consisting of: compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a method of broadly covering immune responses against diseases that are independent of BCMA and TACI targeted therapies.

[0091] In some embodiments, the method further comprises administering an immune agonist to the subject. In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0092] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to the human subject. In certain embodiments, the method further comprises administering lenalidomide to the human subject. In some embodiments, the method further comprises administering one or more of the following to the subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0093] In one aspect, the present disclosure relates to a method of identifying a T cell antigen receptor sequence of a TACI, the method comprising

[0094] (a) generating and / or proliferating TACI-specific cytotoxic T cells by the method described herein;

[0095] (b) Determination of the T cell antigen receptor sequence of TACI in TACI-specific cytotoxic T cells.

[0096] In another aspect, the present disclosure relates to a method for treating a human subject having a disease or cancer that expresses TACI, comprising: administering to the human subject a composition comprising a chimeric antigen receptor T cell (CAR-T cell), wherein the CAR-T cell expresses a chimeric antigen receptor, wherein the chimeric antigen receptor binds TACI.

[0097] In some embodiments, the cancer expresses TACI. In some embodiments, the human subject has multiple myeloma.

[0098] In some embodiments, the method further comprises administering to a human subject a peptide comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 15-17.

[0099] In some embodiments, the method further comprises administering to the human subject an agent selected from the group consisting of: compounds that enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines; (2) checkpoint inhibitors, including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists, including anti-OX40 and anti-GITR; (3) immunomodulators, including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapies, and / or antibodies; and (6) therapeutic agents that have a method of broadly covering immune responses against diseases that are independent of BCMA and TACI targeted therapies.

[0100] In some embodiments, the method further comprises administering an immune agonist to the subject. In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0101] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to the human subject. In certain embodiments, the method further comprises administering lenalidomide to the human subject. In some embodiments, the method further comprises administering lenalidomide to the subject. In some embodiments, the method further comprises administering one or more of the following to the human subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0102] In one aspect, the disclosure provides a composition comprising a nanoparticle and a peptide comprising a sequence at least 60% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 13 or consists of the sequence of SEQ ID NO: 13. In some cases, the peptide has the sequence of SEQ ID NO: 13, but has 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9. The length of the peptide can be 9 to 30 amino acids. In other embodiments, the sequence comprises the sequence of SEQ ID NO: 16 or consists of the sequence of SEQ ID NO: 16. In some cases, the peptide has the sequence of SEQ ID NO: 16, but has 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2 or 9. The length of the peptide can be 9 to 30 amino acids.

[0103] In some embodiments, the peptide is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0104] In some embodiments, the nanoparticles comprise a biodegradable polymer. In some embodiments, the nanoparticles comprise poly(D,L-lactide-co-glycolide) (PLGA). In some embodiments, the nanoparticles comprise poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) copolymers.

[0105] In some embodiments, the amino acid sequence is SEQ ID NO: 13. In some embodiments, the amino acid sequence is SEQ ID NO: 14. In some embodiments, the amino acid sequence is SEQ ID NO: 15. The amino acid sequence is SEQ ID NO: 16. In some embodiments, the amino acid sequence is SEQ ID NO: 17. In some cases, the peptide has the sequence of any one of SEQ ID NOs: 13 to 17, but has 1 to 4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2, or 9. The peptide can be 9 to 30 amino acids in length.

[0106] In some embodiments, the nanoparticle comprises an adjuvant. In some embodiments, the nanoparticle comprises a Toll-like receptor agonist (eg, R848 or an unmethylated CpG oligodeoxynucleotide).

[0107] In one aspect, the disclosure provides a method for treating a human subject suffering from cancer. The method comprises administering a composition (e.g., nanoparticle) as described herein to the human subject. In some embodiments, the human subject suffers from multiple myeloma.

[0108] In some embodiments, the cancer expresses BCMA. In other embodiments, the cancer expresses TACI.

[0109] In some embodiments, the method further comprises administering to a human subject a peptide comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 15-17. In some embodiments, the peptide has a sequence as set forth in one of SEQ ID NOs.: 15-17, except having 1-4 amino acid substitutions. The substitutions may be at one or more of positions 1, 2, or 9. The peptide may be 9 to 30 amino acids in length.

[0110] In some embodiments, the method further comprises administering to a human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to a human subject a CTL specific for TACI. In some cases, the CTL is a CTL obtained by exposure to a peptide, the peptide comprising one or more of SEQ ID NO: 13 or 14 or consisting of one or more of SEQ ID NO: 13 or 14. In other cases, the CTL is a CTL obtained by exposure to a peptide, the peptide comprising one or more of SEQ ID NO: 15-17 or consisting of one or more of SEQ ID NO: 15-17. In some cases, the CTL is a memory CD8+CTL. In some cases, the CTL is a memory CD8+CD45RO+CTL. In some cases, the CTL is an effector CD8+CTL. In some cases, the CTL is an activated CD8+CTL. In some cases, the CTL is a Tetramer positive CD8+CTL. In some cases, the CTL is a CD8+CTL that has upregulated the expression of costimulatory molecules. In some cases, the CTL is a CD8+CTL that has upregulated the expression of a checkpoint molecule. In some embodiments, the method further comprises administering an immune agonist to the subject. In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0111] In some embodiments, the method further comprises administering a checkpoint inhibitor (e.g., an anti-LAG3 antibody) to a human subject. In certain embodiments, the method further comprises administering lenalidomide to a human subject. In some embodiments, the method further comprises administering lenalidomide to a subject. In some embodiments, the method further comprises administering one or more of the following to the subject: an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0113] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figures 1A-1I BCMA expression on multiple myeloma cell lines is shown.

[0116] Figure 2 The binding affinity of native BCMA peptide to HLA-A2 is shown.

[0117] Figure 3 The binding affinity of the native TACI peptide to HLA-A2 is shown.

[0118] Figure 4 The binding affinity of BCMA peptides to HLA-A2 is shown: native peptide versus heteroclitic peptide.

[0119] Figure 5 The binding affinity of TACI peptides to HLA-A2 is shown: native peptide versus mutant peptide.

[0120] Figure 6 HLA-A2 stability of BCMA #4 and #5 peptides is shown: native peptide vs. mutant peptide (50 ug / ml).

[0121] Figure 7 HLA-A2 stability of TACI #1, #3 and #4 peptides is shown: native peptide vs. mutant peptide (50 ug / ml).

[0122] Figures 8A-8C The results show that CD8 + Cytotoxic T cells (CTL).

[0123] Figures 9A-9C Shown is the reduction of naive CTLs upon stimulation with mutant BCMA#4 peptide.

[0124] Figures 10A-10C Shown are increased memory CTLs upon stimulation with mutant BCMA#4 peptide.

[0125] Figures 11A-11C Shown are the kinetics of CM versus effector cells in the setting of stimulation with mutant BCMA#4 peptide.

[0126] Fig.12 It was shown that the mutant BCMA#4 peptide induced memory CD8+ CTL.

[0127] Fig.13 It was shown that the mutant TACI#3 peptide induced memory CD8+CTL.

[0128] Fig.14 The anti-tumor activity of mutant BCMA#4 peptide-CTL is shown (N=5).

[0129] Fig.15 The antitumor activity of mutant TACI#3 peptide-CTL is shown (n=4).

[0130] Fig.16 HLA-A2-specific proliferation of mutant BCMA#4 peptide-CTLs was shown.

[0131] Fig.17 The enhanced α-tumor activity of central memory cells of BCMA-specific CTLs treated with α-OX40 or α-GITR was shown.

[0132] Figures 18A-18C Upregulation of key T cell markers on BCMA peptide-specific CTLs stimulated with mutant BCMA peptides is shown.

[0133] Figures 19A-19F BCMA 72-80 Specific CTL to HLA-A2 + HLA-A2-restricted and antigen-specific immune responses of MM cell lines.

[0134] Figures 20A-20H Shows mutated BCMA 54-62 Specific CTL or mutated BCMA 72-80 Antitumor activity of specific CTL against patients' MM cells.

[0135] Figures 21A-21C BCMA 72-80 Specific Tetramer + CTLs exhibited a unique phenotype and high levels of antitumor activity against MM cells.

[0136] Figures 22A-22E . Using mutant BCMA 72-80 Memory CD8 of BCMA-specific CTLs after peptide stimulation + T cell differentiation.

[0137] Figures 23A-23C Characterization of high antitumor activity of BCMA-specific memory CTLs ( Fig.23A ) and the highest level of central memory CTLs ( Fig. 23B , Fig.23C ).

[0138] Fig.24A Results of BCMA peptide-specific CTLs co-cultured with 25 U266 cells (7 days).

[0139] Figures 24B-24C Enhanced anti-myeloma activity of memory CD8+ T cells of mutant BCMA72-80 CTLs [generated from an HLA-A2+ individual] upon treatment with anti-LAG3 or anti-OX40.

[0140] Fig.24D In the treatment of myeloma cells with anti-OX40 in an HLA-A2 restricted manner [from HLA-A2 + Mutated BCMA from donor 1, donor 2, or donor 3 72-80 Enhanced anti-tumor activity of CTLs.

[0141] Figures 25A-25B . Using mutant BCMA 72-80 Percentage of CD3+CD8+ T cells after peptide stimulation.

[0142] Figures 26A-26B . Using mutant BCMA 72-80 Percentage of CD3+CD4+ T cells after peptide stimulation.

[0143] Figures 27A-27C High BCMA expression was shown on H929, MMIS, U266 and OPM1 cell lines, but not on a breast cancer cell line (MDA-MB231).

[0144] Figures 28A-28B showed that mutant BCMA 72-80 Percentage of CD3+CD8+ T cells expressing PD-1 and LAG-3 after peptide stimulation.

[0145] Figures 29A-29C Shows the use of mutant TACI 154-162 After peptide stimulation, naive cells differentiated into memory CTLs.

[0146] Figures 30A-30F Shows mutation TACI 154-162 Antitumor activity of specific CTL against HLA-A2+ multiple myeloma cells (McCAR).

[0147] Figures 31A-31E Shows mutation TACI 154-162 Antitumor activity of specific CTL against HLA-A2+ multiple myeloma cells.

[0148] Fig.31F Demonstrated mutant TACI targeting HLA-A2+ multiple myeloma cells 154-162 Induction of peptide-specific Tetramer+CTLs and antitumor activity and proliferation of peptides.

[0149] Fig.32A The morphology of BCMA peptide-loaded PLGA nanoparticles is shown.

[0150] Fig.32B The morphology of BCMA peptide-loaded liposome nanoparticles is shown.

[0151] Fig.32C BCMA peptide quantification is shown.

[0152] Fig.33A A higher loading efficiency of BCMA peptide on dendritic cells when encapsulated in nanoparticles (PLGA, liposomes) was shown in a time-dependent manner.

[0153] Fig.33B A higher loading efficiency of BCMA peptide-FITC on dendritic cells when encapsulated in nanoparticles (PLGA, liposomes) was shown in a time-dependent manner.

[0154] Fig.33C Showing higher PLGA / peptide uptake by dendritic cells.

[0155] Fig.33D Higher uptake of BCMA peptide-FITC by dendritic cells when encapsulated in PLGA was shown in a time-dependent manner.

[0156] Fig.33E Higher uptake of BCMA peptide-FITC by T2 cells when encapsulated in PLGA was shown in a time-dependent manner.

[0157] Fig.34A The highest anti-MM activity of BCMA-CTLs generated with PLGA / BCMA peptides against MM cell lines in an HLA-A2 restricted manner was shown.

[0158] Fig.34B The highest IFN-γ production by BCMA-CTLs generated with PLGA / BCMA peptides against MM cell lines in an HLA-A2 restricted manner was shown.

[0159] Fig.34CHighest IL-2 production by BCMA-CTLs generated with PLGA / BCMA peptides in an HLA-A2 restricted manner against MM cell lines is shown.

[0160] Fig.34D Highest TNF-α production by BCMA-CTLs generated with PLGA / BCMA peptides in an HLA-A2 restricted manner against MM cell lines is shown.

[0161] Fig.34E The results show that PLGA / BCMA peptides are used to target MM cell lines in an HLA-A2 restricted manner from different HLA-A2 + The highest anti-MM activity and Th1-type cytokine (IFN-γ, IL-2, TNF-γ) production of BCMA-CTLs were generated by individuals (N=3).

[0162] Fig.35A Baseline activity of BCMA-specific CTLs generated with BCMA peptide itself, PLGA / BCMA peptide, or liposome / BCMA peptide in the absence of tumor cells is shown.

[0163] Fig.35B Shows response from HLA-A2 + Primary HLA-A2 of myeloma patient #1 + CD138 + Tumor cells, highest anti-MM activity by BCMA-CTL generated with PLGA / BCMA peptide.

[0164] Fig.35C Shows response from HLA-A2 + Primary HLA-A2 of myeloma patient #2 + CD138 + Tumor cells, highest anti-MM activity by BCMA-CTL generated with PLGA / BCMA peptide.

[0165] Fig.35D The highest anti-MM activity and Th1-type cytokine (IFN-γ, IL-2, TNF-γ) production of BCMA-CTLs generated with PLGA / BCMA peptides against tumor cells of myeloma patients in an HLA-A2 restricted manner were shown.

[0166] Fig.36A Tetramer showing higher frequency of BCMA-CTL generated with PLGA / BCMA peptide than with BCMA peptide alone + CD8 + T cells and co-stimulatory molecules expression (CD28 + )CD8 + T cells.

[0167] Fig.36B The results showed that BCMA-CTLs generated with PLGA / BCMA peptides had higher peptide-specific CD8 + Increased T cell proliferation.

[0168] Fig.36C It was shown that BCMA-CTLs generated with PLGA / BCMA peptide had a higher increase in peptide-specific IFN-γ production than those generated with BCMA peptide itself.

[0169] Fig.37A The highest myeloma-specific CD8 expression of BCMA-CTLs generated with PLGA / BCMA peptides compared with BCMA peptides alone was shown. + Induction of T cell proliferation.

[0170] Fig.37B The results show that CD45RO+ memory / CD3 in BCMA-CTLs increased after repeated stimulation with PLGA / BCMA peptide compared with BCMA peptide itself. + CD8 + Higher increases in T cell subsets [Representative Results].

[0171] Fig.37C The results showed that after repeated stimulation with PLGA / BCMA peptide, CD45RO+ memory / CD3 + CD8 + Higher increases in T cell subsets [N=3 results].

[0172] Fig.37D The results showed that after repeated stimulation with PLGA / BCMA peptide, central memory / CD3 in BCMA-CTLs was significantly higher than that with BCMA peptide itself. + CD8 + Higher induction and maintenance of T cell subsets.

[0173] Fig.38A It was shown that BCMA-CTLs generated with PLGA / BCMA peptide induced higher central memory and effector memory CTLs and their anti-MM activities against myeloma cells in an HLA-A2 restricted manner than BCMA peptide itself.

[0174] Fig.38B It was shown that BCMA-CTLs generated with PLGA / BCMA peptide induced higher central and effector memory CTLs and their IFN-γ production in an HLA-A2 restricted manner against myeloma cells than BCMA-CTLs generated with BCMA peptide itself.

[0175] Fig.38CIt was shown that BCMA-CTL generated with PLGA / BCMA peptide induced higher central memory and effector memory CTL and their anti-MM activity and IFN-γ, IL-2, and TNF-α production against myeloma cells in an HLA-A2 restricted manner than BCMA-CTL generated with BCMA peptide itself. DETAILED DESCRIPTION OF THE INVENTION

[0177] B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) are key antigens specific to many cancers, including, for example, multiple myeloma and other hematological malignancies. The present disclosure is based, at least in part, on the identification of HLA-A2-specific immunogenic peptides derived from BCMA and TACI antigens, which can be used to generate, for example, multiple myeloma (MM)-specific T cell immune responses. Therefore, the present disclosure relates to BCMA-derived peptides and TACI-derived peptides (and pharmaceutical compositions thereof), which can be used, for example, to induce an immune response against tumor cells in a subject (e.g., stimulating a cytotoxic T cell (CTL) response), or to stimulate the production of cytokines or antibodies. The peptides can be used in a variety of applications, such as methods of inducing an immune response, methods of producing antibodies, methods of producing cytokines involved in an anti-tumor immune response, and methods of treating cancer (e.g., multiple myeloma). The peptides can also be included in MHC molecule multimer compositions and used, for example, in methods of detecting T cells (e.g., antigen-specific T cells) in a cell population.

[0178] The present disclosure also provides information for various types of therapeutic applications, including peptide-based vaccination, peptides constituting various approaches to vaccines (including nanoparticle-based and virus-based peptides), adoptive transfer of BCMA-specific T cells or TACI-specific T cells and antigen-specific T cells generated ex vivo using engineering techniques (including CAR-TCR therapy and induced pluripotent stem cell-based techniques) or infusion of peptide-pulsed dendritic cells, as monotherapy or combination therapy as described herein, in cancer patients, including multiple myeloma, its premalignant diseases or other cancers, or any disease that uniquely expresses and / or overexpresses BCMA and TACI antigens.

[0179] BCMA-derived peptides and TACI-derived peptides

[0180] B cell maturation antigen (BCMA) (NM_001192.2→NP_001183.2), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a protein encoded by the TNFRSF17 gene in humans. BCMA is a cell surface receptor of the TNF receptor superfamily that recognizes B cell activating factor (BAFF). BCMA is expressed in mature B lymphocytes. This receptor has been shown to specifically bind to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B / TALL-1 / BAFF) and leads to NF-kappaB and MAPK8 / JNK activation. This receptor also binds to various TRAF family members and can therefore transduce signals for cell survival and proliferation. BCMA is often overexpressed in various cancer cells, such as in subjects with leukemia, lymphoma, and multiple myeloma.

[0181] Transmembrane activator and CAML interactor (TACI) (NM_012452.2→NP_036584.1), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), is a protein encoded by the TNFRSF13B gene in humans. TACI is a transmembrane protein of the TNF receptor superfamily found primarily on the surface of B cells. TACI recognizes three ligands: APRIL, BAFF, and CAML. TACI is also frequently overexpressed in various cancer cells, such as in subjects with leukemias, lymphomas, and multiple myeloma.

[0182] The amino acid sequences of human BCMA and human TACI are shown below.

[0183] Human BCMA (NP_001183.2; SEQ ID NO: 18)

[0184] 1 mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk gtnailwtcl

[0185] 61 glsliislav fvlmfllrki nseplkdefk ntgsgllgma nidleksrtg deiilprgle

[0186] 121 ytveectced cikskpkvds dhcfplpame egatilvttk tndyckslpa alsateieks

[0187] 181 isar

[0188] Human TACI (NP_036584.1; SEQ ID NO: 19)

[0189] 1 msglgrsrrg grsrvdqeer fpqglwtgva mrscpeeqyw dpllgtcmsc kticnhqsqr

[0190] 61 tcaafcrsls crkeqgkfyd hllrdcisca sicgqhpkqc ayfcenklrs pvnlppelrr

[0191] 121 qrsgevenns dnsgryqgle hrgseaspal pglklsadqv alvystlglc lcavlccflv

[0192] 181 avacflkkrg dpcscqprsr prqspakssq dhameagspv stspepvetc sfcfpecrap

[0193] 241 tqesavtpgt pdptcagrwg chtrttvlqp cphipdsglg ivcvpaqegg pga

[0194] The present disclosure provides peptides (e.g., native peptides) derived from BCMA or TACI antigens and variant peptides derived from the peptides (native peptides). These peptides can be any part or fragment of BCMA or TACI peptides. In some embodiments, the length of these peptides is greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. In some embodiments, the length of these peptides is less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. In some embodiments, the length of these peptides is 8 to 12 amino acid residues, 8 to 15 amino acid residues, 9 to 13 amino acid residues, 9 to 12 amino acid residues or 11 to 30 amino acid residues. In some embodiments, the length of the peptide is 9, 10, 11 or 12 amino acid residues (e.g., 9 amino acid residues).

[0195] In some embodiments, the peptide derived from BCMA or TACI includes the following:

[0196] #1.BCMA 64-72<h2 style=";text-align:left;direction:ltr">(LIISLAVFV)(SEQ ID NO:1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0197] <h2 style=";text-align:left;direction:ltr"> #2.BCMA<h2 style=";text-align:left;direction:ltr"> 69-77 <h2 style=";text-align:left;direction:ltr"> (AVFVLMFLL)(SEQ ID NO:2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0198] <h2 style=";text-align:left;direction:ltr"> #3.BCMA<h2 style=";text-align:left;direction:ltr"> 9-17 <h2 style=";text-align:left;direction:ltr"> (SQNEYFDSL)(SEQ ID NO:3)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0199] <h2 style=";text-align:left;direction:ltr"> #4.BCMA<h2 style=";text-align:left;direction:ltr"> 72-80 <h2 style=";text-align:left;direction:ltr"> (VLMFLLRKI)(SEQ ID NO:4)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0200] <h2 style=";text-align:left;direction:ltr"> #5.BCMA<h2 style=";text-align:left;direction:ltr"> 54-62 <h2 style=";text-align:left;direction:ltr"> (AILWTCLGL)(SEQ ID NO:5)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0201] <h2 style=";text-align:left;direction:ltr"> #6.BCMA<h2 style=";text-align:left;direction:ltr"> 114-122 <h2 style=";text-align:left;direction:ltr"> (ILPRGLEYT)(SEQ ID NO:6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0202] <h2 style=";text-align:left;direction:ltr"> #1.TACI<h2 style=";text-align:left;direction:ltr"> 178-186 <h2 style=";text-align:left;direction:ltr"> (FLVAVACFL)(SEQ ID NO:7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0203] <h2 style=";text-align:left;direction:ltr"> #2.TACI<h2 style=";text-align:left;direction:ltr"> 174-182 <h2 style=";text-align:left;direction:ltr"> (VLCCFLVAV)(SEQ ID NO:8)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0204] <h2 style=";text-align:left;direction:ltr"> #3.TACI<h2 style=";text-align:left;direction:ltr"> 154-162 <h2 style=";text-align:left;direction:ltr"> (KLSADQVAL)(SEQ ID NO:9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0205] <h2 style=";text-align:left;direction:ltr"> #4.TACI<h2 style=";text-align:left;direction:ltr"> 166-174 <h2 style=";text-align:left;direction:ltr"> (TLGLCLCAV)(SEQ ID NO:10)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0206] <h2 style=";text-align:left;direction:ltr"> #5.TACI<h2 style=";text-align:left;direction:ltr"> 161-169 <h2 style=";text-align:left;direction:ltr"> (ALVYSTLGL)(SEQ ID NO:11)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0207] <h2 style=";text-align:left;direction:ltr"> #6.TACI<h2 style=";text-align:left;direction:ltr"> 155-163 <h2 style=";text-align:left;direction:ltr"> (LSADQVALV)(SEQ ID NO:12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0208] These peptides can bind to major histocompatibility complex (MHC) molecules. MHC is a large gene family that plays an important role in the immune system, autoimmunity and reproduction. MHC molecules play a role in the presentation of peptides (including self and non-self (antigenicity) on its surface) to T cells. MHC class I molecules bind short peptides, and their N and C terminal ends are anchored to the pockets located at the ends of the peptide binding groove. Although many of these peptides are 9 in length, longer peptides can be accommodated by the protrusions in their central parts, thereby forming binding peptides with a length of, for example, 8 to 15. The size of the peptides bound to class II proteins is not limited and can vary, for example, 11 to 30 amino acids in length. The peptide binding groove in MHC class II molecules is open at both ends, which enables the binding of peptides with relatively long lengths. Although the nine residue long chain segments of the "core" contribute the most to the recognition of peptides, the flanking regions are also important for the specificity of peptides to class II alleles.

[0209] Therefore, the present invention also provides a peptide with the following sequence, which comprises any sequence described in the present disclosure, is composed of any sequence described in the present disclosure or is substantially composed of any sequence described in the present disclosure. In some embodiments, the peptide can be combined with class I MHC molecules and / or class II MHC molecules. In some embodiments, the MHC class I molecule is HLA-A (e.g., HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A30, HLA-A26, HLA-A68 or HLA-A11), HLA-B or HLA-C.

[0210] In order to improve the stability of peptide binding to MHC molecules (e.g., MHC class I molecules, HLA-A2) molecules, increase immunogenicity, and / or increase immune response, various modifications can be made to the peptide. For example, to increase immunogenicity, the amino acid residues can be modified by changing, for example, the TCR interaction sites in positions 3, 4, 5, 6, 7, and / or 8 of any peptide described herein (e.g., SEQ ID NO: 1-17) to enhance affinity for T cell receptors (TCR).

[0211] Dibasic amino acid residues (e.g., Arg-Arg, Arg-Lys, Lys-Arg or Lys-Lys) can also be added to the N and C termini of the peptide. In some embodiments, amino acids are substituted to enhance major histocompatibility complex (MHC) binding by modifying anchor residues ("fixed anchor epitopes"), or to enhance binding to T cell receptors (TCR) by modifying TCR interaction sites (e.g., positions 1, 2 and / or 9 of SEQ ID NO: 1-17). In some embodiments, epitopes described herein can be modified at any position (e.g., at one, two, three, four, five or six positions). Peptides can also include internal mutations that make them "superantigens" or "superagonists" for T cell stimulation. Superantigenic peptides can be generated by screening T cells with position scanning synthetic peptide combinatorial libraries (PS-CSLs), as described in Pinilla et al, Biotechniques, 13(6):901-5, 1992; Borras et al, J. Immunol. Methods, 267(1):79-97, 2002; U.S. Publication No. 2004 / 0072246; and Lustgarten et al., J. Immun. 176:1796-1805, 2006. In some embodiments, superagonist peptides are peptides as described herein with one, two, three or four amino acid substitutions that render the peptide more potently immunogenic. In some embodiments, the first amino acid residue of a peptide derived from BCMA or TACI can be changed to tyrosine.

[0212] Some variant peptides derived from BCMA or TACI include:

[0213] Mutation #4: BCMA 72-80 (YLMFLLRKI) (SEQ ID NO: 13)

[0214] Mutation #5: BCMA 54-62 (YILWTCLGL) (SEQ ID NO: 14)

[0215] Mutation #1: TACI 178-186 (YLVAVACFL) (SEQ ID NO: 15)

[0216] Mutation #3: TACI 154-162 (YLSADQVAL) (SEQ ID NO: 16)

[0217] Mutation #4: TACI 166-174 (YLGLCLCAV) (SEQ ID NO: 17)

[0218] As used herein, the term "variant" (e.g., a variant peptide) refers to a form of a peptide in which one or more amino acid residues have been modified from the wild-type or native sequence to produce a peptide that is more immunogenic than the corresponding peptide having the wild-type or native sequence.

[0219] The present disclosure further provides variants of the peptides as described herein (e.g., SEQ ID NOs: 1-17). Variants of the peptides described herein may include forms of the peptides having no more than five, no more than four, no more than three, no more than two, no more than one amino acid substitution (e.g., 5, 4, 3, 2, or 1 amino acid substitution). In some embodiments, variants of the peptides described herein may include forms of the peptides having at least one, at least two, at least three, or at least four substitutions.

