Immunogenic peptides specific for the BCMA antigen and uses thereof

Abstract

To provide immunogenic peptides specific to BCMA antigens and use thereof.SOLUTION: This disclosure relates to immunogenic peptides specific to a B-cell maturation antigen (BCMA), a transmembrane activator and CAML interactor (TACI), and methods of use thereof. In one aspect, there is provided a peptide comprising an amino acid sequence that is identical to the amino acid sequence set forth in any one of SEQ ID NO: 13-17, or differs by 1 to 4 amino acid residues, where the amino acid at position 1 of SEQ ID NOs: 13-17 is unaltered. In another aspect, there is provided a peptide comprising: a first amino acid sequence consisting of an amino acid sequence that is at least 60% identical to any one of SEQ ID NOs: 1-17; and a second amino acid sequence that is heterologous to the first amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 553,669, filed Sep. 1, 2017, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT OF FEDERALLY FUNDED RESEARCH 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 the invention.

[0003] The present disclosure relates to immunogenic peptides specific for B-cell maturation antigen (BCMA) and transmembrane activator and interactor of CAML (TACI) and methods of use thereof. [Background technology]

[0004] Cancer is currently one of the diseases with the highest human mortality rates. According to World Health Organization statistical data, the number of cancer incidence and death cases worldwide reached 14 million and 8.2 million, respectively, in 2012. In the United States, cancer is responsible for at least 25% of all deaths.

[0005] In recent years, new therapies have been developed to treat various types of cancer.Patients suffering from cancer are often treated by, for example, surgery, chemotherapy and / or immunotherapy.The prognosis of these patients may still be unsatisfactory.Therefore, there is an urgent need for efficient therapeutic and / or preventive regimens for treating cancer. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure relates in part to immunogenic peptides that encapsulate peptides specific for B cell maturation antigen (BCMA) or transmembrane activator and interactor of CAML (TACI), T cells (e.g., CD8 + Cytotoxic T cells (CTL) and / or CD4 + The present invention relates to a method for treating pulmonary arterial disease (PE) and / or pulmonary arterial disease (PAS ...).

[0007] In one aspect, the disclosure relates to a peptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to, or differing by 1-6 amino acid residues from, an amino acid sequence set forth in any one of SEQ ID NOs: 13-17. In some embodiments, the amino acid at position 1 of SEQ ID NOs: 13-17 is not altered. 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 peptide is 9-30 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 amino acids in length).

[0008] 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.

[0009] In some embodiments, the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-17, comprising 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.

[0010] In another aspect, the disclosure relates to a peptide comprising, consisting essentially of, or consisting of a first amino acid sequence 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, and a second amino acid sequence that is heterologous to the first amino acid sequence. 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 substituted. In certain embodiments, the peptides are 9-30 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 amino acids in length).

[0011] 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, an 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, substituted at positions 3, 4, 5, 6, 7 or 8, substituted at positions 3 and 4, substituted at positions 3 and 5, substituted at positions 3 and 6, substituted at positions 3 and 7, substituted at positions 3 and 8, substituted at positions 4 and 5, substituted at positions 4 and 6, substituted at positions 4 and 7, substituted at positions 4 and 8, substituted at positions 5 and 6, substituted at positions 5 and 7, substituted at positions 5 and 8, substituted at positions 6 and 7, substituted at positions 6 and 8, substituted at positions 7 and 8, any combination of three different, four different, five different, or six different positions from the group of 3, 4, 5, 6, 7 and 8 are substituted.

[0012] In some embodiments, the peptide binds to a major histocompatibility complex (MHC) molecule. In some embodiments, the peptide is recognized by an antigen-specific T cell receptor on a T cell in association with the MHC molecule. In some embodiments, the MHC molecule is an MHC class I molecule or an MHC class II molecule. 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. In some embodiments, the MHC molecule is an HLA-A2 molecule or an HLA-A24 molecule.