[0220] In some embodiments, the amino acid at position 1, 2 and / or 9 of SEQ ID NO: 1-17 contributes to HLA binding affinity and can therefore be substituted without affecting peptide-specific T cell responses. Therefore, the immunogenicity of the peptide can still be maintained by substitutions at position 1, 2 and / or 9 of SEQ ID NO: 1-17. In some embodiments, the amino acid at position 1 of SEQ ID NO: 1-17 (e.g., SEQ ID NO: 13 or SEQ ID NO: 16) is substituted. In some embodiments, the amino acid at position 2 of SEQ ID NO: 1-17 (e.g., SEQ ID NO: 13 or SEQ ID NO: 16) is substituted. In some embodiments, the amino acid at position 9 of SEQ ID NO: 1-17 (e.g., SEQ ID NO: 13 or SEQ ID NO: 16) is substituted. In some embodiments, the amino acid at position 1 and 2, position 1 and 9, position 2 and 9, or position 1, 2 and 9 of SEQ ID NO: 1-17 is substituted. In some embodiments, the disclosure provides peptides comprising a sequence as described herein (e.g., one of SEQ ID NOs: 1-17, with or without substitutions as described herein), and having up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids. Thus, in some embodiments, the length of the peptide can be between 9 and 20 amino acids, between 9 and 30 amino acids, or between 9 and 40 amino acids.

[0221] Substitutions can be any type of amino acid substitution, such as conservative or non-conservative. Conservative substitutions include substitutions in the following groups: (1) valine, alanine, and glycine; leucine, valine, and isoleucine; (2) aspartic acid and glutamic acid; (3) asparagine and glutamine; (4) serine, cysteine, and threonine; lysine and arginine; (5) phenylalanine and tyrosine. Non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above polar, basic, or acidic groups by another member of the same group can be considered a conservative substitution. In contrast, non-conservative substitutions are substitutions of one amino acid with another having different properties, such as substitution of one amino acid with another within another group.

[0222] In some embodiments, one or more (e.g., one, two, three, four or all five) of positions three, four, five, six, seven and eight of any peptide are not substituted. In some embodiments, one or more (e.g., one, two, three, four or all five) of positions three, four, five, six, seven and eight of a peptide are identical to a sequence selected from SEQ ID NOs: 1-17. As used herein, the term position refers to the position starting from the N-terminus of a peptide. For example, position 3 of a peptide refers to the third amino acid residue starting from the N-terminus of a peptide. In some embodiments, residues at positions three, four, five, six, seven and eight of SEQ ID NOs: 1-17 contribute the most to the recognition of the peptide.

[0223] The present disclosure further provides amino acid sequences or nucleotide sequences comprising, consisting of, or consisting essentially of a sequence that is at least 50% (e.g., 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to any sequence as described in the present disclosure, e.g., SEQ ID NOs: 1-17, SEQ ID NOs: 18 and 19, and the nucleotide sequences encoding SEQ ID NOs: 1-17.

[0224] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments at least 90%, 95%, or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which need to be introduced to achieve optimal alignment of the two sequences). For the purposes of the present disclosure, comparison of sequences and determination of percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0225] Also provided herein are peptides comprising a first amino acid sequence and a second amino acid sequence heterologous to the first amino acid sequence or consisting of a first amino acid sequence and a second amino acid sequence heterologous to the first amino acid sequence. An amino acid sequence that is "heterologous" to the first amino acid sequence or the term "heterologous amino acid sequence" is an amino acid sequence flanking the first amino acid sequence, wherein the flanking sequence does not exist in nature (e.g., the flanking sequence is not connected to the first amino acid sequence in nature). The first amino acid sequence may comprise, consist essentially of, or consist of any sequence as described herein, such as SEQ ID NO: 1-17, or any sequence derived from SEQ ID NO: 1-17 (e.g., a sequence having no more than four substitutions of SEQ ID NO: 1-17). Peptides having heterologous flanking amino acid sequences generally do not (and are selected so as not to) adversely affect the production of immunogenic peptides of SEQ ID NO: 1-17 in cells. It is expected that the cellular machinery will remove any other sequences in the peptide to generate immunogenic peptides of SEQ ID NOs: 1-17 that are presented by class I or class II MHC molecules to stimulate an immune response against cancer cells expressing BCMA or TACI.

[0226] The heterologous flanking sequence can be, for example, a sequence used to purify a recombinant protein (e.g., FLAG, polyhistidine (e.g., hexa-histidine), hemagglutinin (HA), glutathione-S-transferase (GST), or maltose binding protein (MBP)). The heterologous sequence can also be a protein that can be used as a diagnostic or detectable marker, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). In some embodiments, the peptide can comprise all or part of an immunoglobulin molecule (e.g., all or part of an immunoglobulin heavy chain constant region).

[0227] In some embodiments, the heterologous sequence may comprise a therapeutic or immunostimulatory polypeptide sequence (e.g., a T helper epitope (e.g., a PADRE epitope or a tetanus toxoid universal T helper epitope) or all or part of a cytokine or chemokine) and / or a carrier (e.g., KLH) that can be used, for example, to elicit an immune response (e.g., for antibody production). In some embodiments, the peptide may comprise one or more linker peptide sequences. The peptide may also comprise a targeting polypeptide. The heterologous sequence may be of varying lengths, and in some cases may be a longer sequence than the first amino acid sequence to which the heterologous amino acid sequence is attached. It is understood that a peptide comprising a first amino acid sequence and a second amino acid sequence heterologous to the first amino acid sequence does not correspond in sequence to a naturally occurring protein.

[0228] As used herein, targeting polypeptides are polypeptides that target the portion (or portions) to which they are attached (e.g., a first amino acid sequence) to a specific tissue (e.g., to a lymph node) or cell (e.g., to an antigen presenting cell or other immune cell) or a specific isolated molecule or molecular complex in vitro. The targeting polypeptide can be, for example, an antibody (immunoglobulin) or an antigen binding fragment thereof or a ligand of a cell surface receptor. The antibody (or its antigen binding fragment) can be, for example, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, a single-chain antibody, a chimeric antibody, or an antibody Fab fragment, F(ab')2 fragment, Fab' fragment, Fv fragment, or scFv fragment. Antibody fragments comprising or as Fc regions (with or without antigen binding regions) can also be used to target agents to Fc receptor expressing cells (e.g., antigen presenting cells, such as interdigitating dendritic cells, macrophages, mononuclear cells, or B cells). The ligand for a cell surface receptor can be, for example, a chemokine, a cytokine (e.g., interleukin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16), or a death receptor ligand (e.g., FasL or TNFα).

[0229] In some embodiments, the heterologous sequence may include, for example, a "transport sequence" that aids in the delivery of the peptide to a cell or a specific compartment of a cell (e.g., the endoplasmic reticulum or the Golgi apparatus). The transport sequence may include, for example, a membrane translocation sequence, a transporter sequence, an antennapedia sequence, a cyclic integrin binding peptide, and a Tat-mediated peptide or a modified form thereof.

[0230] The joint peptide can connect the first amino acid sequence to one or more heterologous amino acid sequences. For example, the joint peptide can connect the first amino acid sequence to the second amino acid sequence. In certain embodiments, the joint peptide can connect / connect the peptide of any one of SEQ ID NO:1-17 to the second peptide selected from SEQ ID NO:1-17. The joint peptide can be or include, for example, an amino acid segment in which at least four to six amino acids are glycine. (See, for example, Mancebo et al. (1990) MoI. Cell. Biol. 10: 2492-2502). The joint can also be or include six or more (for example, seven, eight, nine, ten, eleven or twelve or more) histidine residues. The joint peptide can include or be at least one (for example, one, two, three, four, five, six, seven or eight or more) protease cleavage site. The protease site can be, for example, a trypsin, chymotrypsin or factor Xa cleavage site. Such protease sites can be used, for example, to separate the first amino acid sequence from the heterologous sequence. For example, following expression and purification of a peptide comprising a first amino acid sequence linked to a polyhistidine sequence (eg, for purification) via a trypsin protease cleavage site, the polyhistidine sequence can be removed from the first amino acid sequence by contacting the peptide with trypsin.

[0231] In some embodiments, the disclosure provides peptides (e.g., any one of SEQ ID NOs: 1-17) that can have up to 200 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino and / or carboxyl terminal ends that are heterologous to or present in the native protein. , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) amino acids.

[0232] In some embodiments, the peptide may comprise a sequence that is at least 40%, at least 50%, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any sequence as described herein (e.g., any one of SEQ ID NOs: 1-17). In some embodiments, the sequence may have equal to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids selected from any sequence as described herein (e.g., any one of SEQ ID NOs: 1-17). In some embodiments, the sequence is SEQ ID NO: 13. In some embodiments, the sequence is SEQ ID NO: 14. In some embodiments, the sequence is SEQ ID NO:16.

[0233] In some embodiments, the peptide may have an additional sequence. The additional sequence may be located at the amino terminal end or the carboxyl terminal end of the peptide. 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids. In some embodiments, the additional sequence can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids.

[0234] In certain cases, the present disclosure encompasses any combination of two or more peptides selected from SEQ ID NOs: 13-17.

[0235] The peptides described herein can bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I molecule or an MHC class II molecule). In humans, MHC is referred to as an HLA complex. As used herein, "HLA supertype or family" refers to a collection of HLA molecules grouped based on shared peptide binding specificity. HLA class I molecules that have similar binding affinities for peptides with certain amino acid motifs are grouped as HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where xx represents a specific HLA type) are synonyms. Types of HLA class I molecules include, for example, HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-B7, HLA-B27, HLA-B44, HLA-B58, or HLA-B62. Such HLA molecules are described in detail in U.S. Patent No. 7,026,443, the entire disclosure of which is incorporated herein by reference in its entirety.

[0236] The peptide can bind to the MHC molecule with high affinity or moderate affinity. As used herein, "high affinity" binding of a peptide to an HLA class I molecule is defined as binding with a dissociation constant (KD) of less than 50 (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1, or less than 0.05) nM. By "moderate affinity" is a peptide that binds to an HLA class I molecule with a KD of about 50 nM to about 500 nM (e.g., 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nM). "High affinity" binding of a peptide to an HLA class II molecule is defined as binding with a KD of less than 100 (e.g., 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1, or less than 0.05) nM. 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nM). Methods for determining the binding affinity of a peptide to an MHC molecule are known in the art. Suitable methods are also described, for example, in U.S. Patent No. 7,026,443.

[0237] The peptides described herein can also be bound to MHC molecules and recognized by antigen-specific T cell receptors on T cells. A variety of suitable methods can be used to determine whether a peptide bound to an MHC molecule is recognized by a T cell receptor on a T cell. For example, peripheral blood lymphocytes (PBL) from normal subjects can be cultured in vitro with a test peptide in the presence of antigen presenting cells over a period of several weeks. T cells specific for the peptide are activated during this time and can be detected using, for example, a proliferation assay (carboxyfluorescein succinimidyl ester (CFSE) assay of H-thymidine assay), a limiting dilution assay, a cytotoxicity assay (e.g., calcein release assay), or a cytokine (e.g., IFNγ), lymphokine release, or 51Cr release assay (see, e.g., Wentworth, P.A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998, each of which is incorporated by reference in its entirety). A suitable in vivo method includes immunization of HLA transgenic mice, wherein the peptide in adjuvant is injected subcutaneously into the HLA transgenic mice, and several weeks after immunization, spleen cells are removed and cultured in vitro for about one week in the presence of the test peptide, and peptide-specific T cells are detected using, for example, a 51Cr-release assay (see, e.g., Wentworth, PA et al., J. Immunol. 26:97, 1996; Wentworth, PA et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997, the disclosures of each of which are incorporated by reference in their entirety).

[0238] In addition, direct quantification of antigen-specific T cells can be performed by staining the T cells with detectably labeled MHC complexes, such as any of the MHC molecule multimeric compositions described herein or HLA-I tetramers (e.g., as described in Altman, J.D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993 and Altman, J.D. et al., Science 274:94, 1996, the disclosures of each of which are incorporated by reference in their entireties).

[0239] In some embodiments, the peptide can be further modified (e.g., the amino acids of the peptide can be substituted) to modulate (e.g., increase or decrease) one or more properties of the peptide. For example, one or more (e.g., two, three or four) amino acids of one of the peptides described herein can be substituted to increase the affinity of the peptide for the MHC molecule. In some embodiments, the amino acids of one of the peptides described herein (e.g., the T cell receptor that contacts the amino acid residues of the peptide) can be modified to enhance the binding interaction between the T cell receptor and the peptide (in the context of the MHC molecule). Such modified peptides are generally referred to as "altered peptide ligands". (See, e.g., Kalergis et al. (2000) J Immunol. 165 (1): 280; Conlon et al. (2002) Science 1801; and International Publication No. WO02070003, the disclosures of each of which are incorporated by reference in their entirety). Suitable methods for modifying peptides and determining the effects of the modifications are described, for example, in Collins et al. (Immunlogical Reviews (1998) 163: 151-160, the disclosure of which is incorporated by reference in its entirety).

[0240] The present disclosure further provides a composition comprising any peptide as described herein. In addition, in order to counteract the ability of tumors to escape therapy directed against it, the composition can include a variety of specific peptides to induce an immune response. For example, more than one epitope from the same protein can be included in the composition, for example, the composition can contain at least one, at least two, at least three, at least four, at least five, at least six or at least one seven peptides derived from BCMA or TACI. In addition, a combination or mixture of at least one (e.g., at least two, three, four, five) peptides derived from BCMA and at least one (e.g., at least two, three, four, five) peptides derived from TACI can also be used. Therefore, in some embodiments, the present disclosure provides a composition comprising at least 2, 3, 4, 5, 6, 7 or 8 BCMA-derived peptides (e.g., selected from at least two BCMA-derived peptides such as SEQ ID NO: 1-6 and 13-14). In some embodiments, the present disclosure provides a composition comprising at least 2, 3, 4, 5, 6, 7, or 8 TACI-derived peptides (e.g., at least two TACI-derived peptides selected from, e.g., SEQ ID NOs: 7-12 and 16-17). In some embodiments, the present disclosure provides a composition comprising at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) BCMA-derived peptide and at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) TACI-derived peptide.

[0241] In some embodiments, the composition further comprises an immunostimulant (e.g., a cytokine or a T helper cell epitope). The T helper cell epitope can be a PADRE sequence or a universal tetanus toxoid T helper (TT Th) epitope. In some embodiments, the composition comprises an adjuvant, such as Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand of a Toll receptor, QS21, RIBI or a similar immunostimulant. Adjuvants also include, for example, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185: 1203-1210) and mutant Escherichia coli heat-labile toxin (MLT) (Di Tommaso et al. (1996) Infect. Immunity 64: 974-979). In some embodiments, the composition comprises a toll-like receptor-3 ligand (e.g., Poly ICLC), interferon alfa (IFNα), interferon gamma (IFNγ), or granulocyte-macrophage colony stimulating factor (GM-CSF). In some embodiments, the composition further comprises an immune agonist, such as an anti-OX40 antibody or an anti-GITR antibody.

[0242] In some embodiments, the composition comprises a chemotherapeutic agent (e.g., lenalidomide). In some embodiments, the composition comprises a histone deacetylase 6 (HDAC6) inhibitor (e.g., ACY241; see Niesvizky, et al. "ACY-241, a novel, HDAC6 selective inhibitor: synergy with immunomodulatory drugs in multiple myeloma (MM) cells and early clinical results (ACE-MM-200 Study)." (2015): 3040-3040;Bae et al."Histone deacetylase (HDAC) inhibitor ACY241 enhances anti-tumor activities of antigen-specific central memory cytotoxic T lymphocytes against multiple myeloma and solid tumors." Leukemia (2018): 1). In some embodiments, the composition comprises a checkpoint inhibitor (e.g., an anti-LAG3 antibody) or an immune agonist (e.g., anti-OX40). In some embodiments, the composition comprises an antibody (e.g., a human antibody) that specifically binds to PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT, TIM-3, GITR (CD357), OX40, CD20, EGFR, or CD319.

[0243] In some embodiments, the composition comprises a peptide comprising a sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 16 and lenalidomide. In some embodiments, the composition comprises a peptide comprising a sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 16 and an anti-OX40 antibody, an anti-LAG3 antibody and / or an anti-GITR antibody.

[0244] Nucleic acids and methods for producing peptides

[0245] The present disclosure also features nucleic acid sequences (and nucleic acid vectors comprising nucleic acid sequences) encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of any peptide described herein, and methods of producing nucleic acid sequences (and nucleic acid vectors comprising nucleic acid sequences) of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of any peptide described herein. Such methods may include the following steps: optionally, providing a cell (or group of cells) comprising a nucleic acid vector, the nucleic acid vector comprising one or more nucleic acid sequences encoding any peptide described herein, the nucleic acid sequence being operably linked to an expression control sequence, and culturing the cell under conditions that allow expression of the peptide. The method may also include the step of isolating one or more peptides from the cell or from the culture medium in which the cell is cultured. Thus, in one aspect, the present disclosure provides RNA-based therapeutics and DNA-based therapeutics, including, for example, cancer vaccines. In some embodiments, the cancer vaccine may have a polynucleotide as described herein (e.g., a polynucleotide encoding SEQ ID NO: 1-17). In some cases, the polynucleotide encodes a peptide that is identical to one of SEQ ID NOs: 1-17, but has 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2, or 9. In some cases, the length of the peptide is 2 to 30 amino acids. In some cases, the nucleic acid may include regulatory sequences (e.g., start codons, stop codons, polyA tails). In some embodiments, the RNA / DNA cancer vaccine can be formulated in a polymer or liposome nanocarrier (e.g., nanoparticle).

[0246] Suitable methods for constructing nucleic acid sequences and vectors (e.g., expression vectors) for recombinant expression of one or more peptides described herein are well known to those skilled in the art and are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition Vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989, the disclosure of which is incorporated by reference in its entirety. Nucleic acids and vectors can be used, for example, to express peptides in a wide variety of host cells, including, for example, bacteria, yeast or mammalian cells. Nucleic acids and vectors can also be used, for example, in vivo and in vitro methods as described below. The peptide coding sequence can be operably linked to a promoter, a regulatory element or an expression control sequence. Promoters and / or enhancer elements can direct the expression of the peptides encoded by the nucleic acid. Enhancers provide expression specificity in terms of time, position and level. Unlike promoters, if there is a promoter, enhancers can function when located at a variable distance from the transcription start site. Enhancers can also be located downstream of the transcription start site or in the exons of related genes. In order to place the coding sequence under the control of the promoter, it is necessary to position the translation start site of the peptide translation reading frame between 1 and about 50 nucleotides downstream (3') of the promoter. Promoters of interest include, but are not limited to, the cytomegalovirus hCMV immediate early gene, the early or late promoter of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter region of phage A, the control region of the fd capsid protein, the promoter of 3-phosphoglycerate kinase, the promoter of acid phosphatase and the promoter of yeast α matching factor, the adenovirus EIb minimal promoter or the thymidine kinase minimal promoter.

[0247] The peptide coding sequence or the vector comprising the peptide coding sequence may include a leader sequence encoding a signal peptide. The leader sequence may be at the 5' end of the sequence encoding one or more peptides described herein. The signal peptide may be directly located at the N-terminus of a given peptide, or may be separated therefrom by one or more (e.g., 2, 3, 4, 6, 8, 10, 15 or 20) amino acids, provided that the leader sequence and the nucleic acid sequence encoding the peptide meet the reading frame. The signal peptide is usually cut from the peptide before secretion (unless of course the signal peptide guides the insertion of a transmembrane protein), and the signal peptide guides the peptide attached thereto to the lumen of the host cell endoplasmic reticulum (ER) during translation, and then the peptide is secreted into the environment of the host cell by secretory vesicles. Useful signal peptides include, for example, natural leader sequences of cytokines or growth factors, KDEL (Lys-Asp-Glu-Leu), or, for example, any signal sequence described in U.S. Patent No. 5,827,516 (the disclosure of which is incorporated herein by reference).

[0248] In some embodiments, the 5' end of the peptide coding sequence may include a non-natural ATG "start sequence". That is, for example, an ATG sequence may be added to a nucleic acid encoding a peptide to ensure proper transcription and translation of the peptide. Although the leader sequence typically includes an ATG start sequence, in embodiments where it does not include an ATG start sequence, an ATG sequence may be added to the 5' end of the nucleic acid encoding the leader sequence.

[0249] Suitable methods for constructing peptide coding sequences and expression vectors are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Edition, Vols. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989; the disclosure of which is incorporated herein by reference in its entirety.

[0250] A variety of methods can be used to introduce recombinant vectors into cells, which methods may depend at least in part on the type of cell into which the nucleic acid is introduced. For example, bacterial cells can be transformed using methods such as electroporation or heat shock. Methods for transfecting yeast cells include, for example, spheroplast technology or whole-cell lithium chloride yeast transformation methods (see, for example, U.S. Patent No. 4,929,555; Hinnen et al. (1978) Proc. Nat. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163; U.S. Patent No. 4,879,231; and Sreekrishna et al. (1987) Gene 59:115, the disclosures of each of which are incorporated herein by reference in their entirety). Transfection of animal cells can have the following features: For example, using calcium phosphate, electroporation, heat shock, liposomes or transfection reagents (such as or ) or by contacting the naked nucleic acid vector with the cells in solution (see, e.g., Sambrook et al., supra).

[0251] Expression systems that can be used for small-scale or large-scale production of the peptides described herein include, but are not limited to, microorganisms, such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors; fungi (e.g., yeast (e.g., Saccharomyces and Pichia)) transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) containing recombinant expression constructs, which recombinant expression constructs contain promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, CMV promoter, SV40 promoter, or vaccinia virus 7.5K promoter). Also useful as host cells are primary or secondary cells obtained directly from mammals, transfected with a plasmid vector, or infected with a viral vector (e.g., a viral vector such as herpes virus, retrovirus, vaccinia virus, attenuated vaccinia virus, canarypox virus, adenovirus, adeno-associated virus, etc.).

[0252] After expressing any peptide described herein, the peptide can be isolated from the cultured cells or from the culture medium of the cultured cells using standard techniques. Methods for isolating proteins are known in the art and include, for example, liquid chromatography (e.g., HPLC), affinity chromatography (e.g., metal chelate or immunoaffinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography, precipitation, or differential solubilization.

[0253] Smaller peptides (e.g., peptides having less than 200 (e.g., less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 70, or less than 60) amino acids) can be chemically synthesized by standard chemical means (e.g., FMOC solid phase synthesis).

[0254] The peptides described herein can but need not be separated. As applied to any peptide described herein, the term "isolated" refers to a peptide, fragment thereof (or for compositions, macromolecular complexes) that has been separated or purified from its naturally accompanying components (e.g., proteins or other naturally occurring biological or organic molecules). It should be understood that recombinant molecules (e.g., recombinant peptides) will always be "isolated". Typically, a peptide (or fragment or macromolecular complex) is isolated when it accounts for at least 60%, 70%, 80% or 90% by weight of the total molecules of the same type in a preparation, such as at least 60%, 70%, 80% or 90% of the total molecules of the same type in a sample. For example, a peptide described herein is considered to be isolated when it accounts for at least 60%, 70%, 80% or 90% by weight of the total protein in a preparation or sample. In some embodiments, the molecules in the preparation are composed of at least 75%, at least 90% or at least 99% by weight of the total molecules of the same type in the preparation.

[0255] Similarly, the peptide coding sequence or the vector comprising the peptide coding sequence described herein can also be separated. As applied to any peptide coding sequence or vector described herein, the term "isolated" refers to a peptide coding sequence or vector that has been separated or purified from its naturally accompanying components (e.g., nucleic acids, proteins, or other naturally occurring biological or organic molecules), or a fragment thereof. It should be understood that recombinant molecules (e.g., recombinant vectors or peptide coding sequences) will always be "isolated". Typically, when a peptide coding sequence or vector (or its fragment) accounts for at least 60%, 70%, 80% or 90% by weight of the total molecules of the same type in a preparation, such as at least 60%, 70%, 80% or 90% of the total molecules of the same type in a sample, it is isolated. For example, when a peptide coding sequence or vector described herein accounts for at least 60%, 70%, 80% or 90% by weight of the total nucleic acid in a preparation or sample, it is considered to be isolated. In some embodiments, the molecules in the preparation are composed of at least 75%, at least 90% or at least 99% by weight of the total molecules of the same type in the preparation.

[0256] In some embodiments, an isolated peptide, peptide encoding sequence, or vector can be frozen, lyophilized, or immobilized and stored under appropriate conditions that allow the molecule to retain activity (e.g., the ability of the peptide to bind to an MHC molecule, such as a class I MHC molecule or a class II MHC molecule, or the ability of the vector to support expression of the peptide in a cell).

[0257] Peptide processing

[0258] After expressing or synthesizing any peptide described herein, the peptide can be further processed. Further processing can include chemical or enzymatic modification of the peptide, or in the case of modified peptides, the processing can include enzymatic or chemical changes to existing modifications or both. Additional processing of the peptide can include adding (covalently or non-covalently linked) a heterologous amino acid sequence, such as, but not limited to, any heterologous amino acid sequence described herein. Enzymatic treatment can involve contacting the peptide with, for example, one or more proteases, phosphatases, or kinases under conditions that allow the peptide to be modified. Enzymatic treatment can involve contacting the peptide with one or more enzymes (e.g., oligosaccharyl transferases or mannosidases) that are capable of glycosylation of the peptide or glycosylation of the modified peptide.

[0259] Processing can include adding, for example, a detectable label to the peptide. For example, the peptide can be labeled with an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase), a fluorescent substance (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride, phycocyanin (APC), or phycoerythrin), a luminescent material (e.g., a lanthanide or a chelate thereof), a bioluminescent material (e.g., luciferase, luciferin, or aequorin), or a radionuclide (e.g., 3 H. 32 P. 33 P. 125 I. or 35 S) The peptide is detectably labeled.

[0260] Processing can also involve coupling the peptide to a polymer (e.g., a polyalkylene glycol moiety, such as a polyethylene glycol moiety) or a nanoparticle. In some embodiments, the polymer is coupled to the polypeptide at a site at the N-terminus of the peptide. In some embodiments, the peptide may contain one or more internal amino acid insertions that provide an internal polymer conjugation site that can be conjugated to the polymer.

[0261] Methods of inducing an immune response

[0262] The disclosure also provides a variety of methods for inducing an immune response in a subject (e.g., peptide-based vaccination, nanoparticle-based immunotherapy, APC-based immunotherapy, T cell-based immunotherapy, CART cell-based immunotherapy, or induced pluripotent stem cell methods). The method of inducing an immune response in a subject may include, for example, administering one or more compositions described herein (e.g., any peptide described herein (or an expression vector comprising a nucleic acid sequence encoding the peptide) (or any pharmaceutical composition comprising one or more peptides described herein (or vectors)) to the subject. In some embodiments, the compositions described herein are used as vaccines.

[0263] Any peptide described herein can be used to stimulate an immune response by using a nucleic acid expression system encoding one or more peptides described herein. That is, the method for inducing an immune response in a subject may include the following steps: administering to the subject an expression vector (or a pharmaceutical composition comprising an expression vector) comprising a nucleic acid sequence encoding one or more peptides described herein. The immune response can be a combination of CD8+T cells, CD4+T cells, cytotoxic T lymphocytes (CTL), TH1 responses, TH2 responses, or two types of responses.

[0264] Any of the above methods may also be a method of treating or preventing (guarding against) cancer (e.g., a plasma cell disorder, such as multiple myeloma, or any other cancer expressing BCMA and / or TACI) in a subject. When the terms "prevent," "prevent," or "prevent" are used herein in conjunction with a given treatment for a given condition, they refer to the treated subject not developing clinically observable levels of the condition at all (e.g., the subject does not exhibit one or more symptoms of the condition, or in the case of cancer, the subject does not develop detectable levels of cancer), or the condition develops more slowly and / or to a lesser extent in the subject than it would in the absence of treatment (e.g., fewer symptoms or fewer numbers of cancer cells in the subject). These terms are not limited to situations where the subject does not experience any condition. For example, if treatment is given during, for example, early diagnosis of a cancer (e.g., detection of a few cancer cells in a sample from a subject), the disease is considered to have "prevented" the condition, which is expected to have produced a given manifestation of the condition (advanced cancer) and causes the subject to experience fewer and / or milder symptoms of the condition than would otherwise be expected. Treatment may "prevent" a cancer (eg, a plasma cell disorder such as multiple myeloma) when the subject shows only mild overt symptoms of the cancer. "Prevent" does not mean that the subject so treated must be free from the development of even a single cancer cell.

[0265] Typically, the peptide delivered to the subject will be suspended in a pharmaceutically acceptable carrier (e.g., saline) and administered orally, rectally, or parenterally, such as by intravenous, subcutaneous, intramuscular, intrathecal, intraperitoneal, rectal, vaginal, intranasal, intragastric, intratracheal, or intrapulmonary injection.

[0266] Administration can be performed by regular injection of a bolus of the pharmaceutical composition or can be uninterrupted or continuous by intravenous or intraperitoneal administration from a reservoir externally (e.g., an IV bag) or internally (e.g., a bioerodible implant, a bioartificial organ, or an implanted colony of agent-producing cells). See, e.g., U.S. Pat. Nos. 4,407,957, 5,798,113 and 5,800,828, each of which is incorporated herein by reference in its entirety.

[0267] Conventional and pharmaceutically acceptable routes of administration of therapeutic nucleic acids include, but are not necessarily limited to, intramuscular, subcutaneous, intradermal, transdermal, intravenous, rectal (e.g., enema, suppository), oral, intragastric, intranasal and other effective routes of inhalation and other parenteral routes of administration. If desired, routes of administration may be combined or adjusted according to the desired effect of the nucleic acid molecule and / or on the immune response. Methods for administering nucleic acids to a subject may include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and techniques for facilitating "naked" polynucleotide delivery (e.g., electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides into a subject or subject's cells).

[0268] In general, the required dosage of the peptide or nucleic acid depends on the choice of administration route; the nature of the formulation; the nature or severity of the subject's disease; the subject's immune status; the subject's size, weight, surface area, age and sex; other drugs being administered; and the judgment of the attending medical professional.

[0269] The suitable dosage of the peptide for inducing an immune response is within the range of 0.000001 to 10 mg of reagent or antigen / immunogenic composition per kg subject. Considering the different efficiencies of the type of reagent and various routes of administration, it is expected that the wide variation of the desired dosage will be achieved. For example, nasal or rectal administration may require a higher dosage than that of intravenous injection. The variation of these dosage levels can be regulated using standard empirical procedures for optimization known in the art. Administration can be single or multiple (e.g., 2 times, 3 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 150 times or more). For example, the peptide can be administered as an initial immunization, and then administered once or multiple times as a booster immunization.

[0270] In the context of the methods described herein, the nucleic acid dosage administered to the subject should be sufficient to achieve a beneficial therapeutic response in the subject over time, or to alleviate symptoms. Although the dosage used will vary according to, for example, the subject or the clinical goal to be achieved. Suitable dosage ranges are those that provide up to about 1 μg, up to about 1,000 μg, up to about 5,000 μg, up to about 10,000 μg, up to about 25,000 μg, or about 50,000 μg of nucleic acid per ml of carrier in a single dose.

[0271] In order to optimize the therapeutic efficacy (for example, the efficacy of one or more peptides or nucleic acids encoding peptides in inducing an immune response in a subject), the compositions comprising peptides or nucleic acids can be first administered with different dosing regimens. The unit dose and regimen depend on factors, including, for example, the species of the mammal, its immune status, the body weight of the mammal. The frequency of administration of a pharmaceutical composition (for example, a pharmaceutical composition comprising one or more peptides or one or more nucleic acid sequences encoding one or more peptides described herein) is within the skill and clinical judgment of a medical practitioner (for example, a doctor or nurse). Typically, an administration regimen is established by a clinical trial, which can establish optimal administration parameters. However, practitioners can change such an administration regimen according to the age, health status, weight, sex, and medical condition of the subject.

[0272] In some embodiments, the pharmaceutical composition can be administered to a subject at least twice (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 or more times). For example, the pharmaceutical composition can be administered to a subject once a month for 3 months; once a week for 1 month; once a year for 3 years; once a year for 5 years; once every five years; once every ten years; or once every three years for a lifetime.

[0273] As defined herein, a "therapeutically effective amount" of a peptide or a nucleic acid encoding a peptide is an amount of a peptide or nucleic acid that can produce an immune response in a subject being treated. The therapeutically effective amount (i.e., effective dose) of a peptide includes milligrams, micrograms, nanograms, or picograms of reagents per kilogram of subject or sample weight (e.g., about 1 nanogram per kilogram to about 500 micrograms per kilogram, 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). The therapeutically effective amount of nucleic acids also includes micrograms, nanograms, or picograms of reagents per kilogram of subject or sample weight (e.g., about 1 nanogram per kilogram to about 500 micrograms per kilogram, about 1 microgram per kilogram to about 500 micrograms per kilogram, about 100 micrograms per kilogram to about 500 micrograms per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0274] As defined herein, a "prophylactic effective amount" of a peptide or a nucleic acid encoding a peptide refers to an amount of a peptide or nucleic acid that can generate an immune response against a cancer cell (e.g., multiple myeloma) in a treated subject, which can prevent the formation of cancer in the subject, or can substantially reduce the chance that the subject forms cancer or continues to form cancer. A prophylactic effective amount (i.e., an effective dose) of a peptide includes milligrams, micrograms, nanograms, or picograms of an agent per kilogram of subject or sample weight (e.g., about 1 nanogram per kilogram to about 500 micrograms per kilogram, 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). Prophylactically effective amounts of nucleic acids also include microgram, nanogram, or picogram amounts of the agent per kilogram weight of the subject or sample (e.g., about 1 nanogram per kilogram to about 500 micrograms per kilogram, about 1 microgram per kilogram to about 500 micrograms per kilogram, about 100 micrograms per kilogram to about 500 micrograms per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0275] In some embodiments, the method may also include determining whether an immune response occurs in the subject after the composition is applied to the subject. Suitable methods for determining whether an immune response occurs in the subject include using an immunoassay to detect, for example, the presence of antibodies specific to the peptide in a biological sample from the subject. For example, after the peptide is applied to the subject, a biological sample (such as a blood sample) can be obtained from the subject, and the presence of antibodies specific to the peptide can be tested. An immune response can also be detected by determining the presence or amount of activated T cells in the sample. Such assays include, for example, proliferation assays, limiting dilution assays, cytotoxicity assays (e.g., lymphokine release or 51Cr release assays).

[0276] In some embodiments, the methods described herein (e.g., therapeutic agents, vaccines, cell therapies, antibodies, and / or therapeutic methods targeting some targets other than BCMA and TACI that increase BCMA- and TACI-specific responses) may also include administering to the subject various types of compounds that enhance BCMA- and TACI-specific responses (e.g., cytokines and chemokines), checkpoint inhibitors (e.g., anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3 and / or anti-TIM3 antibodies), immune agonists (e.g., anti-OX40 or anti-GITR antibodies), immunomodulators (e.g., lenalidomide, pomalidomide, HDAC inhibitors such as ACY241), and / or adjuvants.

[0277] The method may also include the following steps: administering one or more chemotherapeutic agents, one or more forms of ionizing radiation or one or more immunomodulators to the subject. One or more forms of ionizing radiation may be gamma radiation, X radiation or beta radiation. One or more forms of chemotherapeutic agents may be selected from the group consisting of cisplatin, carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, doxorubicin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, thalidomide, lenalidomide, proteosome inhibitors (e.g., bortezomib), hsp90 inhibitors (e.g., tenespinmycin), anti-platinum, 5-fluorouracil, vinblastine, vinblastine, methotrexate or analogs of the foregoing. Immunomodulators include, for example, various chemokines and cytokines, such as interleukin 2 (IL-2), granulocyte / macrophage colony stimulating factor (GM-CSF) and interleukin 12 (IL-12). In some embodiments, the peptide or nucleic acid encoding the peptide can be administered together with an immunomodulator such as a Toll receptor ligand or an adjuvant.

[0278] In some embodiments, the additional therapeutic agent is a histone deacetylase 6 (HDAC6) inhibitor (eg, ACY241).

[0279] In some embodiments, the one or more additional therapeutic agents may be an immunomodulator, a checkpoint inhibitor (eg, an anti-LAG3 antibody), or an immune agonist (eg, an anti-OX40 antibody, an anti-GITR antibody).

[0280] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: inhibitors of B-Raf, inhibitors of EGFR, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0281] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of activated hedgehog signaling pathways, and agents that selectively degrade estrogen receptors.

[0282] In some embodiments, the combination therapy includes one or more of the following:

[0283] (A) Compounds that can enhance BCMA and TACI specific responses, such as (1) cytokines and chemokines, (2) checkpoint inhibitors, including, for example, anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3 and / or anti-TIM3 antibodies; (2) immune agonists, including, for example, anti-OX40 and / or anti-GITR antibodies; (3) immunomodulators, including, for example, lenalidomide, pomalidomide, HDAC inhibitors, such as ACY241; (4) adjuvants;

[0284] (B) therapeutic agents that can increase BCMA- and TACI-specific responses, including any therapeutic agent as described herein (e.g., vaccines, cell therapies, and / or antibodies) or independent approaches targeting other targets (e.g., non-BCMA or non-TACI-targeted therapies); and

[0285] (C) other compounds, such as one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0286] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, Everolimus, Fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, Ivitro Limulus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0287] In some embodiments, the additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0288] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0289] In some embodiments, the additional therapeutic agent is an antibody (e.g., a human antibody) that specifically binds to PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT, TIM-3, GITR (CD357), OX40, CD20, EGFR, or CD319. In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0290] The present disclosure also provides viruses comprising nucleic acids encoding one or more peptides as described herein or viral particles comprising one or more peptides as described herein. Various viruses can be used, such as retroviruses, slow viruses, adenoviruses, adeno-associated viruses, alpha viruses, etc. These viruses can be used to deliver peptides or nucleic acids encoding them to subjects to induce an immune response. Similarly, liposomes comprising one or more peptides as described herein and / or nucleic acids encoding them can also be used to deliver peptides or nucleic acids to subjects in need thereof to induce an immune response.

[0291] In some embodiments, the methods described herein can increase the number of immune responses, activities (e.g., production of cytokines, IFN-γ, IL-2 or TNF-α) or immune cells (e.g., T cells, CD8+T cells, cytotoxic T cells and / or CD4+T cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold or 50-fold. In some embodiments, the methods described herein can increase CD107a degranulation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold or 50-fold. In some embodiments, the methods described herein can increase the number of CD8+ effector T cells that specifically target cancer cells or recognize the peptides described herein (e.g., the total number of CD8+ effector T cells, or, for example, the percentage of CD8+ in CD45+ cells) by at least 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold or 50-fold.

[0292] Nanoparticles

[0293] The present disclosure further provides nanoparticles or nanocarriers comprising one or more peptides as described herein (e.g., SEQ ID NO: 1-17) or one or more polynucleotides as described herein (e.g., sequences encoding SEQ ID NO: 1-17). In some cases, the nanocarrier comprises a peptide identical to the amino acid sequence of SEQ ID NO: 1-17 but having 1-4 amino acid substitutions. In some cases, the substitution is at one or more of positions 1, 2, and 9. Polynucleotides encoding such peptides (e.g., mRNA, DNA) can also be encapsulated in nanocarriers. The nanoparticles or nanocarriers can be administered to a subject in need thereof to induce an immune response.

[0294] Peptides can be attached to nanoparticles or nanocarriers by various attachment mechanisms. This attachment mechanism can be electrostatic attraction, covalent coupling or hydrophobic interaction. In some embodiments, nanoparticles can be loaded with adjuvants. Adjuvants can be dendritic cell targeting molecules, such as Toll-like receptor agonists, such as R-848 (which is considered to be an effective synthetic agonist of TLR7 / TLR8), or unmethylated CpG oligodeoxynucleotides (it is a TLR-9 immunostimulatory agonist) or monophosphoryl lipid A (it is an immunostimulatory agonist of TLR-4), or endosomal membrane targeting agents, such as Endo-Porter peptides.

[0295] The polymer forming the nanoparticles can be any biodegradable or non-biodegradable synthetic or natural polymer. Preferably, the polymer is a biodegradable polymer. Examples of useful biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA) or poly (lactic acid-co-glycolic acid) (PLGA). These polymers have established safety records and can be used in human subjects (Jiang, et al., Adv. Drug Deliv. Rev., 57 (3): 391-410, 2005; Aguado and Lambert, Immunobiology, 184 (2-3): 113-25, 1992; Bramwell, et al., Adv. Drug Deliv. Rev., 57 (9): 1247-65, 2005). Other amphiphilic poly (amino acid) nanoparticles, amphiphilic polysaccharide nanoparticles or polyion nanoparticles can be used in the vaccine compositions disclosed herein (see Akagi et al., Adv Polym Sci. 247: 31-64, 2012). The aforementioned polymers can be used alone, as a physical mixture or by forming copolymers. In certain embodiments, the nanoparticles are formed by a mixture of poly (lactic acid-co-glycolic acid)-block-poly (L-histidine)-block-poly (ethylene glycol) (PLGA-PLH-PEG) triblock copolymer; PLGA-PEG diblock copolymer and PLA. These copolymers can be synthesized using standard techniques. For example, the copolymer PLGA-PLH-PEG can be synthesized using a block end grafting strategy.

[0296] As used herein, a "nanoparticle" is a particle ranging between 500 nm and 0.5 nm, for example having a diameter between 50 and 500 nm, having a diameter between 100 and 400 nm, or having a diameter between 200 and 400 nm.

[0297] Nanoparticles and how to prepare and use nanoparticles are known in the art and are described, for example, in US2016 / 0008451, US 2010 / 0129439, US2018 / 0021258, each of which is incorporated herein by reference in its entirety. In some embodiments, the nanoparticle is a liposome. In some embodiments, the nanoparticle is a polymer particle.

[0298] The polymer forming the nanoparticles can be any biodegradable or non-biodegradable synthetic or natural polymer. In some embodiments, the polymer is a biodegradable polymer. Examples of useful biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA) or poly (lactic acid-co-glycolic acid) (PLGA). These polymers have established safety records and can be used for human subjects (Jiang, et al, Adv. Drug Deliv. Rev., 57 (3): 391-410, 2005; Aguado and Lambert, Immunobiology, 184 (2-3): 113-25, 1992; Bramwell, et al., Adv. Drug Deliv. Rev., 57 (9): 1247-65, 2005). Other amphiphilic poly(amino acid) nanoparticles, amphiphilic polysaccharide nanoparticles, or polyion nanoparticles can be used in the compositions disclosed herein (see Akagi et al, Adv Polym Sci. 247:31-64, 2012).

[0299] These polymers can be used alone, as physical mixtures or by forming copolymers. In some embodiments, nanoparticles are formed by a mixture of poly (lactic acid-co-glycolic acid)-block-poly (L-histidine)-block-poly (ethylene glycol) (PLGA-PLH-PEG) triblock copolymer; PLGA-PEG diblock copolymer and PLA. These copolymers can be synthesized using standard techniques. For example, a block end grafting strategy can be used to synthesize the copolymer PLGA-PLH-PEG. Linear structures (e.g., PLGA-PLH-PEG) can provide nanoparticles with several features compatible with extended circulation and charge-mediated targeting.

[0300] In some embodiments, natural polymers can be used. Examples of natural polymers include alginate and other polysaccharides, collagen, albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. Typically, these materials are degraded by enzymatic hydrolysis or exposure to water in vivo or by surface or bulk erosion.

[0301] Other suitable biodegradable polymers include, but are not limited to, poly(hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co-caprolactone).

[0302] The polymer can be a hydrophilic or hydrophobic bioadhesive polymer. Hydrophilic polymers include CARBOPOL TM(a high molecular weight, cross-linked acrylic acid-based polymer manufactured by Noveon), polycarbophil, cellulose esters, and dextran.

[0303] These polymers are available from sources such as Sigma Chemical Co., St. Louis, Mo.; Polysciences, Warrenton, Pa.; Aldrich, Milwaukee, Wis.; Fluka, Ronkonkoma, NY; and BioRad, Richmond, Calif., or can be synthesized using standard techniques from monomers obtained from these or other suppliers.

[0304] Traditionally known extremely many polymers and methods for forming polymer matrices therefrom. Typically, the polymer matrix comprises one or more polymers. The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or copolymer comprising two or more monomers. In terms of sequence, the copolymer may be random, block or a combination comprising random and block sequences. Typically, the polymer according to the present invention is an organic polymer.

[0305] Examples of suitable polymers for use in the compositions described herein include, but are not limited to, polyethylene, polycarbonates (e.g., poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxy acids (e.g., poly(-hydroxyalkanoates))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0306] In some embodiments, polymers according to the present invention include polymers approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 CFR §177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxane-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates and polycyanoacrylates.

[0307] In some embodiments, the polymer can be hydrophilic. For example, the polymer can contain anionic groups (e.g., phosphate groups, sulfate groups, carboxylate groups); cationic groups (e.g., quaternary amine groups); or polar groups (e.g., hydroxyl groups, thiol groups, amine groups). In some embodiments, the polymer can be hydrophobic. The choice of the hydrophilicity or hydrophobicity of the polymer can have an impact on the properties of the material incorporated (e.g., coupled) within the synthetic nanoparticle.

[0308] In some embodiments, polymer can be modified with one or more parts and / or functional groups. According to the present invention, a variety of parts or functional groups can be used. In some embodiments, polymer can be modified with polyethylene glycol (PEG), carbohydrates and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786: 301). The general teachings of U.S. Patent No. 5,543,158 of Gref et al. or WO of Von Andrian et al. WO2009 / 051837 can be used to complete certain embodiments.

[0309] In some embodiments, the polymer can be modified with a lipid or fatty acid group. In some embodiments, the fatty acid group can be one or more of the following: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid group can be one or more of the following: palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linoleic acid, γ-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid.

[0310] In some embodiments, the polymer can be a polyester, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers comprising glycolic acid units, referred to herein as "PGA", and lactic acid units such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA". In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof. In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine esters), poly(4-hydroxy-L-proline esters), poly[α-(4-aminobutyl)-L-glycolic acid] and derivatives thereof. The degradation rate of PLGA can be adjusted by varying the lactic acid:glycolic acid ratio. In some embodiments, the PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of about 85:15, about 75:25, about 60:40, about 50:50, about 40:60, about 25:75, or about 15:85.

[0311] In some embodiments, the polymer can be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. Acrylic polymers may include fully polymerized copolymers of acrylic acid and methacrylate with a low content of quaternary ammonium groups.

[0312] In some embodiments, the polymer can be a cationic polymer. Typically, a cationic polymer is capable of condensing and / or protecting negatively charged chains of nucleic acids (eg, DNA or a derivative thereof). Amine-containing polymers, such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) is positively charged at physiological pH, forms ion pairs with nucleic acids, and mediates transfection in a variety of cell lines.

[0313] In some embodiments, the polymer can be a degradable polyester with cationic side chains (Putnam et al, 1999, Macromolecules, 32:3658; Barrera et al, 1993, J. Am. Chem. Soc, 115:11010; Kwonet al, 1989, Macromolecules, 22:3250; Lim et al, 1999, J. Am. Chem. Soc, 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al, 1993, J. Am. Chem. Soc, 115: 11010), poly(serine ester) (Zhou et al, 1990, Macromolecules, 23: 3399), poly(4-hydroxy-L-proline ester) (Putnam et al, 1999, Macromolecules, 32: 3658; and Lim et al, 1999, J. Am. Chem. Soc, 121: 5633) and poly(4-hydroxy-L-proline ester) (Putnam et al, 1999, Macromolecules, 32: 3658; and Lim et al, 1999, J. Am. Chem. Soc, 121: 5633).

[0314] The properties of these and other polymers and methods for their preparation are well known in the art (see, e.g., U.S. Patents 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al, 2001, J. Am. Chem. Soc, 123:9480; Lim et al, 2003, J. Am. Chem. Soc, 124:1154; Wang et al, 2003, J. Am. Chem. Soc, 124:1154 ... al, 2001, J. Am. Chem. Soc, 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al, 1999, Chem. Rev., 99: 3181). More generally, various methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Goethals, ed., Pergamon Press, 1980; Principles of Polymerization, Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry, Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446 and 6,818,732. Each of the foregoing is incorporated herein by reference in its entirety.

[0315] In some embodiments, the polymer can be a linear or branched polymer. In some embodiments, the polymer can be a dendritic polymer. In some embodiments, the polymer can be substantially cross-linked to each other. In some embodiments, the polymer can be substantially free of cross-linking. In some embodiments, the polymer can be used according to the present invention without undergoing a cross-linking step. It should also be understood that the synthetic nanoparticles of the present invention can include block copolymers, graft copolymers, blends, mixtures and / or adducts of any of the aforementioned polymers and other polymers. Those skilled in the art will recognize that the polymers listed herein represent exemplary, rather than comprehensive, lists of polymers that can be used according to the present invention.

[0316] In some embodiments, synthetic nanoparticles may optionally include one or more amphiphilic entities. In some embodiments, amphiphilic entities may promote the production of synthetic nanoparticles with increased stability, improved uniformity or increased viscosity. In some embodiments, amphiphilic entities may be associated with the inner surface of a lipid membrane (e.g., a lipid bilayer, a lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for preparing synthetic nanoparticles according to the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleoyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexadecanol; fatty alcohols, such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface active fatty acids, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; trioleyl sorbitan ( 85) Glycocholate; Sorbitan Laurate 20); Polysorbate 20( 20); Polysorbate 60( 60); Polysorbate65( 65); Polysorbate 80( 80); Polysorbate 85( 85); polyoxyethylene monostearate; surfactin; poloxomer; sorbitan fatty acid esters, such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebroside; dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; acetyl palmitate; ricinoleylglycerol; hexadecyl stearate; isopropyl myristate; tyloxapol; poly(ethylene glycol) 5000-phosphatidylethanolamine; poly(ethylene glycol) 400-monostearate; phospholipids; synthetic and / or natural detergents with high surfactant properties; deoxycholate; cyclodextrin; chaotropic salts; ion pairing agents; and combinations thereof. The amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will appreciate that this is an exemplary, rather than a comprehensive, list of materials having surfactant activity.Any amphiphilic entity may be used to produce synthetic nanoparticles to be used in accordance with the present invention.

[0317] In some embodiments, the synthetic nanoparticles may optionally include one or more carbohydrates. The carbohydrates may be natural or synthetic. The carbohydrates may be derivatized natural carbohydrates. In certain embodiments, the carbohydrates include monosaccharides or disaccharides, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine and neuraminic acid. In certain embodiments, the carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextrin, glycogen, hydroxyethyl starch, carageenan, glycosyl, amylose, chitosan, N, O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, psoralen, heparin, hyaluronic acid, curdlan and xanthan gum. In embodiments, the synthetic nanoparticles of the present invention do not contain (or explicitly exclude) carbohydrates, such as polysaccharides. In certain embodiments, the carbohydrate may include carbohydrate derivatives, such as sugar alcohols, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol and lactitol.

[0318] In some embodiments, the nanoparticle comprises a peptide comprising a sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 16 (e.g., SEQ ID NO: 13). In some cases, the nanoparticles disclosed herein can be administered in combination with another therapy described below (e.g., APC-based therapy, CTL-based therapy, peptide vaccine therapy). In some cases, the nanoparticles disclosed herein can be administered to human subjects in combination with immune agonists (e.g., anti-OX40 antibodies; anti-GITR antibodies), checkpoint inhibitors (e.g., anti-LAG3 antibodies) and / or lenalidomide.

[0319] Antigen presenting cell (APC)-based immunotherapy

[0320] In vitro strategies for inducing an immune response in a subject may involve contacting a suitable antigen presenting cell (e.g., a dendritic cell, a monocyte, or a macrophage) obtained from the subject with any of the peptides described herein. Alternatively, the cell may be transfected with a nucleic acid (e.g., an expression vector) encoding one or more peptides and optionally cultured for a period of time under conditions that allow expression of the peptide. The transfection method will depend on the type of cell and the nucleic acid being transfected into the cell. After contact or transfection, the cell is then returned to the subject.

[0321] The cell can be any of a large number of types expressing MHC class I or II molecules. For example, the cell can include a myeloid cell, a macrophage, a monocyte, a dendritic cell, a T cell (e.g., a T helper cell, a CD4+ cell, a CD8+ cell, or a cytotoxic T cell), or a B cell.

[0322] Thus, the present disclosure provides a composition comprising an APC (e.g., a dendritic cell), wherein the APC presents a peptide sequence on its surface, wherein the peptide sequence comprises at least one major histocompatibility complex (MHC) class I or class II peptide epitope of one or both of the BCMA antigen (SEQ ID NO: 18) and the TACI antigen (SEQ ID NO: 19). In some embodiments, the APC is a dendritic cell. In some embodiments, the MHC peptide epitope is an MHC class I peptide epitope (e.g., an HLA-A2 or HLA-A24 peptide epitope). In some embodiments, the APC obtains the peptide sequence in vitro by exposure to a synthetic peptide comprising the peptide sequence.

[0323] In some embodiments of any of the ex vivo methods, cells obtained from the subject or from a subject of the same species other than the subject (allogeneic) can be contacted with an agent (or immunogenic / antigenic composition) and administered to the subject.

[0324] In some embodiments, the composition comprises at least 104 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 APCs (e.g., dendritic cells). In some embodiments, the composition comprises less than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 APCs (eg, dendritic cells).

[0325] a. Preparation of antigen presenting cells

[0326] Antigen presenting cells (APCs) such as dendritic cells (DCs) suitable for administration to a subject (e.g., a multiple myeloma patient) can be isolated or obtained from any tissue in which such cells are found, or can be cultured and provided in other ways. For example, APCs (e.g., DCs) can be found in the bone marrow or peripheral blood mononuclear cells (PBMCs) of a mammal, in the spleen of a mammal, or in the skin of a mammal (i.e., Langerhans cells can be found in the skin, which have certain properties similar to those of DCs). For example, bone marrow can be harvested from a mammal and then cultured in a culture medium that promotes the growth of DCs. GM-CSF, IL-4, and / or other cytokines (e.g., TNF-α), growth factors, and supplements can be included in this culture medium. After culturing for an appropriate amount of time in a medium containing appropriate cytokines (e.g., suitable for expanding and differentiating DC into mature DC, e.g., 4, 6, 8, 10, 12, or 14 days), DC clusters are cultured in the presence of an antigen of interest (e.g., in the presence of one or more peptide epitopes of BCMA or TACI, or a combination of at least two peptides of SEQ ID NOs: 13-17) and harvested for use in cancer vaccines using standard techniques. Antigens (e.g., isolated or purified peptides or synthetic peptides) can be added to the culture at a concentration of 1 μg / ml-50 μg / ml per antigen (e.g., 2, 5, 10, 20, 30, or 40 μg / ml per antigen).