[0013] The present disclosure also provides compositions comprising a peptide as described herein and a second agent. In some embodiments, the second agent is selected from the group consisting of (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) immune modulators including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone, (4) BCMA and other anti-cancer drugs such as adjuvants. (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapy and / or antibodies; (6) therapeutic agents that modify BCMA and TACI specific responses, including peptide-based vaccines, different types of vaccines (RNA vaccines, DNA vaccines), cell therapy, specific modulators and / or specific inhibitors; and (7) therapeutic agents that have an approach independent of BCMA and TACI targeting therapies, broadly covering immune responses to diseases, including biological and non-biological approaches. In some embodiments, the second agent is an immunostimulatory agent (e.g., a cytokine or a helper T epitope). In some embodiments, the second agent is a helper T epitope. In some embodiments, the helper T epitope is a PADRE sequence or a universal tetanus toxoid helper T (TT Th) epitope. In some embodiments, the second agent is an adjuvant. The adjuvant may be selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand for the Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat-labile toxin (LT), mutant CT (MCT) and mutant E. 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 inhibitor, anti-OX40 antibody, anti-GITR antibody. In some embodiments, the second agent is a checkpoint inhibitor (e.g., anti-LAG3 antibody). In some embodiments, the second agent is an immune modulator, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone.

[0014] In one aspect, the present disclosure also relates to a pharmaceutical composition comprising a peptide as described herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an agent selected from the group consisting of: (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) immune modulators including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone, (4) compounds that enhance BCMA and TACI specific responses, such as adjuvants, (5) therapeutics that augment BCMA and TACI specific responses, including vaccines, cell therapies and / or antibodies, and (6) therapeutics with a therapy independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease. In some examples, the pharmaceutical composition comprises one or more of an adjuvant (e.g., Freund's complete adjuvant, Freund's incomplete adjuvant, alum, ligands for the Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat labile toxin (LT), mutant CT (MCT) and mutant E. coli heat labile toxin (MLT)), an immune agonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpoint inhibitor (e.g., anti-LAG3 antibody) or an immune modulator (e.g., lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone).

[0015] 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, a thalidomide analog, an IMiDS compound and / or an HDAC inhibitor (e.g., ACY241) as a single agent and / or in combination with dexamethasone.

[0016] 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. In particular examples, the CTLs are CTLs obtained by exposure to a peptide comprising or consisting of one or more of SEQ ID NO: 13 or 14. In other examples, the CTLs are CTLs obtained by exposure to a peptide comprising or consisting of any one or more of SEQ ID NO: 15-17. In some examples, the CTLs are memory CD8 + In some instances, the CTLs are memory CD8 + CD45RO + In some instances, the CTLs are non-memory CD8 + In some instances, the CTL is an effector CD8 + In some instances, the CTL is an activated CD8 + In some instances, the CTL is a tetramer-positive CD8 + In some instances, the CTL is a CD8 + In some instances, the CTL is a CD8 + In some instances, CTLs are CD8+ T cells that produce cytokines and / or have upregulated critical cytolytic marker (e.g., CD107, Granzyme, Perforin) expression and / or production. +In some instances, the CTL is a CD8 + It is CTL.

[0017] In one aspect, the present disclosure relates to the nucleic acid that codes for the peptide as described herein.In certain examples, the nucleic acid is RNA (e.g., mRNA).In other embodiments, the nucleic acid is DNA.In some examples, the RNA or DNA is encapsulated in nanocarrier (e.g., polymer such as PLGA or liposome).RNA and DNA can include other regulatory sequences (e.g., start codon, stop codon, polyA tail).

[0018] In one aspect, the present disclosure also relates to a vector comprising a nucleic acid encoding a peptide as described herein. In some embodiments, the sequence of the nucleic acid is operably linked to a promoter, a regulatory element, or an expression control sequence.

[0019] In another aspect, the present disclosure also relates to a cultured cell comprising the vector as described herein. In some embodiments, the cell is a mammalian cell, a human cell, or an immune cell.