[0327] In some embodiments, APC is separated from a subject (e.g., a human). Mononuclear cells are isolated from blood using leukapheresis (e.g., using COBE Spectra Apheresis System). Mononuclear cells are adhered by incubation at 37°C for 2 hours in a tissue culture bottle. Non-adherent cells are removed by washing. Adherent cells are cultured in a culture medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) (800 units / ml, clinical grade, Immunex, Seattle, Wash.) and interleukin 4 (IL-4) (500 units / ml, R&D Systems, Minneapolis, Minn.) for five days. On the fifth day, TNF-α is added to the culture medium for another 3-4 days. On the 8th or 9th day, cells are harvested and washed, and incubated on a tissue rotator with peptide antigens for 16-20 hours. Peptide antigens are added to the culture at a concentration of about 10 μg / ml (each antigen).

[0328] As will be appreciated by those skilled in the art, various other methods may be used to isolate APCs. DCs are present in small quantities in all tissues in which they reside, requiring isolation and enrichment of DCs. Any of a number of procedures requiring repeated density gradient separation, fluorescence activated cell sorting techniques, positive selection, negative selection, or a combination thereof are routinely used to obtain an enriched population of isolated DCs. Guidance on such methods for isolating DCs can be found in O'Doherty, U. et al., J. Exp. Med., 178:1067-1078, 1993; Young and Steinman, J. Exp. Med., 171:1315-1332, 1990; Freudenthal and Steinman, Proc. Nat. Acad. Sci. USA, 57:7698-7702, 1990; Macatonia et al., Immunol., 67:285-289, 1989; Markowicz and Engleman, J. Clin. Invest., 85:955-961, 1990; Mehta-Damani et al., J. Immunol., 153:996-1003, 1994; and Thomas et al. al., J. Immunol., 151:6840-6852, 1993. A method for isolating DCs from human peripheral blood is described in US Pat. No. 5,643,786.

[0329] Dendritic cells prepared according to the methods described herein present epitopes corresponding to antigens at an average density higher than the epitopes present on dendritic cells exposed to tumor lysates (e.g., neural tumor lysates). The relative density of one or more antigens on antigen presenting cells can be determined by both indirect and direct means. The primary immune response of untreated animals is roughly proportional to the antigen density of antigen presenting cells (Bullock et al., J. Immunol., 170: 1822-1829, 2003). Therefore, the relative antigen density between two antigen presenting cell populations can be estimated by immunizing animals with each population, isolating B or T cells, and monitoring the specific immune response to a specific antigen by, for example, tetramer assay, ELISPOT or quantitative PCR.

[0330] Relative antigen density can also be measured directly. In one method, antigen presenting cells are stained with antibodies that specifically bind to MHC-antigen complexes, and then the cells are analyzed to determine the relative amount of antibodies bound to each cell (see, e.g., Gonzalez et al., Proc. Natl. Acad. Sci. USA, 102: 4824-4829, 2005). Exemplary methods for analyzing antibody binding include flow cytometry and fluorescence activated cell sorting. The results of the analysis can be reported as, for example, the proportion of cells that are positive for individual MHC-antigen complex staining or the average relative amount of staining for each cell. In some embodiments, a histogram of the relative staining amount of each cell can be created.

[0331] In some embodiments, antigen density can be measured directly by direct analysis of peptides bound to MHC, such as by mass spectrometry (see, e.g., Purcell and Gorman, Mol. Cell. Proteomics, 3: 193-208, 2004). Typically, MHC-bound peptides are separated by one of several methods. In one method, cell lysates of antigen-presenting cells are typically analyzed after ultrafiltration to enrich for small peptides (see, e.g., Falk et al., J. Exp. Med., 174: 425-434, 1991; Rotzhke et al., Nature, 348: 252-254, 1990). In another method, MHC-bound peptides are separated directly on the cell surface, such as by acid elution (see, e.g., Storkus et al., J. Immunother., 14: 94-103, 1993; Storkus et al., J. Immunol., 151: 3719-27, 1993). In another method, MHC-peptide complexes are immunoaffinity purified from antigen presenting cell lysates, and then the MHC-bound peptides are eluted by acid treatment (see, e.g., Falk et al., Nature, 351: 290-296). After separation of the MHC-bound peptides, the peptides are usually analyzed by mass spectrometry after a separation step (e.g., liquid chromatography, capillary gel electrophoresis, or two-dimensional gel electrophoresis). Mass spectrometry can be used to both identify and quantify individual peptide antigens to determine the relative average ratio of each antigen in an antigen presenting cell population. In some methods, stable isotope labeling of a population is used, and then the relative amount of peptides in two antigen presenting cell populations is compared by mass spectrometry (see Lemmel et al., Nat. Biotechnol., 22: 450-454, 2004).

[0332] b. Administration of Antigen Presenting Cells

[0333] APC-based cancer vaccines can be delivered to a patient or test animal by any suitable delivery route, which may include injection, infusion, vaccination, direct surgical delivery, or any combination thereof. In some embodiments, a cancer vaccine is administered to a human in the deltoid region or axillary region. For example, the vaccine is administered as an intradermal injection into the axillary region. In some embodiments, the vaccine is administered intravenously.

[0334] Those skilled in the art can select a suitable carrier for administering cells by conventional techniques. For example, the pharmaceutical carrier can be a buffered salt solution, such as a cell culture medium, and can include DMSO for preserving cell viability.

[0335] The amount of APCs suitable for administration as a cancer vaccine to a patient to achieve the methods of the present invention, as well as the most convenient route of such administration, can be based on a variety of factors, as can the formulation of the vaccine itself. Some of these factors include the patient's physical characteristics (e.g., age, weight, and sex), the physical characteristics of the tumor (e.g., location, size, growth rate, and accessibility), and the extent to which other treatments (e.g., chemotherapy and radiation therapy) are being implemented in conjunction with the overall treatment regimen. Although a variety of factors should be considered when practicing the methods of the present invention to treat a disease condition, about 10% of the APCs can be administered to a mammal in a single administration in about 0.05 mL to about 2 mL of a solution (e.g., saline). 5 to about 10 8 APCs (e.g. 10 7 Depending on the above and other factors, such as the severity of the tumor pathology, additional administrations may be performed. In one embodiment, about 10 administrations are performed at two-week intervals. 6 The invention relates to about 1 to about 5 administrations of each APC.

[0336] DC vaccination can be accompanied by other treatments. For example, patients receiving DC vaccination can also receive chemotherapy, radiation and / or surgery at the same time. Methods for treating cancer using DC vaccination in combination with chemotherapy are described in Wheeler et al., U.S. Patent Publication No. 2007 / 0020297. In some embodiments, patients receiving DC vaccination have already received chemotherapy, radiation and / or surgery for cancer. In one embodiment, patients receiving DC vaccination are treated with a COX-2 inhibitor as described in Yu and Akasaki, WO 2005 / 037995.

[0337] T cell-based immunotherapy

[0338] Ex vivo methods for stimulating an immune response can also include contacting T cells (e.g., from a lymphocyte population obtained from a subject) with antigen presenting cells (APCs) expressing MHC molecules that bind to one of the peptides described herein in vitro for an amount of time (and under certain conditions) sufficient to activate the T cells (e.g., cytotoxic T cells and / or CD4+ helper T cells). Thus, the present disclosure provides methods for producing and / or proliferating BCMA-specific and / or TACI-specific T cells (e.g., cytotoxic T cells and / or CD4+ helper T cells). The method involves contacting one or more T cells (e.g., cytotoxic T cells and / or CD4+ helper T cells) with one or more antigen presenting cells pulsed with a peptide as described herein. These T cells can be cytotoxic T cells, such as memory cytotoxic T cells, effector cytotoxic T cells, or CD4+ helper T cells.

[0339] Activated T cells can be used to kill target cells. In some embodiments, the method involves contacting the target cell with one or more BCMA-specific cytotoxic T cells, wherein the target cell expresses or overexpresses BCMA and expresses HLA-A. In some embodiments, the method involves contacting the target cell with one or more TACI-specific cytotoxic T cells, wherein the target cell expresses or overexpresses TACI and expresses HLA-A.

[0340] In some embodiments, BCMA or TACI specific T cells (e.g., cytotoxic T cells and / or CD4+ helper T cells) are administered in combination with peptides disclosed herein (e.g., SEQ ID NO: 13-17, one or more), APCs presenting BCMA (e.g., SEQ ID NO: 13 or 14) or TACI (SEQ ID NO: 16) peptides, lenalidomide, immunomodulators, checkpoint inhibitors (e.g., anti-LAG3 antibodies) or immune agonists (e.g., anti-OX40, anti-GITR). In some embodiments, the additional therapeutic agent administered with BCMA or TACI specific CTL T cells is an antibody (e.g., human antibody) that specifically binds to PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT, TIM-3, GITR (CD357), OX40, CD20, EGFR, or CD319. In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, T cells are administered in combination with an immune agonist, such as an anti-OX40 or anti-GITR antibody.

[0341] Activated T cells can also be reintroduced into the subject from which the cells were obtained. In some embodiments, T cells can be obtained from a subject of the same species other than the subject (allogeneic), can be contacted with an agent (or immunogenic / antigenic composition) and administered to the subject.

[0342] In some embodiments, T cells are derived from in vitro induction of peripheral blood mononuclear cells (PBMC) derived from patients. The following protocol can be used to generate antigen-specific CTLs in vitro from patient-derived PBMCs. To generate dendritic cells, plastic adherent cells from PBMCs are cultured in AIM-V medium supplemented with recombinant human GM-CSF and recombinant human IL-4 at 37°C in a humidified CO2 (5%) incubator. After 6 days, immature dendritic cells in the culture are stimulated with recombinant human TNF-α for maturation. Mature dendritic cells are then harvested on day 8 and grown at 1x106 / mL was resuspended in PBS with peptide (2 μg / mL) and incubated at 37°C for 2 hours. Autologous CD8+ T cells were enriched from PBMC using magnetic microbeads (Miltenyi Biotech, Auburn, Calif.). CD8+ T cells (2x10 per well) were cultured in 2 mL / well of AIM-V medium in each well of a 24-well tissue culture plate. 6 ) and 2x10 per hole 5 The dendritic cells pulsed with peptides were co-cultured, and the AIM-V culture medium was supplemented with 5% human AB serum and 10 units / mL rhIL-7 (Cell Sciences). 2 days after each restimulation, about 20U / ml of IL-2 was regularly added 24 hours later. On the 7th day, lymphocytes were restimulated with autologous dendritic cells pulsed with peptides in the AIM-V culture medium supplemented with 5% human AB serum, rhIL-2 and rhIL-7 (each 10 units / mL). 2 days after each restimulation, about 20U / ml of IL-2 was regularly added 24 hours later. On the seventh day, cells were harvested after the third round of restimulation and the activity of CTL was tested. Stimulated CD8+ cultured cells (CTL) were co-cultured with T2 cells (human TAP-deficient cell line), and the T2 cells were pulsed with 2μg / ml Her-2, gp100, AIM-2, MAGE-1 or IL13 receptor α2 peptides. After 24 h of incubation, IFN-γ in the culture medium was measured by ELISA assay.

[0343] Chimeric Antigen Receptor (CAR) T Cell-Based Immunotherapy

[0344] The present disclosure further provides adoptive transfer of T cells expressing chimeric antigen receptors to treat cancer methods. CAR-modified T cells can be engineered to target almost any tumor-associated antigen (e.g., BCMA and / or TACI). Typically, T cells are genetically engineered to express CARs specific for antigens on patient tumor cells and then infused back into the patient.

[0345] A common form of CAR is a fusion of a single-chain variable fragment (scFv) fused to the CD3-zeta transmembrane domain and the intracellular domain. scFV can be derived from an antigen-specific receptor of a T cell (e.g., BCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells) or an antibody that specifically binds to an antigen.

[0346] In some embodiments, the sequence of the T cell receptor in the BCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells is determined, for example, by sequencing. The sequence of the T cell receptor in the BCMA-specific cytotoxic T cells and / or TACI-specific cytotoxic T cells can be used to generate CAR.

[0347] In some embodiments, the T cells are collected from a patient. In some embodiments, the T cells are obtained from induced pluripotent stem cells (iPSCs).

[0348] Viral vectors such as retrovirus, slow virus or transposon are usually used to integrate transgene (e.g., CAR) into the host cell genome. Alternatively, non-integrating vectors such as plasmids or mRNA can be used to transfer the CAR gene to T cells, and T cells are made to express CAR under appropriate conditions.

[0349] Induced Pluripotent Stem Cell Methods

[0350] Adoptive T cell therapy that administers a large number of ex vivo amplified activated antigen-specific cytotoxic T lymphocytes (CTLs) targeting tumor-specific antigens has induced lasting remission in selected malignant diseases. Although the use of TCRs that mainly recognize intracellular antigens that have been processed and presented as peptide complexes with MHC molecules (Johnson et al. 2009; Morgan et al. 2006) can further enhance tumor selectivity, the introduction of exogenous TCR genes can lead to mismatching of transferred and endogenous α and β chains, leading to severe autoimmune adverse events (Bendle et al. 2010, Hinrichs et al. 2013). In contrast, CAR-T recognizes antigens expressed on the cell surface in a non-MHC restricted manner. So far, the most successful CAR-T therapy targeting B cell antigen CD19 has achieved minimal residual disease negative complete response in patients with relapsed and chemorefractory B cell malignancies (Kochenderfer et al. 2010, Grupp et al. 2013). However, ongoing efforts are directed toward minimizing adverse effects, including cytokine release syndrome, and improving the durability of responses (Brentjens et al. 2011, Kalos et al. 2011, Kochenderfer et al. 2012, Porter et al. 2011). Importantly, CTLs are continuously exposed to tumor antigens during long-term expansion for use in TCR-based or CAR-based therapies, and may lose their proliferative capacity ("exhaustion") and their functional activity as they terminally differentiate.

[0351] To overcome these limitations, the technology currently being developed is to utilize CTLs fully revived from "induced pluripotent stem cells (iPSC)". These iPSCs are a special type of pluripotent cells that are derived from adult somatic cells after ectopically expressing a set of defined transcription factors. Importantly, tumor antigen-specific CTLs can be reprogrammed from antigen-specific CTLs by iPSC technology (Vizcardo et al. 2013, Ando et al. 2015, Timmermans et al. 2009, Kennedy et al. 2012). These iPSC-CTLs are functionally revived and show longer telomeres (1.5 times longer) and higher proliferation capacity (5 to 50 times longer) than the initial CTLs from which they originated (Nishimura et al. 2013). This powerful reprogramming treatment method has the potential to significantly improve the efficacy and durability of antigen-specific cancer immunotherapy. Therefore, the present disclosure provides a method for resuscitating cytotoxic T cells. In some embodiments, the methods can increase proliferation capacity by at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold.

[0352] Activation of tumor-specific CTLs is a major goal of many cancer immunotherapies. Isolation of tumor-specific T cells from cancer patients, in vitro preparation (activation and expansion), and infusion of these T cells into patients are fundamental steps in adaptive immunotherapy with T cells. iPSC technology can be used to improve the efficacy of adoptive cell transfer immunotherapy (ACT).

[0353] iPSCs can be obtained from differentiated cells (e.g., fibroblasts, immune cells, T cells, B cells) induced by retroviral transfection with Yamanaka factors (a combination of Oct3 / 4, Sox2, Klf4, and c-Myc) and differentiated into the T cell lineage by culturing it on a monolayer OP9-DL1 cell system in addition to Flt-3 ligand and IL-7.

[0354] In some embodiments, iPSC can be produced from T cells. After amplification, these cells differentiate into T cells again. Human T lymphocytes can act as a cell source for iPSC production. Peripheral blood mononuclear cells (PBMC) can be separated from whole blood by leukocyte apheresis or venipuncture, and CD3+ T cells can then be amplified by stimulating with IL-2 and anti-CD3 antibodies. When exposed to retroviral transduction of reprogramming factors, T cell-derived iPSC (TiPS) can be produced from activated T cells. These T-iPSCs retain their initial T cell receptor (TCR) gene rearrangements, so they can be used as an unlimited source of hematopoietic stem cells carrying endogenous tumor-specific TCR genes for cancer ACT therapy.

[0355] Therefore, in some embodiments, iPSC is produced from antigen-specific cytotoxic T cells. These antigen-specific T cells are produced by methods such as those lost herein, for example, by contacting one or more T cells with one or more antigen presenting cells pulsed with peptides comprising amino acid sequences as described herein (e.g., SEQ ID NO: 1-17). When T-iPSC retains its initial T cell receptor (TCR) gene rearrangement, after these T-iPSCs differentiate into T cells, these T cells can recognize BCMA and / or TACI on cancer cells.

[0356] In some embodiments, nucleic acids encoding CARs that specifically recognize BCMA and / or TACI can be introduced into T-iPSCs. After these T-iPSCs are differentiated into T cells, these T cells can recognize BCMA and / or TACI on cancer cells.

[0357] In some embodiments, the differentiated T cells are administered to a subject. In some embodiments, T-iPSCs are administered to a subject and then differentiated into cytotoxic T cells within the subject's body.

[0358] Subjects

[0359] The subject can be any animal capable of producing an immune response to an antigen. The terms "subject" and "patient" are used interchangeably throughout the specification, and describe an animal, human or non-human to which the method according to the present disclosure provides treatment. The present invention contemplates veterinary and non-veterinary applications. Human patients can be adult humans or adolescents (e.g., people under the age of 18 years old). In addition to humans, subjects include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorpha, pigs (e.g., pigs, mini-pigs), horses, dogs, cats, cattle and other domestic, farm and zoo animals.

[0360] The subject may be a subject who has, is suspected of having, or is at risk of developing cancer. As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. As used herein, the term "tumor" refers to cancerous cells, such as a large number of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignant diseases of various organ systems, such as those affecting the lungs, breasts, thyroids, lymphoids, gastrointestinal tracts, and genitourinary tracts, as well as adenocarcinomas, including malignant diseases such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, small intestinal cancers, and esophageal cancers. In some embodiments, the reagents described herein are designed to treat or diagnose cancer in a subject. The term "carcinoma" is recognized in the art and refers to malignant diseases of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer and melanoma. In some embodiments, cancer is renal cancer or melanoma. Exemplary carcinomas include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcoma, for example, including malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from mesenchyme. In some embodiments, the subject suffers from hematological cancers, such as multiple myeloma, leukemia, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

[0361] In some embodiments, the subject has a disease that expresses / overexpresses BCMA or a disease that expresses / overexpresses TACI, including, for example, multiple myeloma, B cell-related malignant diseases, plasma cell-related malignant diseases, premalignant diseases (e.g., premalignant diseases of MM, such as SMM or MGUS). In some embodiments, the subject may be a subject suffering from, suspected of suffering from, or at risk of developing a plasma cell disorder. As used herein, the term "plasma cell disorder" refers to a group of diseases or disorders characterized by proliferation of clonal plasma cells (PCs) and paraproteins (e.g., excessive secretion of monoclonal immunoglobulins and / or free light chains (FLCs)).

[0362] Non-limiting examples of plasma cell disorders include monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), Macroglobulinemia (WM), light chain amyloidosis (AL), solitary plasmacytoma (e.g., solitary bone plasmacytoma or extramedullary plasmacytoma), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes syndrome (POEMS) and heavy chain disease. Other plasma cell disorders include, for example, monoclonal gammopathy of renal significance (MGRS), MGUS-related neuropathy and other paraproteinemia neuropathies.

[0363] MGUS, smoldering MM (SMM), and symptomatic MM represent a group of identical diseases. Symptomatic or active multiple myeloma is characterized by greater than 10% BM infiltration of clonal plasma cells and / or biopsy-confirmed plasmacytomas in addition to: any level of monoclonal protein and the presence of end-organ damage consisting of any of the myeloma-defining events in the CRAB criteria table (hypercalcemia, renal insufficiency, anemia, or bone lesions thought to be related to the plasma cell clone), or any new biomarker of malignancy (BM involvement equal to or greater than 60% clonal plasma cells; ratio of involved to uninvolved FLCs equal to or greater than 100; and / or presence of more than one bone lesion on MRI (Kyle RA et al., Leukemia, 23:3-9 (2009); Rajkumar VS et al, Lancet Oncology, 15:12, 2014). MM is a plasma cell malignancy that characteristically involves widespread infiltration of the bone marrow (BM), occasionally forming plasmacytomas as discrete clusters of malignant plasma cells within or outside the BM space (Kyle RA et al., Leukemia, 23:3-9 (2009); Rajkumar VS et al, Lancet Oncology, 15:12, 2014). al., N. Engl. J. Med., 351: 1860-73 (2004)). The consequences of this disease are numerous and involve multiple organ systems. Disruption of the BM and normal plasma cell function leads to anemia, leukopenia, hypogammaglobulinemia, and thrombocytopenia, which variously lead to fatigue, increased susceptibility to infection, and less commonly increased bleeding tendency. Disease in the bone is involved in the production of osteolytic lesions, produces bone pain, and can be associated with hypercalcemia (Kyle RA et al., Blood, 111: 2962-72 (2008)).

[0364] Smoldering MM (SMM) is characterized by serum immunoglobulin (Ig) G or IgA monoclonal protein of 30 g / L or higher and / or 10% or more plasma cells in the bone marrow, but without evidence of end-organ damage or malignant disease-defining biomarkers (Rajkumar et al, Lancet, 2014). Studies of the natural history of SMM have shown that there are 2 different types: evolving smoldering MM and non-evolving smoldering MM (Dimopoulos M. et al., Leukemia, 23 (9): 1545-56 (2009)). Evolving SMM is characterized by a gradual increase in M ​​protein and a shorter median time to progression (TTP) of 1.3 years before active multiple myeloma. Non-evolving SMM has a more stable M protein, which may then change suddenly when it progresses to active multiple myeloma, with a median TTP of 3.9 years.

[0365] Monoclonal gammopathy of undetermined significance (MGUS) is a disorder in which abnormal immunoglobulins (called paraproteins) are found in the blood during standard laboratory blood tests. MGUS is similar to multiple myeloma and similar disorders, but the antibody levels are lower, the number of plasma cells (white blood cells that secrete antibodies) in the bone marrow is lower, and it has no symptoms or major problems.

[0366] In some embodiments, the subject has multiple myeloma, SMM or MGUS. In some embodiments, the subject may be a subject in remission from multiple myeloma. In some embodiments, the subject has a pre-malignant disease (e.g., a pre-malignant disease of MM, such as SMM or MGUS).

[0367] In some embodiments, the subject may have a cancer that expresses or overexpresses BCMA or TACI. Therefore, the method may also include the following steps: before administering one or more peptides (or nucleic acids) to the subject, determine whether one or more cancer cells of the subject's cancer (e.g., multiple myeloma) express or overexpress BCMA or TACI. The expression of these proteins includes both mRNA and protein expression. Methods for detecting protein and mRNA expression in cells are known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA), western blotting and dot blotting techniques or immunohistochemistry techniques for detecting proteins and reverse transcription-polymerase chain reaction (RT-PCR) or Northern blotting techniques for detecting mRNA. In some embodiments, the average expression level of BCMA or TACI in cancer cells is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher than the average expression level of BCMA or TACI in normal cells (e.g., normal tissue cells in the same subject, normal plasma cells in the same subject, or tissue cells or plasma cells in healthy subjects). In some embodiments, the average expression level of BCMA or TACI in cancer cells is at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or 50-fold higher than the average expression level of BCMA or TACI in normal cells (e.g., normal tissue cells in the same subject, normal plasma cells in the same subject, or tissue cells or plasma cells in a healthy subject).

[0368] The subject may have, be suspected of having or be at risk of developing cancer (e.g., multiple myeloma). A subject "suspected of having cancer" is a subject with one or more cancer symptoms. The symptoms of cancer are well known to those skilled in the art, and generally include, but are not limited to, pain, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening dyspnea, hemoptysis, hematuria, blood in the stool, nausea, vomiting, abdominal pain and falling (abdominal fullness), flatulence, abdominal effusion, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, dysphagia, etc. The symptoms of multiple myeloma particularly include, for example, bone pain (e.g., in the back or ribs), high calcium levels in the blood, excessive thirst or urination, constipation, nausea, loss of appetite, confusion, leg weakness or numbness, weight loss or repeated infections.

[0369] As used herein, a subject "at risk of developing cancer" is one that has a predisposition to developing cancer, i.e., a genetic predisposition to developing cancer, such as a mutation in a tumor suppressor gene (e.g., a mutation in BRCA1, p53, RB, or APC), has been exposed to a condition that can lead to cancer, or is currently affected by such a condition. Thus, a subject may also be "at risk of developing cancer" when the subject has been exposed to mutagenic or carcinogenic levels of certain compounds (e.g., carcinogenic compounds in cigarette smoke, such as acrolein, 4-aminobiphenyl, aromatic amines, benz(o)anthracene, benz(o)pyrene, formaldehyde, hydrazine, polonium-210 (radon), urethane, or vinyl chloride). A subject may be "at risk of developing cancer" when the subject has been exposed to, for example, large doses of ultraviolet light or X-rays, or has been exposed to (e.g., infected with) a tumorigenic / associated virus (e.g., papillomavirus, Epstein-Barr virus, hepatitis B virus, or human T-cell leukemia-lymphoma virus). In addition, a subject may be "at risk of developing cancer" when the subject suffers from inflammation (e.g., chronic inflammation). If, for example, the subject suffers from monoclonal gammopathy of undetermined significance (MGUS), the subject may be at risk of developing multiple myeloma. Therefore, it should be understood that subjects who are "suspected of having cancer" or "at risk of developing cancer" are not all subjects within the species of interest.

[0370] In some embodiments, the method may also include a step of determining whether the subject suffers from cancer. Suitable methods for such determinations depend on the type of cancer to be detected in the subject, but are known in the art. Such methods may be qualitative or quantitative. For example, when a subject exhibits two or more (e.g., three, four, five or six or more) symptoms of multiple myeloma (e.g., any of the symptoms described herein), a medical practitioner may diagnose the subject with multiple myeloma. It is also possible to determine that a subject suffers from multiple myeloma by measuring blood calcium levels, white blood cell or red blood cell counts, or the amount of protein in the subject's urine.

[0371] MHC molecule multimer

[0372] The present disclosure also features compositions comprising: (i) any one or more peptides described herein, and (ii) a major histocompatibility complex (MHC) molecule multimer. The multimer comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) complete MHC molecules or peptide binding regions of MHC molecules. One or more peptides may be associated (e.g., covalently or non-covalently bound) with the MHC molecule multimer.

[0373] The multimeric MHC molecule can be a class I MHC molecule (e.g., an HLA-A2 molecule) or a class II MHC molecule. The MHC molecule can be a mammalian (e.g., a rodent, a non-human primate, a human, or any other mammal described herein) MHC molecule.

[0374] The two or more MHC molecules (or peptide binding regions of MHC molecules) in a multimer can be from the same MHC molecule or from different MHC molecules. For example, an MHC molecule multimer can contain five MHC molecules, three of which are identical MHC molecules, and two of which are different from the first three. In another example, each MHC molecule of the multimer is different. At least one of the MHC molecules can bind to at least one peptide.

[0375] In some embodiments, the above compositions can include at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 15 or more) of any of the peptides described herein.

[0376] The composition can also be associated with a detectable marker. For example, one or more MHC molecules of the polymer can be covalently or non-covalently bound to a detectable marker. Suitable detectable markers (e.g., enzymes, fluorescent materials, luminescent materials, bioluminescent materials, or radionuclides) and methods of attaching detectable markers to peptides or MHC molecules are also provided.

[0377] The peptides described herein can be used to generate MHC multimer compositions as follows: the peptide bound to the HLA molecule is refolded in the presence of the corresponding HLA heavy chain and β2-microglobulin to produce a trimolecular complex. The complex is then biotinylated at the carboxyl terminal end of the heavy chain at a site previously engineered into the heavy chain. Multimer formation is then induced by adding streptavidin.

[0378] Since T cell receptors can recognize a specific peptide-MHC complex on a target cell among a wide variety of other peptide-MHC complexes, the MHC multimer compositions described herein can be used, for example, to detect antigen-specific T cells in an unrelated T cell population. For such assays, the multimers will typically be detectably labeled.

[0379] For example, multimeric MHC molecules / peptide complexes can be used in assays to assess the presence of antigen-specific CTLs in peripheral blood mononuclear cells after exposure to an immunogen. MHC multimeric complexes can be used to directly visualize antigen-specific CTLs (see, for example, Ogg et al., Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996) and to measure the frequency of antigen-specific CTL populations in peripheral blood mononuclear cell samples. In some embodiments, a detectably labeled streptavidin for polymerizing MHC multimers can be used to label T cells bound to multimeric MHC molecules / peptide complexes. To this end, cells treated with multimers are exposed to, for example, markers (e.g., fluorophores conjugated to biotin). Cells can then be easily separated or detected, for example, using flow cytometry.

[0380] The peptides (and pharmaceutical compositions thereof), compositions containing MHC multimers, kits and articles described herein can be used in a variety of methods. For example, the peptides can be used to: (i) induce an immune response in a subject (e.g., a subject with cancer); (ii) activate T cells in culture; and / or (iii) treat or prevent cancer, such as multiple myeloma. As described herein, compositions containing MHC multimers can be used, for example, to detect antigen-specific T cells in an unrelated T cell population.

[0381] Methods of producing antibodies in a subject

[0382] Methods for producing antibodies specific to immunogens (e.g., one or more of any peptides described herein) are known in the art. For example, antibodies or antibody fragments specific to the peptides described herein can be produced, for example, by immunization using an animal or by in vitro methods such as phage display. All or part of the peptides described herein can be used to produce antibodies or antibody fragments.

[0383] Peptides can be used to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal, such as human) with the peptide. Suitable immunogenic preparations may include, for example, any composition described herein. The preparation may also include an adjuvant, such as Freund's complete or incomplete adjuvant, alum, RIBI or a similar immunostimulant. Adjuvants also include, for example, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185: 1203-1210) and mutant Escherichia coli heat-labile toxin (MLT) (Di Tommaso et al. (1996) Infect. Immunity 64: 974-979). MCT and MLT include point mutations that substantially reduce toxicity relative to the parent molecule without substantially compromising adjuvant activity. Immunizing a suitable subject with an immunogenic peptide preparation (e.g., any composition described herein) induces a polyclonal anti-peptide antibody response. In some embodiments, a toll-like receptor-3 ligand (eg, Poly ICLC), interferon alfa (IFNα), interferon gamma (IFNγ), or granulocyte-macrophage colony stimulating factor (GM-CSF) can be administered to the subject, for example, to enhance an immune response.

[0384] As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules having an antigen binding site that specifically binds to a peptide (e.g., a peptide described herein). An antibody that specifically binds to a peptide described herein is an antibody that binds to the peptide but does not substantially bind to other molecules in the sample. Examples of immunologically active portions of immunoglobulin molecules include, for example, F(ab) fragments, F(ab')2 fragments, or any other antibody fragment described herein.

[0385] The isolated antibody or antigen-binding fragment thereof produced by the methods described herein can selectively bind to an epitope within or overlapping the amino acid sequence of any one of SEQ ID NOs: 1-17. The antibody can also be an antibody that cross-blocks the binding of an antibody that binds to an epitope within or overlapping the amino acid sequence of any one of SEQ ID NOs: 1-17. Generally, when the antibody is expressed at less than 10 -6When the antibody binds to the epitope with a KD of M, the binding of the antibody to the epitope is considered to be selective. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting the selective binding. A "cross-blocking" antibody or "cross-blocking antibody" refers to a first antibody that reduces or eliminates the ability of a second antibody to bind to the peptide (relative to the binding of the second antibody to the peptide that occurs in the absence of the first antibody) when bound to an epitope (e.g., an epitope contained within or overlapping any one of SEQ ID NOs: 1-17). It should be understood that the antibodies produced by the methods described herein (e.g., antibodies specific for one or more peptides described herein) can be used, for example, to detect cancer cells expressing BCMA or TACI, and therefore can be used in many of the exemplary methods described herein.

[0386] Immunity Test

[0387] Antigen-specific cellular immune responses in vaccinated subjects can be monitored by a number of different assays, such as tetramer assays, ELISPOT and quantitative PCR. These methods and protocols are described in, for example, Current Protocols in Immunology, Coligan, J. et al., ed. (John Wiley & Sons, Inc.; New York, NY).

[0388] The tetramer assay can be used to detect and quantify T cells specific for a given antigen in a blood sample. Tetramers composed of recombinant MHC molecules complexed with peptides can be used to identify antigen-specific T cell populations. To detect T cells specific for antigens, fluorescent dye-labeled specific peptide tetramer complexes (e.g., phycoerythrin (PE)-tHLA) containing peptides from these antigens are synthesized and provided by Beckman Coulter (San Diego, Calif.). Specific CTL clones CD8 cells are incubated at 10 5 Cells were resuspended in 50 μl FACS buffer (phosphate buffered saline plus 1% inactivated FCS buffer). Cells were incubated with 1 μl tHLA for 30 minutes at room temperature and incubated with 10 μl anti-CD8 mAb (Becton Dickinson, San Jose, Calif.) for another 30 minutes at 4°C. Cells were washed twice in 2 ml cold FACS buffer and analyzed by FACS (Becton Dickinson).

[0389] The ELISPOT assay can be used to detect cells that secrete cytokines, for example, to determine whether cells in vaccinated patients secrete cytokines in response to antigens, thereby demonstrating whether an antigen-specific response has been elicited. The ELISPOT assay kit was provided by R&D Systems (Minneapolis, Minn.) and was performed as described in the manufacturer's instructions. 1×10 5 PBMC cells of each patient were seeded in a 96-well plate with a nitrocellulose membrane insert coated with capture Ab. Stimulation (S) cells (TAP-deficient T2 cells pulsed with antigen) were added at a 1:1 R:S ratio. After incubation for 24 hours, the cells were removed by washing the plate 4 times. The detection antibody was added to each well. The plate was incubated overnight at 4°C, and the washing step was repeated. After incubation with streptavidin-AP for 2 hours, the plate was washed. An aliquot (100 μl) of BCIP / NBT chromogen was added to each well to form spots. The reaction was terminated by washing with water after 60 minutes. Spots were scanned and counted using computer-assisted image analysis (Cellular Technology Ltd, Cleveland, Ohio). When the experimental value was significantly different from the average number of spots (background value) for non-pulsed T2 cells (as determined by a two-tailed Wilcoxon rank sum test), the background value was subtracted from the experimental value.

[0390] Quantitative PCR is another means of evaluating immune responses. To examine IFN-γ production in patients by quantitative PCR, cryopreserved PBMCs from pre- and post-vaccination samples of patients as well as autologous dendritic cells were thawed in RPMI DC medium with 10% patient serum, washed, and counted. PBMCs were plated at 3x10 6 PBMCs were plated in 2 ml of culture medium in a 24-well plate; dendritic cells were plated at 1x10 6 / ml distribution and pulsed with 2 ml of 10 μg / ml tumor peptide in each well of a 24-well plate for 24 hours. Dendritic cells were collected, washed, counted, and diluted to 1x10 6 / ml, then 3x10 5(ie 300 μl solution) was added to the wells with PBMC (DC:PBMC=1:10). 2.3 μl IL-2 (300 IU / mL) was added every 3-4 days, and the cells were harvested between the 10th and 13th days after the start of culture. The harvested cells were then stimulated at 37°C for 4 hours with tumor cells or autologous PBMC pulsed with 10 μg / ml tumor peptide. On days 11-13, the culture was harvested, washed twice, and then divided into four different wells, two wells for control (no target); and if tumor cells are available, the other two wells were co-cultured with tumor cells (1:1) CTL. If tumor cells are not available, 10 μg / ml tumor lysate is added to the CTL. After 4 hours of stimulation, cells were collected, RNA was extracted, and the expression of IFN-γ and CD8 mRNA was evaluated using a thermal cycler / fluorescence photography system. The PCR amplification efficiency followed the natural logarithmic progression, and linear regression analysis demonstrated a correlation coefficient of more than 0.99. Based on empirical analysis, a 1 cycle difference was interpreted as a two-fold difference in mRNA quantities and CD8 normalized IFN-γ quantities were determined. A >1.5-fold increase post-vaccine relative to pre-vaccine IFN-γ is an established criterion for type I positive vaccine responsiveness.

[0391] Methods used to select therapy

[0392] A method for selecting a therapy for a subject having cancer (e.g., a plasma cell disorder such as multiple myeloma or any cancer that expresses or overexpresses BCMA or TACI) comprises the following steps: optionally, determining whether one or more cancer cells of the subject express or overexpress BCMA or TACI; and if the one or more cells express BCMA or TACI, selecting a composition containing at least one peptide as described herein (a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-17, a peptide comprising an amino acid sequence that is at least 50%, 60%, 70%, 80% or 90% identical to SEQ ID NOs: 1-17, or having no more than 4 substitutions of an amino acid sequence of any one of SEQ ID NOs: 1-17) as a therapy for the subject, provided that the amino acid sequence is capable of: (i) inducing an immune response in the subject; (ii) binding to an MHC molecule; and (iii) being recognized by a T cell receptor on a T cell in the context of an MHC molecule.

[0393] In some embodiments, the method further comprises the step of determining whether one or more cancer cells of the subject express an MHC molecule, such as an MHC class I molecule (eg, HLA-A2) or an MHC class II molecule.

[0394] It should be understood that in the case where one or more cells (e.g., plasma cells) of a subject's cancer express or overexpress both BCMA and TACI, a combination of suitable peptides can be delivered to the subject. For example, in the case where one or more cells (e.g., plasma cells) of a subject's cancer are determined to express or overexpress both BCMA and TACI, a method for selecting a therapy may include selecting the following as a therapy for the subject: a composition containing at least one peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-6 and 13-14 and a composition containing at least one peptide comprising the amino acid sequence of any one of SEQ ID NOs: 7-12 and 15-17.

[0395] Methods for determining whether one or more cells express BCMA, TACI and / or MHC molecules are known in the art. For example, BCMA and / or TACI specific antibodies prepared by the methods described herein can be used to test biological samples (e.g., blood samples or lymph node tissue samples) obtained from a subject to detect the presence or amount of BCMA and TACI polypeptides expressed by cells (cell lysates). Methods for determining the presence or content of polypeptides in biological samples include, for example, ELISA, immunohistochemistry, flow cytometry, western blotting, or dot blot assays. In some embodiments, any of the methods described herein may also include providing a biological sample from a subject and / or obtaining a biological sample from a subject. Suitable biological samples for the methods described herein include any biological fluid, cell, tissue, or fraction thereof, which includes an analyte protein of interest. The biological sample may be, for example, a specimen obtained from a subject, or may be derived from such a subject. For example, the sample may be a tissue section obtained by biopsy, or a cell placed in tissue culture or suitable for tissue culture. Biological samples can also be biological fluids containing cells, such as urine, blood, plasma, serum, saliva, semen, sputum, cerebrospinal fluid, tears, mucus or aspirates (e.g., lung or nipple aspirates), or such samples absorbed onto paper or polymer substrates. If desired, biological samples can be further fractionated into fractions containing specific cell types. For example, a blood sample can be fractionated into serum or fractions containing specific types of blood cells, such as red blood cells or white blood cells (leukocytes). If desired, a sample can be a combination of sample types from a subject, such as a combination of tissues and biological fluids.

[0396] Biological samples can be obtained from subjects, such as subjects suffering from, suspected of suffering from, or at risk of developing cancer (e.g., multiple myeloma). Any suitable method for obtaining biological samples can be used, although exemplary methods include, for example, phlebotomy, swabs (e.g., cheek swabs), aspiration or fine needle aspiration biopsy procedures. Non-limiting examples of tissues that are susceptible to fine needle aspiration include lymph nodes, lungs, thyroid glands, breasts, and livers. Samples can also be collected, for example, by microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder washings, smears (PAP smears), or ductal lavage.

[0397] Medical practitioners can also select, prescribe and / or administer one or more other therapeutic agents to treat cancer, or one or more drugs to treat the side effects of anticancer agents. Suitable chemotherapeutic agents for treating multiple myeloma include, for example, melphalan, cyclophosphamide, vincristine, doxorubicin, prednisone, dexamethasone, proteosome inhibitors (e.g., bortezomib), thalidomide or lenalidomide. Side effects of anticancer drugs include, for example, anemia, gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea), leukopenia (a decrease in the number of white blood cells, which can lead to infection), temporary hair loss or thrombocytopenia (e.g., a decrease in the number of platelets, which can lead to bleeding). Therefore, medical practitioners can prescribe or administer to a subject a chemotherapeutic agent such as vincristine and an anti-anemia drug such as epoetin alpha (e.g., or ).

[0398] Nucleic acid vaccines

[0399] The present disclosure provides nucleic acid vaccines (NAVs) comprising one or more polynucleotides encoding one or more polypeptides as described herein, such as polynucleotide constructs. Exemplary polynucleotides include, for example, polynucleotide constructs, including DNA, RNA, RNA polynucleotides encoding antigens, such as mRNA. In some embodiments, polynucleotides, such as RNA polynucleotides encoding antigens, may include at least one chemical modification. In some embodiments, nucleic acid vaccines may be formulated in polymers or liposome nanocarriers (e.g., nanoparticles).

[0400] In some embodiments, adjuvants or immunopotentiators may also be administered with or in combination with one or more NAVs. In some embodiments, the adjuvant acts to elicit co-signals of T cells and / or B cells and / or NK cells.

[0401] NAV can vary in its valency. Valency refers to the number of antigenic components in a NAV or NAV polynucleotide (e.g., RNA polynucleotide) or polypeptide. In some embodiments, NAV is monovalent. In some embodiments, NAV is bivalent. In some embodiments, NAV is trivalent. In some embodiments, NAV is multivalent. A multivalent vaccine may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more antigens or antigenic portions (e.g., antigenic peptides, etc.). The antigenic components of NAV can be on a single polynucleotide or on separate polynucleotides.

[0402] NAVs can be used as therapeutic or prophylactic agents. They are provided for use in medicine and / or for eliciting immune effector cells, such as ex vivo stimulation / transfection of peripheral blood mononuclear cells (PBMCs) and re-infusion of activated cells. For example, NAVs described herein can be administered to a subject, wherein polynucleotides are translated in vivo to produce antigens. Compositions, methods, kits, and reagents for diagnosing, treating, or preventing diseases or conditions in humans and other mammals are provided. Active therapeutic agents can include NAVs, cells containing NAVs, or polypeptides translated from polynucleotides contained in the NAVs.

[0403] Provided herein are methods for inducing translation of a polypeptide (e.g., an antigen or immunogen) in a cell, tissue, or organism using a polynucleotide of a NAV described herein. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue, or organism is contacted with an effective amount of a composition containing a NAV, wherein the NAV contains a polynucleotide having at least one translatable region encoding a polypeptide of interest (e.g., an antigen or immunogen).

[0404] An "effective amount" of a NAV composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical properties of the polynucleotide (e.g., size and extent of modified nucleosides), and other components of the NAV, and other determinants. Typically, an effective amount of a NAV composition provides an induced or enhanced immune response as a function of antigen production in a cell, preferably more effectively than a composition containing a corresponding unmodified polynucleotide encoding the same antigen. Increased antigen production can be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the NAV), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by an increase in the duration of protein translation from a modified polynucleotide), or an altered innate immune response of the host cell.

[0405] The present disclosure also provides methods for inducing in vivo translation of polypeptide antigens in mammalian subjects in need thereof. Wherein, an effective amount of a NAV composition containing a polynucleotide having at least one structural or chemical modification and a translatable region encoding the polypeptide (e.g., an antigen or immunogen) is administered to a subject using the delivery methods described herein. The polynucleotide is provided in an amount and under other conditions so that the polynucleotide is translated in a cell. The cells in which the polynucleotide is located or the tissues in which the cells are present can be targeted with one or more rounds of NAV administration.

[0406] The proteins described herein can be engineered to be localized within a cell, potentially in a specific compartment such as the cytoplasm or nucleus, or can be engineered to be secreted from a cell or transported to the plasma membrane of a cell.

[0407] In some embodiments, nucleic acid (e.g., DNA, RNA) may have one or more modifications. In some embodiments, nucleic acid molecules as defined herein (e.g., RNA molecules) may contain nucleotide analogs / modifications, e.g., backbone modifications, sugar modifications, or base modifications. Main chain modifications in conjunction with the present invention are modifications of the phosphate esters of the nucleotide backbones contained in the nucleic acid molecules as defined herein, wherein the chemical modifications are made. Sugar modifications in conjunction with the present invention are chemical modifications of the sugars of the nucleotides of the nucleic acid molecules as defined herein. In addition, base modifications in conjunction with the present invention are chemical modifications of the base moieties of the nucleotides of the nucleic acid molecules of the nucleic acid molecules. In this context, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and / or translation.

[0408] Modified nucleosides and nucleotides that can be incorporated into nucleic acid molecules can be modified in the sugar moiety. For example, the 2'hydroxyl (OH) of the RNA molecule can be modified or replaced with many different "oxy" or "deoxy" substituents. Examples of "oxy"-2'hydroxyl modifications include, but are not limited to, alkoxy or aryloxy (-OR, such as R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), O (CH2CH2O) nCH2CH2OR; "locked" nucleic acids (LNA), wherein the 2'hydroxyl is connected to the 4' carbon of the same ribose, for example, through a methylene bridge; and amino groups (-O-amino, wherein the amino group, such as NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0409] The sugar group may also contain one or more carbons that possess a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, a modified nucleic acid molecule may include nucleotides containing, for example, arabinose as a sugar.

[0410] The phosphate backbone can be further modified in modified nucleosides and nucleotides, which can be incorporated into nucleic acid molecules (e.g., RNA) as described herein. The phosphate groups of the backbone can be modified by replacing one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include completely replacing the unmodified phosphate moiety with modified phosphates as described herein. The example of modified phosphate groups includes but is not limited to phosphorothioate, selenophosphate (phosphoroselenate), boranophosphates (borano phosphates), boranophosphate esters (boranophosphate esters), hydrogen phosphonates (hydrogen phosphonates), phosphoramidates (phosphoroamidates), phosphonic acid alkyl or aryl esters and phosphotriester. Two non-connected oxygens of dithiophosphates are replaced by sulfur. Phosphate joints can also be modified by replacing the connected oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioate) and carbon (bridged methylene phosphonates).

[0411] Modified nucleosides and nucleotides that can be incorporated into nucleic acid molecules (e.g., RNA molecules) as described herein can be further modified in the core base portion. Examples of core bases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, nucleosides and nucleotides described herein can be chemically modified on the surface of the major groove. In some embodiments, the major groove chemical modification may include amino, thiol, alkyl, or halogen groups.

[0412] In some embodiments, the nucleotide analogs / modifications are selected from base modifications, which can be selected from, for example, 2-amino-6-chloropurine riboside-5′-triphosphate, 2-aminopurine-riboside-5′-triphosphate; 2-aminoadenosine-5′-triphosphate, 2′-amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate, 2 -thiouridine-5′-triphosphate, 2′-fluorothymidine-5′-triphosphate, 2′-O-methylinosine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, 5-propynyl-2′-deoxyuridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurine riboside-5′-triphosphate, 7 -deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate.

[0413] In some embodiments, nucleic acid molecules can be modified by adding a so-called "5' cap" structure. The 5'-cap is an entity that is usually "capped" to the 5'-end of a mature mRNA, typically a modified nucleotide entity. The 5'-cap can usually be formed by a modified nucleotide, particularly a derivative of a guanine nucleotide. Preferably, the 5'-cap is connected to the 5'-end via a 5'-5'-triphosphate connection. The 5'-cap can be methylated, such as m7GpppN, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of the RNA. m7GpppN is a 5'-cap structure that naturally occurs in mRNA transcribed by polymerase II and is therefore not considered a modification included in the modified RNA according to the present invention.

[0414] How to prepare and use nucleic acid vaccines is described in, for example, US20070269451, US20160317647, US9872900, and US2017002984, each of which is incorporated herein by reference in its entirety.

[0415] Pharmaceutical composition

[0416] Any peptide, nucleic acid encoding the peptide, nanoparticle and cell described herein can be incorporated into a pharmaceutical composition. The composition can include one or more peptides (and / or nucleic acid encoding the peptide) and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents compatible with drug administration, etc. One or more peptides can be formulated into pharmaceutical compositions in the form of syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, troche, aqueous solutions, creams, ointments, lotions, gels, emulsions, etc. Supplementary active compounds (e.g., one or more chemotherapeutic agents) can also be incorporated into the composition.

[0417] The pharmaceutical composition is usually formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, rectal and parenteral, for example, intravenous, intramuscular, intradermal, subcutaneous, inhaled, transdermal or transmucosal. Solutions or suspensions for parenteral applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tension such as sodium chloride or dextrose. Acids or bases such as hydrochloric acid or sodium hydroxide may be used to adjust pH. The composition may be enclosed in an ampoule, disposable syringe or multidose vial made of glass or plastic.

[0418] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the pharmaceutical composition must be sterile and should flow to the extent that there is easy injectability. It should be stable under manufacturing and storage conditions, and must be preserved to avoid any contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using such as coatings such as lecithin, in the case of dispersions, by maintaining the required particle size and by using a surfactant to maintain appropriate fluidity. The prevention of microbial contamination can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. The extended absorption of injectable compositions can be promoted by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0419] Can be by the one or more peptides (or one or more nucleic acids encoding peptides) of the required amount be incorporated in a suitable solvent with one or combination of components as required, then filter sterilization to prepare a sterile injection solution.Usually, dispersions are prepared by incorporating one or more peptides (or nucleic acids encoding one or more peptides) into a sterile vehicle comprising a basic dispersion medium and other components required from those components listed above.In the case of sterile powders for the preparation of sterile injection solutions, preparation methods may include vacuum drying or freeze drying, which produces a powder of active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0420] Oral compositions typically include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, one or more peptides can be incorporated with an excipient and used in the form of tablets, troches or capsules (e.g., gelatin capsules). Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or magnesium sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings such as mint, methyl salicylate or orange flavoring.

[0421] Powders and tablets may contain from 1% to 95% (w / w) of an individual peptide or a mixture of two or more peptides. In certain embodiments, the peptide may range from about 5% to 70% (w / w). Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting waxes, cocoa butter, and the like. The term "formulation" is intended to include formulations of peptides (or nucleic acids) with an encapsulating material as a carrier providing a capsule in which the peptides with or without other carriers are surrounded by the carrier, so that the carrier is associated with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0422] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as required. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with a viscous material (e.g., natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents).

[0423] For administration by inhalation, the peptide or nucleic acid can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0424] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be completed by using nasal sprays or suppositories. For transdermal administration, the peptide or nucleic acid can be formulated into an ointment, salves, gel or cream, as generally known in the art.

[0425] The peptides or nucleic acids may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0426] In some embodiments, the peptide or nucleic acid can be prepared with a carrier that will protect the peptide from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such preparations are obvious to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes, which are targeted to APCs, for example, with monoclonal antibodies directed to APC-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0427] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of peptide (or nucleic acid) calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The dosage unit may also be accompanied by instructions for use.

[0428] The nucleic acid molecule encoding the peptide can be inserted into the vector and used as a gene therapy vector. The gene therapy vector can be delivered to the subject by, for example, intravenous injection, local administration (see, for example, U.S. Patent No. 5,328,470) or by stereotactic injection (see, for example, Chen, et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow-release matrix in which the gene delivery vector is embedded. Alternatively, in the case where the complete gene delivery vector can be completely produced from recombinant cells, such as retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0429] Other examples of gene delivery vehicles include, but are not limited to, liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses, such as baculoviruses, adenoviruses and retroviruses; bacteriophage cosmids; plasmids; fungal vectors and other recombinant vehicles commonly used in the art, which have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0430] Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, etc. Liposomes containing targeting moieties such as antibodies or fragments thereof can also be used to prepare pharmaceutical compositions of nucleic acids for delivery to a subject.

[0431] Any of the pharmaceutical compositions described herein may be included in a container, pack, or dispenser together with instructions for administration as described below.

[0432] Kits and Products

[0433] The present disclosure also features multiple kits. The kit may include, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of any peptide described herein (or an expression vector containing a nucleic acid sequence encoding one or more peptides); and instructions for administering the peptide to a subject. The kit may include one or more pharmaceutically acceptable carriers and / or one or more immunostimulants. The immunostimulant may be, for example, a T helper cell epitope, an altered peptide ligand, or an adjuvant. The kit may also include one or more therapeutic agents, diagnostic agents, or prophylactic agents.The one or more therapeutic, diagnostic, or prophylactic agents include, but are not limited to: (i) agents that modulate the inflammatory response (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, sodium cromoglycate, or theophylline); (ii) agents that affect renal and / or cardiovascular function (e.g., furosemide, thiazides, amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine, verapamil, digoxin, isordil, dobutamine, lidocaine, , quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, or mevalonate); (iii) drugs that affect gastrointestinal function (e.g., omeprazole or sucralfate); (iv) antibiotics (e.g., tetracycline, clindamycin, amphotericin B, quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid, fluconazole, or amantadine); (v) anticancer agents (e.g., cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine , vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, or dacarbazine); (vi) immunomodulators (e.g., interleukins, interferons (e.g., interferon gamma (IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), cyclosporine, FK506, azathioprine, steroids); (ix) drugs that act on the blood and / or blood-forming organs (e.g., interleukins, G-CSF, GM-CS F, erythropoietin, heparin, warfarin or coumarin); or (vii) hormones (e.g., growth hormone (GH), prolactin, luteinizing hormone, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgen, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodothyronine); hormone antagonists; agents that affect calcification and bone turnover (e.g., calcium, phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride).

[0434] In some embodiments, the kit may include one or more (e.g., one, two, or three or more) BCMA and / or TACI antibodies described herein. In some embodiments, the kit may include two antibodies, each specific for a different protein. For example, the kit may contain a BCMA-specific antibody (described herein) and a TACI-specific antibody (described herein). The kit may optionally include instructions for use in determining the presence or amount of one or more of the BCMA and / or TACI proteins in a biological sample. The feature is also an article of manufacture, which comprises: a container; and a composition contained in the container, wherein the composition comprises an active ingredient for inducing an immune response in a mammal (e.g., a human), wherein the active ingredient comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of any peptide described herein, wherein the container has a label indicating that the composition is used to induce an immune response in a mammal (e.g., any mammal described herein). The label may further indicate that the composition is to be administered to a mammal having, suspected of having, or at risk of developing multiple myeloma. The composition of the article of manufacture can be dried or lyophilized, and can include, for example, one or more solutions (and / or instructions) for dissolving the dried or lyophilized composition.