[0020] In another aspect, the present 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 poliovirus, a vaccinia virus, a herpes simplex virus or a vesicular stomatitis virus.

[0021] In one aspect, the disclosure relates to a combination of at least two different peptides, the at least two different peptides being selected from the group of peptides having the amino acid sequences set forth in SEQ ID NOs: 13-17. The combination may include peptides having one to four substitutions in one or more of SEQ ID NOs: 13-17. In some examples, the substitutions are at one or more of positions 1, 2, or 9. In some examples, position 1 is unchanged. In some examples, the peptides are 9-30 amino acids in length. In some embodiments, the combination includes at least two, three, four, or all five peptides having the amino acid sequences set forth in SEQ ID NOs: 13-17. In some examples, the combination includes two or more peptides set forth in SEQ ID NOs: 13-17, the two or more peptides having one to fifteen (i.e., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen) amino acids at the N- and / or C-terminus of the amino acid sequence.

[0022] In one aspect, the present disclosure also relates to a pharmaceutical composition comprising a combination of peptides as described herein and a pharma- ceutical acceptable carrier.In some embodiments, the pharmaceutical composition comprises an agent selected from the group consisting of: (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) immune modulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone; (4) compounds that enhance BCMA and TACI specific responses, such as adjuvants; (5) therapeutic agents that increase BCMA and TACI specific responses, including vaccines, cell therapy and / or antibodies; and (6) therapeutic agents that have a therapy-independent approach to target BCMA and TACI, broadly covering immune responses to disease. In some examples, the pharmaceutical composition comprises one or more of an adjuvant (e.g., Freund's complete adjuvant, Freund's incomplete adjuvant, alum, ligands for the Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat labile toxin (LT), mutant CT (MCT) and mutant E. coli heat labile toxin (MLT)), an immune agonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpoint inhibitor (e.g., 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.

[0023] In some embodiments, the pharmaceutical composition further comprises an immune agonist. In some examples, the immune agonist can be an anti-OX40 antibody or an anti-GITR antibody. In some embodiments, the pharmaceutical composition further comprises a checkpoint inhibitor. In one example, the checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the pharmaceutical composition further comprises 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. In one aspect, the present disclosure also provides a composition comprising an isolated dendritic cell, the dendritic cell presenting a peptide sequence on its surface, the peptide sequence comprising at least one major histocompatibility complex (MHC) class I peptide epitope of one or both of the BCMA antigen (SEQ ID NO: 18) and the TACI antigen (SEQ ID NO: 19).

[0024] In some embodiments, the MHC class I peptide epitope is an HLA-A2 peptide epitope.

[0025] In some embodiments, the MHC class I peptide epitope is an HLA-A24 peptide epitope.

[0026] In some embodiments, dendritic cells acquire the peptide sequence in vitro by exposure to a peptide that includes the peptide sequence.

[0027] In some embodiments, the peptide sequence is a synthetic peptide sequence. In some embodiments, the peptide sequence is a sequence set forth in any one of SEQ ID NOs: 1-12 and 13-17. In some examples, the peptide sequence is a sequence set forth in SEQ ID NOs: 13-17, but with one to four amino acid substitutions. In certain cases, the substitutions are at one or more of positions 1, 2, or 9. In one particular embodiment, the peptide sequence is SEQ ID NO: 13. In another embodiment, the peptide is SEQ ID NO: 13, but with one to four amino acid substitutions. In certain cases, the substitutions are at one or more of positions 1, 2, or 9. In another particular embodiment, the peptide sequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ ID NO: 16, but with one to four amino acid substitutions. In certain cases, the substitutions are at one or more of positions 1, 2, or 9.

[0028] In some embodiments, the composition comprises 10 5 ~10 8 Contains dendritic cells between the individuals.