[0435] The article of manufacture may further include instructions for administering the composition to a mammal. Example

[0436] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0437] Example 1: BCMA expression on multiple myeloma cell lines

[0438] A total of 12 cancer cell lines, including 11 MM cell lines and 1 breast cancer cell line (MDA-MB231), were evaluated for their BCMA antigen expression levels by staining with antibodies specific to each of the following clones; #1. ANC3B1 (LifeSpan Biosciences, catalog number LS-C357630), #2. VICKY1 (LifeSpan Biosciences, catalog number LS-C18662) and #3. 19F2 (BioLegend, catalog number 357506). Among these cell lines, H929 (MM cell line) showed the highest level of BCMA expression, while MDA-MB231 (breast cancer cell line; BCMA negative) showed the lowest level of BCMA expression. ( Figures 1A-1I )

[0439] Example 2: Selection of BCMA and TACI natural peptides specific for HLA-A2

[0440] Six natural peptides derived from BCMA or TACI antigens were identified as follows:

[0441] #1.BCMA 64-72 (LIISLAVFV) (SEQ ID NO: 1)

[0442] #2.BCMA 69-77 (AVFVLMFLL) (SEQ ID NO:2)

[0443] #3.BCMA 9-17 (SQNEYFDSL) (SEQ ID NO:3)

[0444] #4.BCMA 72-80 (VLMFLLRKI) (SEQ ID NO:4)

[0445] #5.BCMA 54-62 (AILWTCLGL) (SEQ ID NO:5)

[0446] #6.BCMA 114-122 (ILPRGLEYT) (SEQ ID NO:6)

[0447] #1.TACI 178-186 (FLVAVACFL) (SEQ ID NO:7)

[0448] #2.TACI 174-182 (VLCCFLVAV) (SEQ ID NO:8)

[0449] #3.TACI 154-162 (KLSADQVAL) (SEQ ID NO:9)

[0450] #4.TACI 166-174 (TLGLCLCAV) (SEQ ID NO: 10)

[0451] #5.TACI 161-169 (ALVYSTLGL) (SEQ ID NO: 11)

[0452] #6.TACI 155-163 (LSADQVALV) (SEQ ID NO: 12)

[0453] Example 3: Binding affinity of BCMA or TACI natural peptides to HLA-A2 molecules

[0454] The T2 cell line was used to evaluate the HLA-A2-specific binding ability of the listed BCMA peptides. In the assay, T2 cells were washed and resuspended in serum-free AIM-V medium to a final concentration of 1x10 6 Cells were then added to the wells of a 24-well tissue culture plate. Cells were pulsed with different concentrations of the corresponding BCMA peptide (0-200 μg / ml) and 3 μg / ml human β2-microglobulin (Sigma) and incubated at 37°C in a humidified atmosphere of 5% CO2. After overnight incubation, cells were washed, stained with mouse anti-human HLA-A2-FITC mAb for 15 minutes at 4°C, washed, and analyzed using a FACSort TM CellQuest for Flow Cytometry TM The analysis was performed using the Becton Dickinson v2.1 software (Becton Dickinson, San Jose, CA). The binding of peptides to HLA-A2 was determined by upregulation of HLA-A2 molecules on T2 cells caused by HLA-A2-specific peptide binding and was demonstrated by flow cytometric analysis measuring mean fluorescence intensity (MFI). Among the BCMA peptides evaluated, “#4.BCMA 72-80 (VLMFLLRKI (SEQ ID NO: 4))" showed the highest level of HLA-A2 specificity, "#5.BCMA 54-62 (AILWTCLGL (SEQ ID NO: 5))" showed the second highest specificity level. Figure 2 ). Among the TACI peptides evaluated, all peptides except TACI#5 showed HLA-A2 specificity, but TACI#4. 166-174 (TLGLCLCAV (SEQ ID NO: 10)) measured the highest level ( Figure 3 ).

[0455] Example 4. Stability of BCMA or TACI natural peptides on HLA-A2 molecules

[0456] To improve the stability of peptides binding to HLA-A2 molecules, the following mutant BCMA or TACI peptides were designed:

[0457] Mutation #4: BCMA 72-80 (YLMFLLRKI) (SEQ ID NO: 13)

[0458] Mutation #5: BCMA 54-62 (YILWTCLGL) (SEQ ID NO: 14)

[0459] Mutation #1: TACI 178-186 (YLVAVACFL) (SEQ ID NO: 15)

[0460] Mutation #3: TACI 154-162 (YLSADQVAL) (SEQ ID NO: 16)

[0461] Mutation #4: TACI 166-174 (YLGLCLCAV) (SEQ ID NO: 17)

[0462] The T2 cell line was used to examine HLA-A2 binding stability for native BCMA and mutated BCMA and TACI peptides. T2 cells were pulsed with the corresponding peptides. After overnight incubation, the cells were washed to remove unbound peptides; their binding affinity was assessed as described above and stability was assessed as follows. The cells were incubated with 10 μg / ml Brefeldin A (Sigma) at 37°C and 5% CO2 for 1 hour to prevent cell surface expression of newly synthesized HLA-A2 molecules. Peptide / HLA-A2 binding stability was assessed at 0, 2, 4, 6 and 18 hours after Brefeldin A treatment. After the incubation period, the cells were harvested, washed, stained with mouse anti-human HLA-A2-FITC mAb and analyzed by flow cytometry. "Mutated #4BCMA 72-80 (YLMFLLRKI(SEQ ID NO:13))", "Mutation #5BCMA 54-62 (YILWTCLGL (SEQ ID NO: 14))" and "mutation #3TACI 154-162 The HLA-A2 binding affinity of the peptide (YLSADQVAL (SEQ ID NO: 16))" was increased from that of its native peptide ( Figure 4 and 5 ). In terms of binding stability, “mutant #4BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13))" and "variant #3TACI 154-162 (YLSADQVAL (SEQ ID NO: 16))" peptide showed significant improvement compared to the native peptide at all time points evaluated, including 0, 2, 4, 6 and 18 hours ( Figure 6 and 7 ). Therefore, mutation #4BCMA was selected 72-80 (YLMFLLRKI (SEQ ID NO: 13)) and mutation #3.TACI 154-162 The YLSADQVAL (SEQ ID NO: 16) peptide was used to further evaluate its immunogenic potential for generating MM-specific cytotoxic T cells (CTLs).

[0463] Example 5. Induction of BCMA or TACI peptide-specific CD3 + CD8 +CTL

[0464] From different HLA-A2 + Normal donors generated peptide-specific CTLs for evaluation of functional activity against MM cell lines. To generate peptide-specific CTLs, mature dendritic cells (mDCs) generated from the same donor were resuspended in serum-free AIM-V medium and treated with 50 μg / ml of mutant #4BCMA. 72-80 (YLMFLLRKI (SEQ ID NO: 13)) peptide or "mutation #3TACI 154-162 (YLSADQVAL (SEQ ID NO: 16))" peptide was pulsed overnight at 37°C in a humidified atmosphere of 5% CO2. Peptide-pulsed mDCs were washed, counted, irradiated with 10 Gy, and used for in vitro hybridization at a ratio of 1:20 antigen presenting cells / peptide to CD3 + ratio of T cells to induce CD3 + T cells. The cultures were restimulated every seven days with irradiated T2 cells pulsed with peptide for a total of four cycles. To maintain T cells ex vivo, IL-2 (50 U / ml) was added to the cultures two days after the second stimulation. Control T cell cultures were maintained under the same culture conditions in the presence of IL-2 (50 U / ml) but without peptide stimulation. The phenotype of the resulting CTLs was assessed one week after each peptide stimulation cycle. Flow cytometric analysis showed that with the gradual increase in the population, the mutant #4BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13)) stimulated CD3 + CD8 + Significant changes in the phenotype of T cell subsets. CD3 induced by mutant BCMA peptides + CD8 + T cell enhancement and immunogenicity with CD138 260-268 (GLVGLIFAV (SEQ ID NO: 20)) CD3 + CD8 + T cell increase was similar to the immunogenic CD138 260-268 BCMA peptides have been previously identified as immunogenic peptides, indicating the potential immunogenicity of BCMA peptides. After 4 cycles of peptide stimulation, BCMA peptide-specific CTL cultures contained a higher percentage of CD8 + T cells (about 80%) Figures 8A-8C ).

[0465] Example 6: By mutating BCMA 72-80 Peptide stimulation reduces naive CD3 + CD8 +CTL and increased memory CD3 + CD8 + CTL

[0466] Antigen-specific CTLs can be phenotypically identified from naive T cells to activated / memory T cells by their expression of unique cell surface antigens. By analyzing the phenotypes of naive and memory cells, the phenotype of BCMA-CTLs was examined as potential effector cells. 72-80 (YLMFLLRKI (SEQ ID NO: 13))-pulsed antigen-presenting cells repeatedly stimulate HLA-A2 + CD3 from normal donors + T cells generated BCMA peptide-specific CTLs. One week after each peptide stimulation, the resulting CTLs were evaluated for their phenotypic profile by flow cytometry. Compared with control T cells, BCMA-CTLs showed naive CD3 + CD8 + The frequency of T cells decreased (donor 1: 80% unstimulated to 2% after 4 stimulation cycles; donor 2: 83% unstimulated to 2% after 4 stimulation cycles). 72-80 (YLMFLLRKI (SEQ ID NO: 13)) peptide enables memory CD3 + CD8 + The frequency of T cells increased accordingly (donor 1: 18% unstimulated to 86% after 4 stimulation cycles; donor 2: 10% unstimulated to 92% after 4 stimulation cycles). These phenotypic changes demonstrate that the use of mutant BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13)) Repeated stimulation of CD3 + T cells give rise to CD8 with a memory cell phenotype + CTL expansion indicated the immunogenicity of BCMA peptides ( FIGS. 9 and 10 ).

[0467] Example 7: By mutating BCMA 72-80 Peptide stimulation of central memory and effector CD3 + CD8 + Changes in CTL frequency

[0468] Using mutated BCMA 72-80 After stimulation of T cells with the (YLMFLLRKI (SEQ ID NO: 13)) peptide, central memory and effector cells were further evaluated. After 3 stimulation cycles, central memory CTL expansion achieved by the BCMA peptide was detected, which was consistent with the reduction of effector CTL. After 4 cycles of peptide stimulation, a reduction in central memory CTL and an increase in effector CTL including effector memory cells were also detected.72-80 The pattern of this phenotypic change in CD8+ T cells stimulated with the (YLMFLLRKI (SEQ ID NO: 13)) peptide was similar to that observed with CD138 260-268 (GLVGLIFAV (SEQ ID NO: 20)) stimulated cells similar to ( Figures 11A-11C ).

[0469] Example 8: Using mutated BCMA 72-80 (YLMFLLRKI-SEQ ID NO: 13) peptide or mutant TACI 154-162 Specific CTLs stimulated by the (YLSADQVAL-SEQ ID NO: 16) peptide showed a unique phenotype representing a specific T cell subset.

[0470] We also observed that CD3 + CD3 in T cell culture + Naive (CD45RO - / CCR7 + )、Central Memory(CD45RO + / CCR7 + ), effector memory (CD45RO + / CCR7 - ) and end effectors (CD45RO - / CCR7 - ) cells with mutant BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13)) peptide or mutant TACI 154-162 CD3 in CTL stimulated with (YLSADQVAL (SEQ ID NO: 16)) peptide + CD8 + Distinct phenotypic changes in T cell subsets. Effector memory CD3 + CD8 + The frequency of T cells increased, and naive T cells (CD45RO - CCR7 + / CD3 + CD8 + ) and central memory T cells (CD45RO + CCR7 + / CD3 + CD8 + Therefore, these results demonstrate that repeated stimulation of CD3 with selected mutant BCMA or TACI peptides + T cells resulted in significant phenotypic changes and expansion of CD3+CD8+ T cell subsets characteristic of antigen-specific CTLs. Fig.12 and Fig.13 ).

[0471] Example 9: BCMA-specific CTL and TACI-specific CTL induce cytotoxic activity on MM cells in an HLA-A2-restricted manner, produce Th1-type cytokines (IFN-γ, IL-2, TNF-α) and upregulate 41BB expression.

[0472] Flow cytometry analysis of mutated BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13)) peptide or mutant TACI 154-162 Peptide-specific CTLs stimulated with (YLSADQVAL (SEQ ID NO: 16)) were analyzed for their ability to lyse myeloma cells and produce key cytokines involved in antitumor activity. BCMA-CTL and TACI-specific CTL showed significant differences in recognition of HLA-A2 + U266 cells (which are higher than HLA-A2 - OPM2 cells) after immunization with CD107a, a marker of degranulation (a measure of cytotoxic activity). + MM cells rather than targeting HLA-A2 - Increased levels of IFN-γ, IL-2, and TNF-α were detected in BCMA-specific CTLs and TACI-specific CTLs of MM cells, demonstrating that the immune response was HLA-A2 restricted ( Fig.14 and Fig.15 ).

[0473] Example 10. BCMA-specific CTLs proliferate in response to MM cells in an HLA-A2-restricted and antigen-specific manner.

[0474] The expression of mutant BCMA was further analyzed using CFSE-proliferation assay. 72-80 Functional activity of peptide-specific CTLs stimulated with YLMFLLRKI (SEQ ID NO: 13). CD8 + Proliferation of T cells, which are mediated by HLA-A2 + MM(U266), HLA-A2 + Breast cancer (MDA-MB231) or HLA-A2 - BCMA-CTLs respond to HLA-A2 as evidenced by decreased fluorescence of CFSE-labeled CTLs after stimulation of MM (MM1S) cells (gated CFSE low). + U266 MM cell line induced significant CD8 +T cell proliferation (proliferating cells: 46%). However, there was no response to MDA-MB231 or MM1S induction of CD8 + T cell proliferation was low (11%-14%) due to culturing cells only in culture medium (10%). Together, these results suggest that BCMA-CTLs specifically respond to myeloma cells and that their CD8+ T cell proliferation is HLA-A2 restricted and antigen-specific ( Fig.16 ).

[0475] Example 11: Achieving higher levels of cytotoxicity by BCMA-specific CTLs in combination with immune agonists

[0476] The expression of mutant BCMA was measured in cells treated with anti-OX40 or anti-GITR for 48 h. 72-80 (YLMFLLRKI (SEQ ID NO: 13)) stimulated peptide-specific CTL activity. + CD8 + CD107a degranulation in T cells measured the level of cytotoxicity. It was observed that CD107a degranulation increased after treatment of BCMA peptide-specific CTLs with anti-OX40 (92%) or anti-GITR (55%) compared with the untreated group (43%), suggesting that combined treatment with immune agonists helps to induce the antitumor activity of BCMA-CTLs ( Fig.17 ).

[0477] Example 12: Selective targeting of multiple myeloma by BCMA-specific central memory CD8+ cytotoxic T lymphocytes

[0478] Although new progress has been made in the treatment of multiple myeloma (MM) incorporating novel therapies into the stem cell transplantation paradigm, ongoing DNA damage and genomic evolution have become the basis for relapse in many patients. Therefore, novel treatment methods with different mechanisms of action are needed. The genetic complexity of composition or evolution, coupled with the immune responsiveness of B cell malignancies, has stimulated the development of immunotherapy options in MM, including monoclonal antibodies, bispecific antibodies, immunotoxins and CAR T cells. Although MM patient-specific CAR T cell therapy has achieved significant deep responses, the persistence of responses has not yet been established, and they are labor-intensive, time-consuming and expensive. In order to overcome these limitations, the present embodiment provides a cancer vaccine based on immunogenic peptides as a ready-made immunotherapy for more extensive and more effective treatment of patients. Peptide-based treatment methods are not limited to vaccines based on recombinant proteins, mRNA or DNA, and the vaccine requires internalization, protein degradation to the best immunogenic peptides for HLA, and the process of other steps required for proper translation (for mRNA) or transcription (for DNA). To overcome MHC restrictions and treat a more diverse patient population using an immunogenic epitope vaccine approach, peptide cocktails were combined to include major HLA subtypes. In addition, lenalidomide can enhance peptide vaccine-specific immune responses and memory cytotoxic T cell (CTL) activity, setting the stage for combination therapy with checkpoint inhibitors and / or immune agonists. Additionally, the anti-tumor efficacy triggered by immunogenic peptides can be enhanced by their ability to induce "epitope spreading" upon effector cell generation, whereby targeted lysed cancer cells release new antigenic epitopes that are subsequently taken up, processed and presented to a new repertoire of CTLs by antigen-presenting cells.

[0479] B cell maturation antigen (BCMA) is a member of TNF receptor superfamily 17 (TNFRSF17) and is characterized as a type III transmembrane protein containing a cysteine-rich extracellular domain that plays a central role in regulating B cell maturation and differentiation into plasma cells. As a receptor for MM cell growth and survival factors B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), BCMA is required for the survival of MM cells, making it a promising therapeutic target. Almost all MM tumor cells express BCMA, and it has been proposed as a marker for identifying tumor cells. Its selective expression on mature B cells and long-lived plasma cell subsets further suggests a favorable therapeutic index for BCMA-directed treatment methods. Currently, several immunotherapy strategies are targeting BCMA, including antibodies (naked antibodies, antibody-drug conjugates, and bispecific antibodies) and cell therapies (chimeric antigen receptor T cells), even with promising clinical results in relapsed refractory MM. In addition, serum soluble BCMA is elevated in patients with MM and chronic lymphocytic leukemia, and can serve as a prognostic marker and response monitoring. Finally, recent studies indicate that BCMA is expressed in non-hematopoietic tissues: BCMA is aberrantly expressed in non-small cell lung cancer cell lines and can play a role in tumors through the ERK1 / 2 signaling pathway. These data support targeting BCMA in immunotherapy strategies in MM and potentially also in solid tumors that express BCMA.

[0480] This example provides a peptide-based immunotherapy approach targeting BCMA by generating antigen-specific CD8+ CTLs with effective and durable immunity against MM cells. Novel immunogenic native and mutant HLA-A2-specific BCMA peptides that can elicit MM-specific responses with potent antitumor activity were identified. Importantly, mutant BCMA 72-80 ( Y The LMFLLRKI (SEQ ID NO: 13)) peptide demonstrated the highest level of immunogenicity, had the greatest affinity / stability for HLA-A2 molecules, and robustly induced BCMA-specific memory CTLs against HLA-A2 + The patient's MM cells and MM cell lines have multifunctional activity. The experiment showed that this new engineered immunogenic BCMA 72-80 The peptides provide a framework for clinical application in cancer vaccines and adoptive immunotherapy regimens and provide long-lasting memory antitumor immunity in patients with cancers expressing MM or BCMA.

[0481] In particular, the results showed that CD138 +Tumor-associated antigens on tumor cells can be used to expand the breadth and scope of current multiple myeloma (MM)-specific immunotherapies. These experiments were designed to generate BCMA-specific memory CD8 + CTLs target B cell maturation antigen (BCMA), which promotes the growth and survival of MM cells. The BCMA-specific memory CD8 + CTLs mediate potent and durable immune responses against MM cells. Here, the experiments show that compared with its native peptide, BCMA, 72-80 (YLMFLLRKI (SEQ ID NO:13)) and BCMA 54-62 The novel engineered peptide specific for (YILWTCLGL (SEQ ID NO: 14)) showed improved affinity / stability for HLA-A2 and induced BCMA-specific CTLs in the presence of increased expression of activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules. Importantly, the mutant BCMA 72-80 Specific CTLs showed multifunctional Th1-specific immune activity against MM [IFN-γ / IL-2 / TNF-α production, proliferation, cytotoxicity], and they + and memory CD8 + When combined with anti-OX40 or anti-LAG3, mutated BCMA 72-80 Specific CTLs exhibited increased cytotoxicity against MM, especially by central memory CTLs. These results provide the potential for the use of mutant BCMA alone or in combination with anti-LAG3 and / or anti-OX40 in vaccination and adoptive immunotherapy strategies to generate durable autologous antitumor immunity in patients with MM and other BCMA-expressing tumors. 72-80 A framework for the clinical application of peptides.

[0482] The following materials and methods were used in this example.

[0483] Materials and methods

[0484] Cell lines

[0485] MM cell lines MM1S, OPM2, OPM1, H929, OCIMY5, RPMI, U266, KMS1, HSB2, McCAR and ANBL6 and breast cancer cell line MDA-MB-231 were obtained from ATCC (Manassas, VA). T2 cell line (a human B and T cell hybrid expressing HLA-A2 molecules) was provided by Dr. J. Molldrem (University of Texas MD Anderson Cancer Center, Houston, TX). Cell lines were cultured in DMEM (for MM and T2 cells; Gibco-Life Technologies, Rockville, MD) or Leibovitz's L-15 (for MDA-MB231; ATCC, Manassas, VA) supplemented with 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD), 100 IU / ml penicillin and 100 μg / ml streptomycin (Gibco-Life Technologies).

[0486] Reagents

[0487] Fluorescent dye-conjugated anti-human BCMA, HLA-A2, CD3, CD8, CD38, CD40L, CD69, 41BB, CCR7, CD45RO, CD107a, IFN-γ, IL-2, TNF-, PD1, LAG3, OX40, and GITR monoclonal antibodies (mAbs) were purchased from Becton Dickinson (BD) (San Diego, CA), LifeSpan Bioscience (Seattle, WA), or BioLegend (San Diego, CA). Live / Dead Aqua staining kit was purchased from Molecular Probes (Grand Island, NY). Recombinant human GM-CSF was obtained from Immunex (Seattle, WA); human IL-2, IL-4, IFN-γ, and TNF-β were purchased from R&D Systems (Minneapolis, MN). BCMA peptide-specific tetramer-PE was synthesized by MBL International Corporation (Woburn, MA). Clinical-grade mAbs against LAG3 or OX40 were provided by Bristol-Myers Squibb (New York, NY).

[0488] Synthetic peptides

[0489] Natural BCMA peptide [BCMA 64-72(LIISLAVFV(SEQ ID NO:1)), BCMA 69-77 (AVFVLMFLL(SEQ IDNO:2)), BCMA 9-17 (SQNEYFDSL(SEQ ID NO:3)), BCMA 72-80 (VLMFLLRKI(SEQ ID NO:4)), BCMA 54-62 (AILWTCLGL(SEQ ID NO:5)), BCMA 114-120 (ILPRGLEYT (SEQ ID NO: 6))], mutant BCM peptides [hBCMA 72-80 ( Y LMFLLRKI (SEQ ID NO:13)), hBCMA 54-62 ( Y ILWTCLGL (SEQ ID NO: 14)), hBCMA 9-17 ( Y QNEYFDSL (SEQ ID NO: 22))] and HIV-Gag 77-85 (SLYNTVATL (SEQ ID NO:21)) was synthesized by standard fmoc (9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >95% using reverse phase chromatography, and the molecular weight was verified by mass spectrometry (Biosynthesis, Lewisville, TX).

[0490] HLA-A2 affinity and stability assay

[0491] T2 cells were pulsed overnight with various doses of peptide plus β2-microglobulin (3 μg / ml) (Sigma, St Louis, MO). After overnight incubation, cells were stained with HLA-A2-PE mAb and analyzed using FACSCanto TM Analysis was performed by flow cytometry (BD). The stability of peptide / HLA-A2 complexes was measured on T2 cells loaded with peptides at 0, 2, 4, 6 and 14 hours after Brefeldin A treatment by staining with HLA-A2-PE mAb and flow cytometric analysis.

[0492] Generation of dendritic cells

[0493] Monocytes isolated from peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS in the presence of 1,000 units / ml GM-CSF and 1,000 units / ml IL-4. Fresh medium plus GM-CSF and IL-4 were added to the culture every other day. Mature DCs (mDCs) were obtained on day 7 after incubation with 1,000 units / ml IFN-α plus 10 ng / ml TNF-α for 3 days.

[0494] Induction of BCMA peptide-specific CTLs

[0495] Repeated stimulation of HLA-A2 antigen presenting cells (APCs) with peptide pulses + Donor-derived CD3 + T cells generated BCMA peptide-specific CTL (BCMA-CTL) ex vivo. Briefly, APCs (10 Gy) pulsed with peptide (50 μg / ml) were irradiated and used to stimulate T cells at a ratio of 1 APC / peptide: 20 T cells. T cell cultures were restimulated every 7 days and maintained in AIM-V medium supplemented with 10% human AB serum (BioWhittaker) in the presence of IL-2 (50 units / ml).

[0496] Phenotypic analysis of BCMA peptide-specific CTLs or tumor cells

[0497] Phenotypic characterization was performed on BCMA-CTLs after staining with a Live / Dead Aqua staining kit and fluorochrome-conjugated anti-human mAbs and tetramer-PE. Alternatively, MM and breast cancer cell lines were stained with fluorochrome-conjugated BCMA or HLA-A2 mAbs. After staining, cells were washed, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0498] Cell proliferation tracked by carboxyfluorescein succinimidyl ester (CFSE)

[0499] BCMA-CTLs were labeled with CFSE (Molecular Probes) and co-cultured with irradiated (10 Gy) tumor cells or peptide-pulsed APCs in the presence of IL-2 (10 units / ml). On day 4, 5, 6, or 8 of co-culture, cells were harvested and stained with a Live / Dead Aqua staining kit and CD3 / CD8 / CD45RO / CCR7 mAbs. CD3 + CD8 + The level of CTL proliferation was measured as a decrease in CFSE fluorescence intensity.

[0500] CD107a degranulation and intracellular IFN-γ / IL-2 / TNF-α cytokine production

[0501] Functional cytolytic activity of BCMA-CTLs was measured by flow cytometry through CD107a degranulation and production of Th1 cytokines. Briefly, BCMA-CTLs were co-incubated with tumor cells or T2 / peptides in the presence of CD107a mAb. After 1 hour of incubation, CD28 / CD49d mAb, Brefeldin A, and Monensin (BD) were added for another 5 hours. Cells were harvested, washed in PBS, and incubated with mAbs specific for T cell antigens. After surface staining, cells were fixed / permeabilized, stained with anti-IFN-γ / IL-2 / TNF-α mAbs, washed with permeabilization / washing solution (BD), fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0502] Statistical analysis

[0503] Results are expressed as mean ± SE. Groups were compared using an unpaired Student's t-test. Differences were considered significant when p < 0.05.

[0504] Binding affinity and stability of BCMA peptides to HLA-A2 molecules.

[0505] The BCMA full-length protein sequence was evaluated using various search software programs (including BIMAS and NetCTL) to predict epitopes with HLA-A2 affinity, prolonged half-life dissociation rate, proteasomal C-terminal cleavage, and TAP trafficking. 64-72 (LIISLAVFV), BCMA 69-77 (AVFVLMFLL), BCMA 9-17 (SQNEYFDSL), BCMA 72-80 (VLMFLLRKI), BCMA 54-62 (AILWTCLGL), BCMA 114-120 (ILPRGLEYT)], BCMA 72-80 (VLMFLLRKI) and BCMA 54-62 (AILWTCLGL) showed the highest HLA-A2 binding affinity in a dose-dependent manner. Among the designed mutant peptides, the mutant BCMA 72-80 (YLMFLLRKI (SEQ ID NO: 13)) and mutant hBCMA 54-62 ( YILWTCLGL (SEQ ID NO: 14)) showed the highest increase in HLA-A2 binding affinity. In contrast, non-HLA-A2 specific BCMA 9-17 The anchor motif in (SQNEYFDSL) was replaced by the heteromorphic BCMA 9-17 ( Y QNEYFDSL (SEQ ID NO: 22)) did not change its HLA-A2 affinity status, indicating improved A2 affinity by modifications only within the HLA-A2 specific peptide.

[0506] Evaluation of BCMA after Brefeldin A treatment of T2 cells pulsed with peptides 72-80 and BCMA 54-62 HLA-A2 stability of HLA-A2-specific peptides. Native BCMA 72-80 and BCMA 54-62 Peptides show prolonged HLA-A2 stability for more than 6 hours engineered to be mutated BCMA 72-80 ( Y LMFLLRKI (SEQ ID NO:13)) and BCMA 54-62 (YILWTCLGL (SEQ ID NO: 14)) was further enhanced. Overall, BCMA 72-80 ( Y LMFLLRKI (SEQ ID NO: 13)) detected the highest level of HLA-A2 affinity and stability, which was higher than the HLA-A2 positive control HIV-Gag 77-85 Peptide.

[0507] Mutated BCMA 72-80 ( Y LMFLLRKI(SEQ ID NO:13)) or BCMA 54-62 ( Y BCMA-specific CTLs generated by ILWTCLGL (SEQ ID NO: 14)) showed increased T cell activation and expression of co-stimulatory molecules.

[0508] Flow cytometry was used to analyze BCMA mutations after the fourth round of peptide stimulation. 72-80 Peptide-specific CTL (hBCMA 72- 80 CTL) or mutated hBCMA 54-62 Peptide-specific CTL (hBCMA 54-62 Both CTL populations showed increased expression of activation markers (CD69, CD38) and hBCMA 72-80The highest upregulation was detected on CTLs: CD38 increased from 23% at baseline to 80%; CD69 and CD69 increased from 7% at baseline to 38% ( Fig.18A ). In addition, hBCMA 72-80 CTL display and hBCMA 54-62 Compared with CTL, the expression of 41BB, CD40L, OX30 and GITR co-stimulatory molecules was higher ( Fig.18B and 18C ).

[0509] exist Figures 18A-18C In the experiment, CD3+T cells obtained from HLA-A2+ individuals were stimulated weekly with irradiated APCs pulsed with the corresponding BCMA peptides BCMA72-80 (YLMFLLRKI (SEQ ID NO: 13)) or BCMA54-62 (YILWTCLGL (SEQ ID NO: 14)). One week after the fourth stimulation cycle, CD3+CD8+T cells were analyzed by flow cytometry. The expression of T cell activation markers (CD69, CD38) and co-stimulatory molecules (41BB, CD40L, OX30, GITR) was evaluated on CD8+T cells. Representative ( Fig.18A and 18B ) or summary of three independent experiments using BCMA-CTL generated from different individuals (N=3) ( Fig. 18C ) indicates the result.

[0510] Mutated BCMA 72-80 Specific CTL responses to MM cell lines showed antigen-specific antitumor activity.

[0511] Assessment of hBCMA after each round of peptide stimulation 72-80 Phenotype and activity of CTLs. In the generated specific CTLs (n=3), the mutant BCMA 72-80 ( Y After stimulation with LMFLLRKI (SEQ ID NO: 13), % CD3 + CD8 + The gradual increase of T cells ( Figures 25A-25B ) and %CD3 + CD4 + The corresponding decrease in T cells ( Figures 26A-26B In parallel, phenotypic analysis of target cells stained with BCMA mAb clones (ANC3B1, VICKY1, 19F2) showed high BCMA expression on H929, MMIS, U266, and OPM1 cell lines but not on a breast cancer cell line (MDA-MB231) ( Figures 27A-27C ). In the functional activity assessment, - BCMA+ MM1S (7%), HLA-A2 + BCMA - hBCMA was significantly higher in MDA-MB231 (9%) or medium alone (6%) compared with 72-80 CTL response to HLA-A2 + BCMA + U266 (49%) showed significantly (*p<0.05) higher CD3 + CD8 + T cell proliferation ( Figures 19A-19D ; histogram). In the + hBCMA produced by an individual 72-80 This HLA-A2-restricted and MM-specific CD8 + CTL proliferation ( Fig.19E ; bar graph). In addition, hBCMA 72-80 CTL demonstrated response to HLA-A2 + U266, not HLA-A2 + MDA-MB231 cells, CD8 expressing the CD107a degranulation marker (47.1%) and producing granzyme B (32.6%) and perforin (29.9%) + Increased T cells ( Fig.19F ). In the + hBCMA produced by individuals (N=5) 72-80 Consistent results were observed for antitumor activity in CTLs, such as by targeting BCMA in an HLA-A2-restricted manner + IFN-γ / IL-2 / TNF-β production, 41BB upregulation, and CD107a degranulation in MM cells were measured. These data further demonstrated that mutated BCMA 72-80 Peptide-induced MM-specific immune responses.

[0512] exist Fig.19A -9F, through the use of mutated BCMA 72-80 ( Y BCMA-specific CTLs generated by repeated stimulation with LMFLLRKI (SEQ ID NO: 13) peptide were examined for their antigen specificity and HLA-A2-restricted CD8 + T cells, which respond to BCMA + MM cells or BCMA - Proliferation, CD107a degranulation, granzyme B / perforin production, IFN-γ / IL-2 / TNF-β production, and 41BB upregulation of breast cancer cells were performed. Figures 19A-19F) or the summary of three independent experiments using BCMA-CTL generated from different individuals (N=3) show the results.

[0513] Targeting HLA-A2 + Mutated BCMA in MM cells of patients 72-80 CTL functional immune response.

[0514] CD138 in MM patients + Tumor cells were used as target cells to evaluate BCMA-specific CTLs generated by the corresponding mutant peptides. 54-62 In comparison, BCMA 72-80 The peptides induced more robust antigen-specific CTL and antitumor activity against patient MM cells, as evidenced by CD107a degranulation (hBCMA 54-62 CTL 13.8% relative to hBCMA 72-80 CTL 21.5%) and IL-2 production (4.4% vs. 16.3%, respectively) were measured ( Fig. 20A ). hBCMA 72-80 CTLs consistently demonstrated higher anti-MM activity against patient cells in an HLA-A2 restricted manner, including CD107a degranulation, granzyme B / IFN-γ / TNF-β upregulation ( Fig. 20B ) and perforin / IL-2 production (n=3) ( Figures 20C-20H ). Therefore, in hBCMA 72-80 The anti-MM responses detected in CTLs were consistent with higher activation (CD69, CD38) and co-stimulatory molecule expression (41BB, CD40L, OX40, GITR) ( Figures 18A-18C These data provide new insights into BCMA mutations. 72-80 Additional evidence of immunogenicity supports its potential clinical application in novel MM treatment.

[0515] exist Figures 20A-20H In the present study, the functional activity of aberrant BCMA peptide-specific CTLs against patient MM cells was evaluated. The responses were measured relative to the baseline response (without tumor cell stimulation) of CD138 isolated cells obtained from HLA-A2 negative or HLA-A2 positive MM patients. + The specific activity of BCMA-CTL on tumor cells. Fig. 20A , Fig. 20B ) or summary of three independent experiments using BCMA-CTL generated from different individuals (N=3) ( Figures 20C-20H ) indicates the result.

[0516] Mutated BCMA 72-80 Specific CTLs are enriched in CD8 with robust anti-MM activity + Tetramer+ T cells.

[0517] Further investigation of hBCMA 72-80 CTL were characterized for their phenotype and antitumor activity within the tetramer-positive population. Tetramer-positive CTL showed a significant increase in CD8 + T cells, with activation (CD38 + :Tetramer + Compared to tetramer - :49.4% vs. 3.2%) and co-stimulatory molecule expression (CD40L + :38.0% vs. 1.2%, 41BB:24.7% vs. 1.9%, OX40:46.2% vs. 1.7% and GITR:34.9% vs. 1.5%) Fig.21A ). From several HLA-A2 + hBCMA produced by individuals (n=3) 72-80 CTLs consistently demonstrated higher levels of anti-MM activity against U266 MM cells achieved by tetramer-positive cells (83%, 97%, 97%; donor A, B, or C BCMA-CTLs) compared to tetramer-negative cells (6%, 18%, 13%; donor A, B, or C BCMA-CTLs) ( Fig. 21B ). CD107a-positive or CD107a-negative CD8 + Frequency of tetramer-positive cells in CTLs. Significantly higher tetramers were observed in degranulating CD107a-positive CTLs (82%, 98%, 98%; donors A, B, or C) compared to CD107a-negative CTLs (1%, 2%, 1%; donors A, B, or C BCMA-CTLs). + The frequency of cells ( Fig. 21C ). Therefore, these results confirm that hBCMA 72-80 The specific anti-MM activity of CTLs was contained within BCMA peptide-specific tetramer-positive cells, which showed CTL activation and upregulation of co-stimulatory molecules.

[0518] exist Figures 21A-21C In the study, the mutated BCMA that recognized tetramer-positive CTLs or did not recognize tetramer-negative CTLs was detected. 72-80 Analysis of CD8 + Expression of CD38, CD40L, 41BB, OX40, and GITR on T cells ( Fig.21A By measuring CD107 upregulation in tetramer-positive CTLs or tetramer-negative CTL subsets ( Fig. 21B ); and by evaluating the tetramer positivity status within CD107a-positive CTLs or CD107a-negative CTLs ( Fig. 21C ), further characterized the mutant BCMA72-80 Antitumor activity of specific CTL (N=3).

[0519] Mutated BCMA 72-80 Peptide-induced MM-specific memory CD8 + CTL.

[0520] To characterize BCMA-specific CTL activity, experiments were performed to assess the composition of naive:memory CTL subsets after 2 cycles and after 4 cycles of peptide stimulation compared to baseline. + Gradual and progressive phenotypic changes were detected in T cells: from naive (CD45RO - CCR7 + ) [donor 1 – naive: 83.0%, CM: 0.4%, donor 2 – naive: 84.1%]; progression to central memory after 2 cycles of peptide stimulation (CM; CD45RO + CCR7 + ) [Donor 1 – naive: 37.4%, CM: 32.1%, Donor 2 – naive: 19.0%, CM: 49.6%]; then progressed to effective memory after 4 stimulation cycles (EM; CD45RO + CCR7 - )(Donor 1 – CM: 44.2%, EM: 54.6%, Donor 2 – CM: 18.3%, EM: 77.6%))( Fig.22A ). In general, in each round of mutation BCMA 72-80 After peptide stimulation (after 1, 2, 3, and 4 cycles), memory CD8 + CTL formation gradually increased ( Figures 22B-22C ), associated with a corresponding decrease in naive T cells ( Figures 22D-22E ). Therefore, these results demonstrate that the use of mutant BCMA 72-80 Induction and progression of memory CTLs after peptide stimulation of T cells.

[0521] exist Figures 22A-22E In the experiment, the expression of mutant BCMA was analyzed at baseline (no peptide stimulation) or one week after each cycle of peptide stimulation. 72-80 Naive: memory phenotype of CTL (donor 1, donor 2). After each cycle of BCMA peptide stimulation, the dot plots ( Fig.22A ) and bar charts ( Figures 22B-22E ) demonstrated a pattern of phenotypic change from naive CD8 + T cells differentiate into memory CD8 + Expansion of T cells and memory CTLs.

[0522] Central Memory hBCMA 72-80 CTL showed maximal anti-MM activity

[0523] Next, eight (N=8) different HLA-A2 + Specific memory T cell subsets within the BCMA-specific CTLs generated by an individual characterize their anti-MM activity. - Compared with non-memory CTL, CD45RO + Memory CTLs show response to HLA-A2 + U266 MM cells (non-memory vs. memory: 7.25% vs. 28.2%) and HLA-A2 + Increased CD107a degranulation in McCAR MM cells (non-memory vs. memory: 4.14% vs. 13.2%) Fig.23A ; donor BCMA-CTL). hBCMA 72-80 Specific tetramer + Cells predominantly and consistently displayed the CM phenotype in BCMA-CTLs generated from different individuals (tetramers + % CM in cells - Donor B: 88.2%, Donor C: 97.4%, Donor D: 100%) ( Fig. 23B CM CTLs were also evaluated for their functional activity against U266 MM cells. Importantly, the level of CD107a degranulation was directly correlated with the frequency of CM cells (CD107a + Intracellular %CM - Donor E: 81.0%, Donor F: 82.6%, Donor G: 67.0%, Donor H: 41.5%) ( Fig.23C In addition, high responders (donor E, donor F) showing higher anti-MM activity showed an increased frequency of BCMA peptide-specific CM CTLs compared to intermediate responders (donor G) or low responders (donor H). Therefore, these results further indicate that BCMA is mutated. 72-80 Peptide-generating CM subsets induced unique peptide specificity and anti-MM activities.

[0524] exist Figures 23A-23C In response to HLA-A2 + MM cells (U266, McCAR; Fig.23A ) in Childish: Memory CD3 + CD8 + Abnormal BCMA was assessed in T cell subsets 72-80 Anti-MM activity of CTL. 72-80 CTL (N = 3) in different CTL subsets; in tetramer-positive or tetramer-negative CTL subsets ( Fig. 23B ) and within CD107a-positive or CD107a-negative CTL subsets ( Fig.23C ) analyzed central memory CD8 + The frequency of T cells.

[0525] Inhibition of LAG3 or stimulation of OX40 enhances hBCMA 72-80 CTL proliferation and anti-MM activity

[0526] Finally, experiments were performed to characterize the specific T cell subsets that were highly responsive to BCMA-CTLs against MM cells. + T cell subsets were gated to demonstrate HLA-A2 restricted MM-specific CTL proliferation and characterize their naive:memory subsets. The most robust response and highest proliferation of hBCMA against U266 MM cells 72-80 CTLs were mainly in the CM subset (donor 1: 97.4%, donor 2: 100%) ( Fig.24A ), confirming the major role of the CM subset within BCMA antigen-specific CTLs in MM activity. Next, experiments were performed to investigate the effects of checkpoint inhibitors (anti-LAG3) or immune agonists (anti-OX40) on these memory T cells. hBCMA treated with anti-LAG3 or anti-OX40 72-80 CTLs showed enhanced cytotoxic activity, especially by targeting HLA-A2 + U266 MM cells (untreated 28.2% vs anti-LAG3 treated 35.8% vs anti-OX40 treated 39.5%); and + Memory CTL of McCAR MM cells (untreated 13.2% vs LAG3 treated 14.5% vs OX40 treated 20.0%) Fig. 24B Interestingly, checkpoint inhibitors and immune agonists did not induce an enhanced anti-MM response of non-memory cells within BCMA-CTLs. 72-80 The beneficial effects of anti-LAG3 and anti-OX40 were further investigated in the CM and EM subsets of CTLs. Either treatment induced a greater effect on BCMA-specific CM cells compared to EM cells, as evidenced by higher CD107a degranulation in response to anti-LAG3 or anti-OX40 treatment ( Fig.24C ). Therefore, these results support the use of anti-LAG3 or anti-OX40 antibodies in combination with hBCMA 72-80 The utility of peptide combinations to induce CTLs to further enhance anti-MM activity within BCMA-specific CM subsets.

[0527] exist Figures 24A-24C In response to the mutated BCMA in U266 cells 72-80 Identification of specific CTLs induced by MM-specific CD8 + Specific subsets of T cell proliferation ( Fig.24A ). In addition, combined with anti-LAG3 or anti-OX40 to inhibit mutated BCMA72-80 CTL evaluates its effect on memory T cells ( Fig. 24B ) or central memory T cell subsets ( Fig.24C ) for its anti-myeloma activity.

[0528] discuss

[0529] Durable responses have been achieved with donor lymphocyte infusion (DLI) even in patients with refractory MM who relapse after allogeneic transplantation. These early encouraging results with DLI provide a framework for evaluating active specific immunotherapy for the treatment of MM. Cancer-targeted vaccines, one such active specific immunotherapy, have been shown to induce highly potent CD8 + The ability of CTL. The success of vaccination depends on the selection of appropriate patient populations, targeting antigens selectively expressed on tumors, and utilizing a combination approach to effectively induce and maintain antigen-specific memory anti-tumor immune responses. The present disclosure provides immunogenic HLA-A2 and HLA-A24 specific peptides derived from XBP1, CD138, and CS1 antigens that are highly overexpressed in MM and solid tumors (including breast cancer, pancreatic cancer, and colon cancer), and demonstrates that they induce anti-tumor immune responses against HLA-A2 in both preclinical and clinical studies. + or HLA-A24 + Peptide-specific CD8 on tumor cells with anti-tumor activity + In addition, combination studies of peptide stimulation / vaccination with immunomodulatory drugs such as lenalidomide or with the histone deacetylase 6 inhibitor ACY241 enhanced peptide-specific CTL activity against tumor cells. Experiments have demonstrated that the combination of peptide stimulation with lenalidomide or ACY241 enhances antigen-specific CD8 + T cell activity, which is associated with upregulation of transcriptional regulators such as T-bet / Eomes and with AKT activation, which links antigen-specific CTL differentiation to FOXO, mTOR, and Wnt / β-catenin signaling pathways. Importantly, these effects are primarily restricted to antigen-specific CD45RO + Memory CTLs, mainly observed in specific CM subsets, responded to tumor cells with IFN-γ and granzyme B production and CD8 + Most robust increase in T cell proliferation.

[0530] Due to encouraging preclinical results, the XBP1 / CD138 / CS1 multi-peptide vaccine has been evaluated alone and in combination with lenalidomide in clinical trials for the treatment of patients with smoldering MM (SMM), and in combination with anti-PD1 in clinical trials for the treatment of patients with triple-negative breast cancer. In SMM patients, the multi-peptide vaccine was well tolerated and immunogenic as a monotherapy, with enhanced tetramer + CD8 + The frequency of CTLs was demonstrated by IFN-γ production; in addition, the combination with lenalidomide triggered higher CD8 + The mean fold increase in T cells was associated with tetramer positivity and IFN-γ production. Importantly, the peptide vaccine induced CD45RO specific for the XBP1 / CD138 / CS1 peptide. + Memory CTLs were generated and further enhanced in combination with lenalidomide. Although stable disease and responses have been observed in SMM, randomized trials are needed to evaluate whether vaccination with peptides can prolong the time from progression to active disease in SMM.

[0531] To expand MM-specific immunotherapy beyond XBP1 / CD138 / CS1 antigens, the present disclosure also identified CD138 from newly diagnosed MM patients (N=616). + Other tumor-associated antigens on tumor cells. Here, the present disclosure provides the identification and characterization of immunotherapy strategies targeting BCMA, which is selectively expressed on normal and malignant plasma cells and is the target of several current immunotherapies in MM. The embodiments provide highly immunogenic engineered BCMA-specific nanopeptides, mutant BCMA 72-80 ( Y LMFLLRKI (SEQ ID NO:13)) and BCMA 54-62 ( Y ILWTCLGL (SEQ ID NO: 14)), highly improved HLA-A2 affinity / stability from their native BCMA peptides. These peptides elicit BCMA-specific CTLs against MM cells in an HLA-A2 restricted manner, with increased BCMA-specific tetramers + cells, enhanced CD107 degranulation, Th1 cytokine (IFN-γ / IL-2 / TNF-α) production, and proliferation. Most importantly, BCMA-specific memory CD8 + The increase in CTLs (both CM and EM cells), as well as the ability to self-renew and respond to MM cells, strongly supports the potential of the mutant BCMA peptide in a new vaccination and / or immunotherapy approach for MM. In fact, the present disclosure provides the following treatment regimen: 72-80Peptide vaccination, ex vivo harvest and expansion of BCMA-specific CM cells were performed, these CM cells were reinfused as adoptive immunotherapy, and then vaccinated with BCMA peptides as needed to ensure their durability for effective treatment of MM patients.

[0532] It has been observed that BCMA-specific memory CD8 + CTLs express key molecules that regulate T cell function, both for co-stimulation and immunosuppression. 72-80 The highest costimulation and immune checkpoint molecule induction were detected on the CM subgroup within peptide-specific CTLs, which is a group that shows highly effective multifunctional activity against MM. Importantly, these findings indicate that the combination therapy of BCMA-CTL with checkpoint inhibitors or immune agonists enhances the potential of its functional anti-MM activity. This may be particularly relevant given recent concerns when combining PD-1 checkpoint inhibitors with immunomodulatory drugs lenalidomide or pomalidomide or with Ab daratumumab (Daratumumab), where toxicity has shortened the study. Here, the embodiments attempt to target alternative inhibitory receptors and inhibitory mechanisms within the MM tumor microenvironment. In particular, LAG3 (CD223) is the third inhibitory receptor clinically targeted after CTLA and PD1 / PD-L1, and is expressed on BCMA-specific CM CTLs. In parallel, immune agonists, especially the co-stimulatory tumor necrosis factor receptors targeting OX40 (CD134), 41BB (CD137), and GITR (CD357), have attracted much attention for their therapeutic utility in enhancing antitumor immune responses; among them, anti-OX40 mAbs have recently demonstrated encouraging efficacy in inducing tumor regression by enhancing effector T cell expansion and functional antitumor activity in several preclinical studies. Importantly, clinical-grade anti-LAG3 and anti-OX40 (provided by Bristol-Myers Squibb; New York, NY) were used to evaluate the expression of mutant BCMA. 72-80 In vitro experiments demonstrated that both anti-LAG3 and anti-OX40 specifically increased the functional activity of memory CTLs within BCMA-CTLs targeting MM cells without affecting the activity of non-memory CTLs. + The impact of BCMA-CTL generated by individual T cells was greater after treatment with OX40 than with anti-LAG3, and was greater on the CM subset than on the EM subset within BCMA-specific CTLs. These studies provide a framework for scientifically informed combination clinical trials of BCMA peptide-specific immunotherapy with immune agonists or checkpoint inhibitors.

[0533] In summary, these experiments identified and validated novel immunogenic HLA-A2-specific engineered BCMA peptides that were able to induce antigen-specific CD8+CTLs with functional antitumor activity against MM cells. These results provide a framework for the therapeutic application of these highly immunogenic mutant BCMA peptides as vaccines and / or as stimulators for the expansion of autologous antigen-specific memory CTLs in MM patients. They further support the potential use of incorporating BCMA peptide vaccines or BCMA-specific adoptive T cell immunotherapy in combination with anti-OX40 and / or anti-LAG3 to enhance BCMA-directed anti-MM responses.

[0534] Example 13: HLA-A2-specific immunogenic TACI peptides for eliciting TACI-specific CD8+ cytotoxic T lymphocytes

[0535] Experiments were performed to demonstrate the immunogenicity of novel engineered mutant TACI 154-162 ( Y LSADQVAL (SEQ ID NO: 16)) peptide can induce antigen-specific memory CD8 with robust multifunctional immune response against MM + These results in this example provide a framework for the therapeutic use of mutant TACI peptides in MM patients and support the therapeutic use of TACI peptide-specific vaccines or TACI peptide-specific adoptive T cell immunotherapy to treat patients with myeloma or other diseases expressing TACI.

[0536] The following materials and methods were used in the examples.

[0537] Materials and methods

[0538] Cell lines

[0539] HLA-A2 + (U266 and McCAR) and HLA-A2 - (OPM2 and RPMI) MM cell lines were obtained from ATCC (Manassas, VA). T2 cell line (human B and T cell hybrid expressing HLA-A2) was provided by Dr. J. Molldrem (University of Texas MD Anderson Cancer Center, Houston, TX). Cell lines were cultured in DMEM (for MM and T2 cells; Gibco-Life Technologies, Rockville, MD) medium supplemented with 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Gibco-Life Technologies).

[0540] Reagent Type

[0541] Fluorescent dye-conjugated anti-human monoclonal antibodies (mAbs) were purchased from Becton Dickinson (BD) (San Diego, CA), LifeSpan Bioscience (Seattle, WA), or BioLegend (San Diego, CA). Live / Dead Aqua staining kit was purchased from (Grand Island, NY). Recombinant human GM-CSF was purchased from Immunex (Seattle, WA), and human IL-2, IL-4, IFN-α, and TNF-α were purchased from R&D Systems (Minneapolis, MN). TACI peptide-specific tetramer-PE was synthesized by MBL International Corporation (Woburn, MA).

[0542] Synthetic peptides

[0543] Natural TACI peptide [TACI 178-186 (FLVAVACFL(SEQ ID NO:7)),TACI 174-182 (VLCCFLVAV(SEQID NO:8)),TACI 154-162 (KLSADQVAL(SEQ ID NO:9)),TACI 166-174 (TLGLCLCAV(SEQ ID NO:10)),TACI 161-169 (ALVYSTLGL(SEQ ID NO:11)),TACI 155-163 (LSADQVALV (SEQ ID NO: 12))], mutant TACI peptide [TACI 178-186 ( Y LVAVACFL (SEQ ID NO:15)), TACI 154-162 ( Y LSADQVAL(SEQ ID NO:16)),TACI 166-174 ( Y LGLCLCAV (SEQ ID NO: 17))] and HIV-Gag 77-85 The (SLYNTVATL (SEQ ID NO:21)) peptide (HLA-A2 specific positive control peptide) was synthesized by standard fmoc (9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >95% using reverse phase chromatography, and the molecular weight was verified by mass spectrometry (Biosynthesis, Lewisville, TX).

[0544] HLA-A2 affinity and stability assay

[0545] T2 cells were pulsed overnight with various concentrations of peptide plus β2-microglobulin (3 μg / ml) (Sigma, St Louis, MO). After overnight incubation, cells were stained with HLA-A2-PE mAb and analyzed using FACSCanto TM Analysis was performed by flow cytometry (BD). The stability of peptide / HLA-A2 binding was measured on T2 cells loaded with peptides at 0, 2, 4, 6 and 14 hours after Brefeldin A treatment by staining with HLA-A2-PE mAb and flow cytometric analysis.

[0546] Generation of dendritic cells

[0547] Monocytes isolated from peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS in the presence of 1,000 units / ml GM-CSF and 1,000 units / ml IL-4. Fresh medium plus GM-CSF and IL-4 were added to the culture every other day. Mature DCs (mDCs) were obtained on day 7 after incubation with 1,000 units / ml IFN-α plus 10 ng / ml TNF-α for 3 days.

[0548] Induction of TACI peptide-specific CTL

[0549] Repeated stimulation of HLA-A2 antigen presenting cells (APCs) with peptide pulses + Donor-enriched CD3 + T cells generated TACI peptide-specific CTL (TACI-CTL) ex vivo. Briefly, APCs pulsed with peptide (50 μg / ml) were irradiated (10 Gy) and used to stimulate T cells at a 1:20 APC / peptide to T cell ratio. T cell cultures were restimulated every 7 days and maintained in AIM-V medium supplemented with 10% human AB serum (BioWhittaker) in the presence of IL-2 (50 units / ml).