[0029] In some embodiments, the composition further comprises a peptide as set forth in any one of SEQ ID NOs: 1-12 and 13-17. In one particular embodiment, the peptide sequence is SEQ ID NO: 13. In another particular embodiment, the peptide sequence is SEQ ID NO: 16. In some embodiments, the composition further comprises an immunomodulator, 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) immune modulators, including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds, and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone, (4) BCMA and T The composition comprises an agent selected from the group consisting of compounds that enhance ACI-specific responses, (5) therapeutic agents that increase BCMA- and TACI-specific responses, including vaccines, cell therapies and / or antibodies, (6) therapeutic agents that modify 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 an approach independent of BCMA- and TACI-targeting therapies that broadly encompass immune responses to diseases, including biological and non-biological approaches. 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).

[0030] In one aspect, the disclosure relates to a method of inducing an immune response against cells expressing BCMA and / or TACI (e.g., cancer cells) in a human subject in need thereof, 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 with one to four amino acid substitutions. In particular cases, the substitutions are at one or more of positions 1, 2 or 9. In another particular embodiment, the peptide sequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ ID NO: 16 but with one to four amino acid substitutions. In particular cases, the substitutions are at one or more of positions 1, 2 or 9.

[0031] In some embodiments, the subject has a cancer that expresses BCMA and / or TACI, and the immune response is directed against such cancer cells.

[0032] 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.

[0033] In some embodiments, the cancer is a hematological cancer (e.g., multiple myeloma). In some embodiments, the cancer cells are plasma cells (e.g., cancerous plasma cells). In some embodiments, the human subject has refractory multiple myeloma. In some embodiments, the human subject has refractory multiple myeloma that relapses after an allogeneic transplant.

[0034] 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 in a healthy human subject.

[0035] In some embodiments, cancer cells express TACI and the level of TACI in 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 in healthy human subjects.

[0036] In some embodiments, the method further comprises administering to the human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to the human subject a CTL specific for TACI. In certain examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of one or more of SEQ ID NO: 13 or 14. In some cases, the CTL is exposed to a peptide of SEQ ID NO: 13 or 14, but with one to four substitutions. 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 other examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of any one or more of SEQ ID NO: 15 to 17. In some cases, the CTL is exposed to a peptide of SEQ ID NO: 15, 16 or 17, but with one to four substitutions. 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 instances, the CTLs are memory CD8 + In some instances, the CTLs are memory CD8 + CD45RO + In some instances, the CTL is an effector CD8 + In some instances, the CTL is an activated CD8 + In some instances, the CTL is a tetramer-positive CD8 + In some instances, CD8 + The CTL is a CTL that upregulates costimulatory molecule expression when stimulated with a peptide described herein. In some examples, the CTL is a CD8+ The CTLs are CTLs that upregulate checkpoint molecule expression when stimulated with the peptides described herein. In some examples, the CTLs are CD8+ CTLs that produce cytokines and / or have upregulated critical cytolytic marker (e.g., CD107, Granzyme, Perforin) expression and / or production. + In some instances, the CTL is a CD8 + It is CTL.

[0037] In some embodiments, the method further comprises administering to the human subject an immune agonist, hi some embodiments, the immune agonist is anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40 and anti-GITR.

[0038] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or an immune modulator.

[0039] In some embodiments, the method further comprises determining whether an immune response against a BCMA and / or TACI expressing cancer has occurred in the human subject after administering the peptide or composition to the human subject.

[0040] In one aspect, the present disclosure relates to a method of treating a human subject having cancer or a pre-malignant disease, comprising administering to the human subject a peptide as described herein or a composition as described herein.

[0041] In some embodiments, the cancer is a hematological cancer, hi some embodiments, the cancer is multiple myeloma, leukemia, lymphoma, or any B-cell or plasma cell malignancy.

[0042] In some embodiments, the pre-malignant disease is monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma.

[0043] In some embodiments, the method further comprises detecting that one or more cancer cells express or overexpress BCMA and / or TACI in the human subject.