[0550] Phenotypic analysis of TACI peptide-specific CTLs or stimulatory tumor cells

[0551] Phenotypic characterization of MM target cells was performed to confirm TACI expression. Phenotypic characterization was performed on TACI-CTLs after staining with a Live / Dead Aqua staining kit and fluorescent dye-conjugated anti-human mAbs. After staining, cells were washed, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0552] Cell proliferation tracked by carboxyfluorescein succinimidyl ester (CFSE)

[0553] TACI-CTLs were labeled with CFSE (Molecular Probes) and co-cultured with irradiated (10 Gy) MM cells or peptide-pulsed APCs in the presence of IL-2 (10 units / ml). On day 4, 5, 6, or 8 of co-culture, cells were harvested and stained with a live / dead Aqua staining kit and fluorescent dye-conjugated anti-human mAbs specific for CD3, CD8, CD45RO, and CCR7. CD3 + CD8 + The level of CTL proliferation was measured as a decrease in CFSE fluorescence intensity.

[0554] CD107a degranulation and intracellular IFN-γ / IL-2 / TNF-α cytokine production

[0555] Functional cytolytic activity of TACI-CTL was measured by flow cytometry through CD107a degranulation (cytotoxicity) and the production of Th1 cytokines. In brief, TACI-CTL was co-incubated with tumor cells or T2 / peptides in the presence of CD107a mAb. After 1 hour, CD28 / CD49d mAb, Brefeldin A and Monensin (BD) were added and incubated for another 5 hours. Cells were harvested, washed in PBS, and incubated with mAbs for key T cell markers conjugated with fluorescent dyes. After surface staining, cells were fixed / permeabilized, stained with anti-IFN-γ / IL-2 / TNF-α mAb, washed with permeabilization / washing solution (BD), fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0556] Statistical analysis

[0557] Results are expressed as mean ± SE. Groups were compared using an unpaired Student's t-test. Differences were considered significant when p < 0.05.

[0558] result

[0559] Identification of the mutant TACI with the highest binding affinity and stability to HLA-A2 molecules 154-162 Peptides

[0560] The TACI full-length protein sequence was evaluated using various search software programs, including BIMAS and NetCTL, to predict epitopes with HLA-A2 affinity, prolonged half-life off-rate, proteasomal C-terminal cleavage, and TAP trafficking. 178-186 (FLVAVACFL(SEQ ID NO:7)),TACI174-182 (VLCCFLVAV(SEQ ID NO:8)),TACI 154-162 (KLSADQVAL(SEQ ID NO:9)),TACI 166-174 (TLGLCLCAV(SEQ IDNO:10)),TACI 161-169 (ALVYSTLGL(SEQ ID NO:11)),TACI 155-163 (LSADQVALV (SEQ ID NO: 12))], except for TACI 161-169 All peptides except TACI showed HLA-A2 specificity, but 166-174 The highest level of affinity was detected. To improve the binding and stability of the peptide to the HLA-A2 molecule, three variant peptides were designed, including TACI 178-186 ( Y LVAVACFL (SEQ ID NO:15)), TACI 154-162 ( Y LSADQVAL (SEQ ID NO: 16)) and TACI 166-174 ( Y LGLCLCAV (SEQ ID NO: 17)) peptides were synthesized and their HLA-A2 affinity was evaluated. At 50 μg / ml and 100 μg / ml peptide doses, the mutant TACI 154-162 The peptide showed greatly enhanced affinity from its native form. 154-162 The peptide's HLA-A2 affinity is similar to HIV-Gag 77-85 (SLYNTVATL (SEQ ID NO: 21)), which served as an HLA-A2-specific positive control peptide. However, compared with their native peptides, the engineered mutant TACI 178-186 and TACI 166-174 The peptides did not show significant improvement in HLA-A2 affinity. The selected TACI peptides (50 μg / ml) were further evaluated for their affinity and stability to HLA-A2 molecules. 154-162 The highest HLA-A2 binding affinity (time = 0) and stability (2, 4, 6, and 18 hours) were seen. Overall, at each time point, the mutant TACI 154-162 The HLA-A2 affinity stability is similar to that of the HLA-A2 positive control HIV-Gag 77-85 Based on the highest level of HLA-A2 affinity and stability among the TACI peptides studied, the mutant TACI was selected 154-162Its immunogenic potential to induce antigen-specific effector T cells against MM was further evaluated.

[0561] By + Antigen-specific memory CD8 cells + Cell development and expansion, mutation TACI 154-162 Antitumor activity of peptide-specific CTLs.

[0562] In the weekly use of mutation TACI 154-162 ( Y LSADQVAL (SEQ ID NO: 16)) stimulates HLA-A2 + Enrichment of donor CD3 + After T cells, CD8 was assessed in naive:memory subsets + Phenotypic changes of T cells. At baseline [before peptide stimulation], it was found that most (84.1%) CD8 + T cells are naive (CD45RO - CCR7 + ) cell subsets. 154-162 After peptide stimulation, naive CTLs were observed to gradually differentiate into mutant TACI 154-162 Peptide-specific CTL (hTACI 154-162 Central memory CTL (CM; CD45RO) + CCR7 + / CD3 + CD8 + ), followed by effector memory CTL (EM; CD45RO + CCR7 - / CD3 + CD8 + ). Use mutant TACI 154-162 The formation of memory CTLs was detected after 3 peptide stimulation cycles [naive: 2.2%, CM: 47.8%, EM: 52.4%]. Further differentiation from naive CTLs into memory CTL subsets was observed after 4 peptide stimulation cycles [naive: 0.72%, CM: 24.4%, EM: 74.1%] and after 5 stimulation cycles [naive: 0.57%, CM: 7.36%, EM: 69.7%] Fig.29A ), supporting the potential of the peptide to form memory T cells. + The mutant TACI was further evaluated in T cells obtained from donors (N=3). 154-162 Immunogenicity of peptides ( Fig.29B ). When using iso-TACI 154-162 After peptide stimulation, naive CD8 +Gradual reduction of CTL and memory CD8 + Therefore, these results confirm that the mutant TACI 154-162 Peptide-forming memory CD8 + T cell capacity.

[0563] Next, experiments were performed to evaluate the mutated TACI 154-162 The ability of specific CTL to respond to myeloma cells. + After co-culture with U266 MM cells, the mutant TACI 154-162 CD8 was detected in peptide-specific CTLs, especially in memory CTLs including central memory and effector memory CTL subsets. + Proliferation of CTL. After more TACI154-162 peptide stimulation cycles [2' stimulation: proliferation CM-38.1%, proliferation EM-19.2%; 3' stimulation: proliferation CM-54.4%, proliferation EM-85.2%; 4' stimulation: proliferation CM-64.4%, proliferation EM-86.9%], the proliferation capacity of TACI-CTL continued to increase ( Fig.29C ). Therefore, these results further support the role of memory CD8 + The formation and continuous expansion of T cells, mutated TACI 154-162 Immune function of peptide-specific CTL.

[0564] In the mutation TACI 154-162 TACI-specific tetramer in specific CTL + The proliferation of CTLs was confirmed by the restriction of HLA-A2 Multifunctional and Th1-specific anti-myeloma activity in a restrictive manner.

[0565] hTACI 154-162 Functional activity of CTLs was examined to examine their polyfunctional immune response against myeloma cells. - Compared with RPMI (8.24%) or culture medium alone (5.03%), hTACI 154-162 CTLs expressed against HLA-A2 + HLA-A2-restricted degranulation (CD107a upregulation) of McCARMM cells directly correlates with cytotoxic activity against tumor cells ( Fig. 30A ). In addition, - Compared with RPMI (IFN-8.26%, TNF-7.80%, IL-2 6.42%) or culture medium alone (IFN-5.76%, TNF-5.52%, IL-2 6.24%), hTACI 154-162 CTLs show response to HLA-A2 +McCAR, higher production of Th1-type cytokines, IFN-(15.0%), TNF-(17.5%), and IL-2(14.9%). - Compared with MM cells (RPMI), targeting HLA-A2 + MM cells (McCAR), as measured by CD107a degranulation and IFN-γ production, in cells derived from different HLA-A2 + hTACI produced by individuals (N=5) 154-162 It was consistently observed in CTLs ( Fig. 30B , 3C). In order to confirm the mutated hTACI 154-162 The HLA-A2-specific anti-MM activity of CTL was evaluated against other myeloma cell lines, including HLA-A2 + U266 and HLA-2 - OPM2 cells) in the immune function activity. + Donor 1 ( Fig.31A ), and a total of five different HLA-A2 + individual( Figures 31B-31E ) 154-162 The same pattern of HLA-A2-restricted functional antitumor activity was observed in hTACI154-162 CTLs against myeloma cells. 154-162 Peptide-specific tetramer + CTL staining was positive, and these tetramers + Cells responding to HLA-A2 + U266 myeloma cells showed high levels of CD107a degranulation and proliferation ( Fig.31F ). Therefore, these results demonstrate that the mutant TACI 154-162 The HLA-A2-restricted immunogenicity of the peptides elicited antigen-specific CTLs with multifunctional activity (cytotoxicity, Th-1 type cytokine production) against MM cells, supporting the immunogenic variant TACI 154-162 ( Y The potential therapeutic application of the LSADQVAL (SEQ ID NO: 16)) peptide in myeloma patients.

[0566] Example 14. BCMA variant peptide-encapsulated nanoparticles enhance antigenic stimulation and tumor-specific CD8 against multiple myeloma + Cytotoxic T lymphocytes.

[0567] B cell maturation antigen (BCMA), a member of the tumor necrosis factor (TNF) receptor superfamily and the receptor bound by B cell activating factor (BAFF) and proliferation inducing ligand (APRIL), is a promising therapeutic target for MM. BCMA has a restricted expression pattern on MM cells and plasma cells and plays a role in promoting MM cell growth, survival and drug resistance.

[0568] The present disclosure has identified nanomedicine-based therapeutics targeting BCMA as a promising area of ​​translational research to effectively elicit and enhance anti-tumor responses in MM patients. Several nanomedicines are available, and more advanced nanoparticle constructs are being developed for antigen encapsulation. To this end, the present example provides novel engineered peptides specific for BCMA and uses mutant BCMA 72-80 ( Y In addition, a nanoparticle-based cancer vaccine was developed to induce more robust BCMA-specific CD8 in MM patients. + Cytotoxic T lymphocyte (CTL) activity, with more sustained antigen release and increased bioavailability and presentation of immunogenic peptides. Here, experiments were performed to examine the potential of a novel nanomedicine-based therapeutic delivery system specific for the BCMA antigen to treat MM patients. The purpose of this example was to design an optimal nanoparticle-encapsulated BCMA antigen construct to effectively elicit BCMA-specific CD8 + CTL.

[0569] The results showed that nanoparticles [liposomes or poly (D, L-lactide-co-glycolide) (PLGA)] with different antigen release kinetics demonstrated their ability to effectively deliver mutated BCMA peptides to antigen presenting cells and elicit BCMA antigen-specific CTLs with anti-MM activity. Nanoparticles encapsulated with mutated BCMA peptides showed higher uptake by human dendritic cells than free peptides, with liposome-based nanoparticles mediated the highest uptake. In contrast, BCMA-specific CTLs induced with PLGA-based nanoparticles demonstrated the highest functional activity and specific immune response against MM cells. Importantly, BCMA-specific CTLs induced by PLGA / BCMA peptide nanoparticles demonstrated greater CD107a degranulation, antigen-specific CD8 in response to tumor cells and MM cell lines from MM patients, compared with BCMA-CTLs generated with free BCMA peptide or liposome / BCMA peptide nanoparticles. +CTL proliferation and Th-1 cytokine (IFN-γ, IL-2, TNF-α) production. CD28 co-stimulatory molecules were upregulated and tetramers were generated in BCMA-CTLs generated by PLGA / BCMA nanoparticles. + CTL generation and peptide-specific responses were also greater than BCMA-CTL generated with free BCMA peptide or liposome / BCMA peptide nanoparticles. In addition, PLGA / BCMA nanoparticles triggered antigen-specific memory CD8 + More robust induction of T cells was demonstrated by the induction of non-memory CD8 T cells compared to BCMA-CTLs. + Significantly higher antitumor activity of T cells, as evidenced by CD107a degranulation and IFN-γ production. Induction of central memory CTLs with antitumor activity by PLGA / BCMA peptides was associated with optimal peptide release kinetics and enhanced immunogenicity.

[0570] Thus, these results demonstrate that the nanoparticle strategy of aberrant BCMA peptide encapsulation enhances peptide delivery to dendritic cells and subsequently to T cells, thereby inducing BCMA-specific central memory CTLs with multifunctional activity against MM.

[0571] These results also demonstrate the utility of nanotechnology using encapsulated heteromutant BCMA peptides to enhance the immunogenicity of BCMA peptide-specific therapeutics for MM. Importantly, the observations provide a framework for the therapeutic application of PLGA nanoparticle-based heteromutant BCMA peptide delivery to enhance BCMA-specific memory T cell immune responses, overcome the limitations of current peptide-based cancer vaccines and adoptive immunotherapy, and improve patient outcomes in MM.

[0572] The following methods and materials were used in this example.

[0573] Materials and methods

[0574] Material

[0575] Poly(D,L-lactide-co-glycolide) (PLGA, molecular weight 23,000, copolymer ratio 50:50) was purchased from Birmingham Polymers (Birmingham, AL). Polyvinyl alcohol (PVA, average molecular weight 30,000-70,000), trifluoroacetic acid, acetonitrile, lipopolysaccharide, and L-15 (Leibovitz) medium were purchased from Sigma-Aldrich (St Louis, MO).

[0576] Formulation and characterization of BCMA peptide-loaded PLGA-NPs.

[0577] Using immunogenic mutant hBCMA 72-80 (Y hBCMA was formulated with PLGA-NPs and poly(vinyl alcohol) (PVA) using double emulsion-solvent technology. 72-80 Peptide to stabilize the emulsion. Mutated hBCMA with PLGA-NP 72-80 Peptides are formulated essentially as previously described (Sahoo SK, et al. 2004). In brief, PLGA (50:50 lactide-co-glycolide ratio) and peptide or blank PLGA itself are emulsified in dichloromethane (DCM), and the mixture is resuspended in 2% PVA to form an oil and water emulsion. The emulsification process is completed in an ice bucket at 55 watts using a micro-tip probe sonicator for 10 minutes. The emulsion is stirred at room temperature for 3 hours to evaporate DCM and form PLGA-NPs. The peptide-loaded PLGA-NPs are recovered by ultracentrifugation at 4 ° C for 30 minutes to eliminate PVA and free peptides, washed and resuspended in PBS. In order to evaluate the PLGA-NP structure, the nanoparticle preparation is freeze-dried for 24 hours and visualized using a scanning electron microscope (Hitachi S-4800 microscope, Schaumburg, IL).

[0578] Formulation and characterization of BCMA peptide-loaded liposome-NPs.

[0579] BCMA peptide-loaded liposome-NPs were synthesized using a thin film hydration method. Liposome lipid bilayers were prepared with a mixture of cholesterol (MW = 386.654), DOPC (MW = 786.113) and DOTAP (MW = 698.542) (Avanti Polar, Alabaster, AL). Briefly, a 1 ml stock solution from a mixture of 3 mM cholesterol, 5 mM DOPC and 5 mM DOTAP was prepared in chloroform. The solvent was evaporated using a rotary evaporator (RV 10, IKA, Wilmington, NC) to produce a thin lipid film at the bottom of the flask. The lipid film was subjected to vacuum drying overnight to remove any residual organic solvent. The next day, hBCMA dissolved in disodium hydrogen phosphate (binary; pH ​​11) buffer and 1% DMSO was used. 72-80Peptide, the lipid film was hydrated using 10 freeze-thaw cycles (-80 ° C and 37 ° C), and then the particle size was reduced to the desired range by probe sonication on ice for 1 minute. The peptide-loaded liposome-NPs were recovered by ultracentrifugation and resuspended in PBS. Blank liposomes were prepared according to the same procedure except for the hydration step, in which a binary buffer containing 1% DMSO was used without any peptide. The surface morphology of the BCMA peptide-loaded liposome-NPs was characterized using transmission electron microscopy (TEM). Uranyl acetate (2%) was used as a negative stain for visualizing the BCMA peptide-loaded liposome-NPs using TEM (JEM-1000, JEOL, Tokyo, Japan).

[0580] BCMA peptide encapsulation in PLGA-NPs or liposome-NPs.

[0581] The level of peptide encapsulation on PLGA-NPs or liposome-NPs was measured using a quantitative fluorescence measurement peptide assay kit (Thermo Fisher) according to the manufacturer's recommended protocol. Briefly, equal amounts of BCMA peptide in suspension, BCMA peptide loaded in PLGA-NPs (10 μl), or BCMA peptide loaded in liposome-NPs (10 μl) were loaded in triplicate into a 96-well fluorescence-compatible microplate. Blank PLGA or blank liposomes were used as negative controls. Next, 1:1 acetonitrile:DMSO (70 μl) and FluoroBrite TM A solution of DMEM (20 μl) was added to each well and incubated at room temperature in the dark for 5 minutes. An acetonitrile:DMSO solution was used as a control to measure background fluorescence. After incubation, fluorescence was measured using a spectrophotometer at Ex / Em of 390 nm / 475 nm. Peptide concentrations were determined based on a standard curve (0–1,000 μg / ml) generated with a linear fit.

[0582] Generation of monocyte-derived dendritic cells

[0583] Monocyte-derived dendritic cells (DCs) were generated essentially as described previously (Bae et al. 2011, Bae et al. 2012). Briefly, HLA-A2 +Monocytes isolated from peripheral blood mononuclear cells (PBMC) of normal donors were cultured in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS in the presence of 1,000U / ml GM-CSF and 1,000U / ml IL-4 for 7 days. Fresh medium containing cytokines was replaced every other day. Immature dendritic cells were collected from the culture on day 7 for uptake studies. When immature dendritic cells were generated on day 7, mature DCs were obtained by adding 1,000U / ml IFN-α plus 10ng / ml TNF-α and fresh GM-CSF and IL-4 in 10...

Claims

1. A peptide consisting of an antigen consisting of the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14, wherein the length of the peptide is 9 to 30 amino acids, and wherein the peptide increases a B cell maturation antigen (BCMA) specific response.

2. The peptide of claim 1, wherein the antigen consists of the amino acid sequence shown in SEQ ID NO:

13.

3. The peptide of claim 1, wherein the antigen consists of the amino acid sequence shown in SEQ ID NO:

14.

4. A peptide consisting of a first amino acid sequence and a second amino acid sequence heterologous to the first amino acid sequence, wherein the first amino acid sequence consists of an antigen consisting of the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14; wherein the peptide increases a B cell maturation antigen (BCMA) specific response.

5. The peptide of claim 1, wherein the peptide: associates with an MHC molecule and is recognized by an antigen-specific T cell receptor on a T cell.

6. The peptide of claim 4, wherein the peptide: associates with an MHC molecule and is recognized by an antigen-specific T cell receptor on a T cell.

7. The peptide of claim 5, wherein the MHC molecule is: (a) a class I MHC molecule, (b) a class II MHC molecule, (c) an HLA-A2 molecule, or (d) an HLA-A24 molecule.

8. The peptide of claim 6, wherein the MHC molecule is: (a) a class I MHC molecule, (b) a class II MHC molecule, (c) an HLA-A2 molecule, or (d) an HLA-A24 molecule.

9. A composition comprising the peptide of any one of claims 1 to 8 and a second agent.

10. The composition of claim 9, wherein the second agent is an immunostimulant.

11. The composition of claim 9, wherein the second agent is a T helper cell epitope.

12. The composition of claim 11, wherein the T helper cell epitope is a PADRE sequence or a universal tetanus toxoid T helper cell (TT Th) epitope.

13. The composition of claim 9, wherein the second agent is an adjuvant.

14. The composition of claim 13, wherein the adjuvant is selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand of a Toll receptor, QS21, RIBI, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), mutant CT (MCT), and mutant Escherichia coli heat-labile toxin (MLT).

15. The composition of claim 9, wherein the second agent is toll-like receptor-3, interferon alfa (IFNα), interferon gamma (IFNγ), anti-OX40 antibody, anti-GITR antibody, or granulocyte-macrophage colony stimulating factor (GM-CSF).

16. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

17. A nucleic acid encoding the peptide of any one of claims 1 to 8.

18. A vector comprising a nucleic acid encoding the peptide of any one of claims 1 to 8.

19. The vector of claim 18, wherein the nucleic acid is operably linked to a promoter, a regulatory element or an expression control sequence.

20. A cultured cell comprising the vector of claim 19.

21. The cultured cell of claim 20, wherein the cell is a mammalian cell.

22. The cultured cell of claim 20, wherein the cell is a human cell.

23. The cultured cell of claim 20, wherein the cell is an immune cell.

24. A virus comprising a nucleic acid encoding a peptide according to any one of claims 1 to 8.

25. The virus of claim 24, wherein the virus is a lentivirus or an adenovirus.

26. The virus of claim 24, wherein the virus is an adeno-associated virus.

27. A combination product of a first peptide and a second peptide, wherein the first peptide consists of the amino acid sequence shown in SEQ ID NO: 13, and the second peptide consists of the amino acid sequence shown in SEQ ID NO: 14, wherein the peptide increases B cell maturation antigen (BCMA) specific response.

28. A pharmaceutical composition comprising The combination product of claim 27; and A pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition further comprises an immune agonist.

30. The pharmaceutical composition of claim 29, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

31. Use of a peptide according to any one of claims 1 to 8, or a composition according to any one of claims 9 to 15, or a pharmaceutical composition according to any one of claims 16, 28 to 30, in the preparation of a medicament for inducing an immune response against multiple myeloma cells in a human subject.

32. The use of claim 31, wherein the human subject suffers from multiple myeloma.

33. The use of claim 31, wherein the medicament is used in combination with an immune agonist.

34. The use of claim 33, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

35. The use of claim 31, wherein the medicament is used in combination with: determining whether an immune response against multiple myeloma occurs in the human subject after the medicament is administered to the human subject.

36. Use of a peptide according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 15 or a pharmaceutical composition according to any one of claims 16 and 28 to 30 for the preparation of a medicament for treating a human subject suffering from multiple myeloma.

37. The method of claim 36, wherein the drug is used in combination with: One or more cancer cells in the human subject are detected to express or overexpress BCMA.

38. The use of claim 36, wherein the human subject has one or more cancer cells expressing MHC molecules.

39. The use of claim 36, wherein the medicament is used in combination with an immune agonist.

40. The use of claim 39, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

41. The use of claim 36, wherein the medicament is used in combination with chemotherapy or radiotherapy.

42. A method for generating and / or expanding BCMA-specific cytotoxic T cells, the method comprising contacting one or more cytotoxic T cells with one or more antigen presenting cells pulsed with the peptide of claim 1.

43. The method of claim 42, wherein the cytotoxic T cells are memory cytotoxic T cells.

44. The method of claim 42, wherein the cytotoxic T cells are effector cytotoxic T cells.

45. The method of claim 42, wherein the antigen presenting cell is a dendritic cell.

46. ​​Use of one or more multiple myeloma-specific cytotoxic T cells in the preparation of a medicament for killing multiple myeloma target cells, wherein the target cells express or overexpress BCMA and express HLA-A, The one or more multiple myeloma-specific cytotoxic T cells are obtained by contacting the one or more cytotoxic T cells with one or more antigen-presenting cells pulsed with the peptide of claim 1.

47. The use of claim 46, wherein the use further comprises contacting the one or more multiple myeloma-specific cytotoxic T cells with an immune agonist.

48. The use of claim 47, wherein the immune agonist is an OX40 agonist or a GITR agonist.

49. The use of claim 47, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

50. Use of a plurality of BCMA-specific cytotoxic T cells in the preparation of a medicament for treating multiple myeloma in a human subject in need thereof, The BCMA-specific cytotoxic T cells are obtained by contacting one or more cytotoxic T cells with one or more antigen-presenting cells pulsed with the peptide of claim 1.

51. The use of claim 50, wherein the medicament is used in combination with an immune agonist.

52. The use of claim 51, wherein the immune agonist is an OX40 agonist or a GITR agonist.

53. The use of claim 51, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

54. The use of claim 50, wherein the one or more cytotoxic T cells are derived from cells of the human subject.

55. The use of claim 50, wherein the one or more cytotoxic T cells are derived from induced pluripotent stem cells.

56. A method for producing an antigen presenting cell that presents a peptide on a major histocompatibility complex (MHC) molecule, comprising (a) obtaining bone marrow-derived mononuclear cells from a subject; (b) culturing the mononuclear cells in vitro under conditions where the mononuclear cells adhere to a culture container; (c) selecting adherent mononuclear cells; (d) culturing the adherent mononuclear cells in the presence of one or more cytokines under conditions whereby the cells differentiate into antigen presenting cells; and (e) contacting the antigen-presenting cell with the peptide according to any one of claims 1 to 8, thereby producing an antigen-presenting cell that presents the peptide on a major histocompatibility complex (MHC) molecule.

57. The method of claim 56, wherein the major histocompatibility complex molecule is an MHC class I molecule.

58. The method of claim 56, wherein the one or more cytokines include granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).

59. The method of claim 56, wherein the one or more cytokines comprise tumor necrosis factor-α (TNF-α).

60. The method of claim 56, wherein the bone marrow-derived cells are obtained from a subject diagnosed with multiple myeloma.

61. A method for identifying a T cell antigen receptor sequence for BCMA, the method comprising (a) generating and / or proliferating BCMA-specific cytotoxic T cells by the method of claim 42; and (b) determining the T cell antigen receptor sequence of BCMA in the BCMA-specific cytotoxic T cells.

62. A composition comprising: Nanoparticles and the peptide of any one of claims 1 to 8.

63. The composition of claim 62, wherein the peptide is encapsulated in the nanoparticle.

64. The composition of claim 62, wherein the nanoparticles comprise a biodegradable polymer.

65. The composition of claim 62, wherein the nanoparticles comprise poly(D,L-lactide-co-glycolide) (PLGA).

66. The composition of claim 62, wherein the nanoparticles comprise poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) copolymer.

67. The composition of claim 62, wherein the nanoparticle is a liposome.

68. The composition of any one of claims 62-66, wherein the nanoparticles comprise an adjuvant.

69. The composition of any one of claims 65-66, wherein the nanoparticles comprise a Toll-like receptor agonist.

70. The composition of any one of claims 62-66, further comprising an adjuvant, an immune agonist, a checkpoint inhibitor, lenalidomide, or any combination thereof.

71. Use of the composition of any one of claims 62-70 in the preparation of a medicament for treating multiple myeloma in a human subject in need thereof.

72. The use of claim 71, wherein the drug is used in combination with one or more of the following: a peptide selected from SEQ ID NO: 13-17; BCMA-specific CTL and TACI-specific CTL.

73. The use of claim 71, wherein the drug is used in combination with a peptide consisting of the amino acid sequence shown in SEQ ID NO: 13 or 16.

74. The use of claim 71, wherein the medicament is used in combination with BCMA-specific CTLs generated by stimulating CTLs with a peptide consisting of the amino acid sequence shown in SEQ ID NO:

13.

75. The use of claim 71, wherein the medicament is used in combination with TACI-specific CTL generated by stimulating CTL with a peptide consisting of the amino acid sequence shown in SEQ ID NO:

16.

76. The peptide of claim 1, wherein the peptide binds to an HLA-A2 molecule.

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