[0044] In some embodiments, a human subject has one or more cancer cells that overexpress BCMA and / or TACI, and the levels of BCMA and / or TACI in the cancer cells are at least 20% greater than in normal cells (e.g., plasma cells in a healthy subject).

[0045] In some embodiments, the subject has one or more cancer cells that express an MHC molecule.

[0046] In some embodiments, the method further comprises administering to the human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to the human subject a CTL specific for TACI. In certain examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of one or more of SEQ ID NO: 13 or 14. In some cases, the CTL is exposed to a peptide of SEQ ID NO: 13 or 14, but with one to four substitutions. 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 other examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of any one or more of SEQ ID NO: 15 to 17. In some cases, the CTL is exposed to a peptide of SEQ ID NO: 15, 16 or 17, but with one to four substitutions. 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 examples, the CTL is a memory CD8+ CTL. In some examples, the CTL is a memory CD8+CD45RO+ CTL. In some examples, the CTL is an effector CD8+ CTL. In some examples, the CTL is an activated CD8+ CTL. In some examples, the CTL is a tetramer positive CD8+ CTL. In some examples, the CTL is a CD8+ CTL that has upregulated costimulatory molecule expression. In some examples, the CTL is a CD8+ CTL that has upregulated checkpoint molecule expression.

[0047] In some embodiments, the method comprises administering to the human subject an agent selected from the group consisting of: (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) immune modulators including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241), (4) compounds that enhance BCMA and TACI specific responses, such as adjuvants, (5) therapeutics that increase BCMA and TACI specific responses, including vaccines, cell therapy and / or antibodies, and (6) therapeutics that have a therapy-independent approach to target BCMA and TACI to broadly encompass immune responses to disease. In some embodiments, the method further comprises administering to the human subject an immune agonist. In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody.

[0048] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or an immune modulator.

[0049] In some embodiments, the method further comprises administering chemotherapy or radiation therapy to the human subject.

[0050] In one aspect, the disclosure relates to a method of generating and / or expanding BCMA-specific cytotoxic T cells, 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 9-30 amino acids in length.

[0051] In some embodiments, the cytotoxic T cells are memory cytotoxic T cells. In some embodiments, the cytotoxic T cells are effector cytotoxic T cells. In some examples, the CD8+ CTL is a CTL that upregulates costimulatory molecule expression when stimulated with the peptides described herein. In some examples, the CD8+ CTL is a CTL that upregulates checkpoint molecule expression when stimulated with the peptides described herein. In some examples, the CTL is a CD8+ CTL that produces cytokines and / or has upregulated critical cytolytic marker (e.g., CD107, Granzyme, perforin) expression and / or production. In some examples, the CTL is a CD8+ CTL that has activity against tumors or other targets.

[0052] In some embodiments, the antigen presenting cells are dendritic cells (DCs). In one particular embodiment, the peptide comprises or consists of SEQ ID NO: 13. In another aspect, the disclosure relates to a method of generating TACI-specific cytotoxic T cells, 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 NOs: 15-17. In some cases, the CTLs are exposed to a peptide of SEQ ID NO: 15, 16 or 17, but with one to four substitutions. In some cases, the substitutions are at one or more of positions 1, 2 or 9. In some cases, the peptide is 9-30 amino acids in length.

[0053] In one particular embodiment, the peptide comprises or consists of SEQ ID NO:16.

[0054] In one aspect, the disclosure also relates to a method of killing a target cell, 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.

[0055] 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: (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) immune modulators including lenalidomide, pomalidomide, thalidomide analogs, IMiDS compounds and / or HDAC inhibitors (e.g., ACY241) as single agents and / or in combination with dexamethasone, (4) compounds that enhance BCMA- and TACI-specific responses, such as adjuvants, (5) therapeutics that augment BCMA- and TACI-specific responses, including vaccines, cell therapy and / or antibodies, and (6) therapeutics with a therapy-independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease.

[0056] In some embodiments, the method further comprises contacting the one or more BCMA-specific cytotoxic T cells with an immune agonist.

[0057] In some embodiments, the method further comprises administering a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13 or 14. In some cases, the peptide can 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- and / or C-terminus of the amino acid sequence of SEQ ID NO: 13 or 14.

[0058] 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.

[0059] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0060] In another aspect, the disclosure relates to a method of killing a target cell, 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.

[0061] In some embodiments, the method further comprises administering a peptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 15-17. In some cases, the peptide can have 1-15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids added to the N- and / or C-terminus of the amino acid sequence of SEQ ID NOs: 15-17.

[0062] 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: (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) immune modulators including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241), (4) compounds that enhance BCMA- and TACI-specific responses, such as adjuvants, (5) therapeutics that augment BCMA- and TACI-specific responses, including vaccines, cell therapy and / or antibodies, and (6) therapeutics that have a therapy-independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease.

[0063] In some embodiments, the method further comprises contacting one or more TACI-specific cytotoxic T cells with an immune agonist.

[0064] In some embodiments, the immune agonist is an OX40 agonist or a GITR agonist.

[0065] In some embodiments, the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0066] In one aspect, the disclosure relates to a method of treating a human subject having a BCMA- or TACI-expressing disease or cancer, comprising administering to the human subject a plurality of BCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells.

[0067] In some embodiments, the method further comprises administering to the human subject a peptide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 13-17. In some examples, the peptide has a sequence set forth in one of SEQ ID NOs: 13-17 except with one to four substitutions. In some cases, the substitutions may be at one or more of positions 1, 2 or 9. In some examples, the peptide is 9-30 amino acids in length.

[0068] In some embodiments, the method further comprises administering to the human subject an agent selected from the group consisting of: (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) immune modulators including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241), (4) compounds that enhance BCMA- and TACI-specific responses, such as adjuvants, (5) therapeutics that augment BCMA- and TACI-specific responses, including vaccines, cell therapies and / or antibodies, and (6) therapeutics with a therapy-independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease.

[0069] In some embodiments, the method further comprises administering to the subject an immune agonist. 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.

[0070] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0071] 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.

[0072] In some embodiments, the cancer expresses BCMA and / or TACI. In some embodiments, the human subject has multiple myeloma.

[0073] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) obtaining bone marrow-derived mononuclear cells from a subject; (b) culturing the mononuclear cells in vitro under conditions that result in the mononuclear cells adhering to the culture vessel; (c) selecting adherent mononuclear cells; (d) culturing the adherent mononuclear cells in the presence of one or more cytokines under conditions in which the cells differentiate into antigen-presenting cells; (e) contacting an antigen-presenting cell with a peptide as described herein (e.g., SEQ ID NOs: 13-17), thereby producing an antigen-presenting cell that presents the peptide on a major histocompatibility complex (MHC) molecule; The present invention relates to a process comprising:

[0074] In some embodiments, the major histocompatibility complex molecule is an MHC class I molecule.

[0075] In some embodiments, the one or more cytokines comprise granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4).

[0076] In some embodiments, the one or more cytokines comprises tumor necrosis factor-α (TNF-α).

[0077] In some embodiments, the bone marrow-derived cells are obtained from a subject diagnosed with multiple myeloma.

[0078] In one aspect, the disclosure provides a method of identifying a T cell antigen receptor sequence for BCMA, comprising: (a) generating and / or expanding BCMA-specific cytotoxic T cells by a method as described herein; (b) Determine the T cell antigen receptor sequence of BCMA in BCMA-specific cytotoxic T cells. The present invention relates to a method comprising the steps of:

[0079] In another aspect, the disclosure relates to a method for treating a human subject having a BCMA-expressing disease or cancer, the method comprising administering to the human subject a composition comprising chimeric antigen receptor T cells (CAR-T cells), where the CAR-T cells express a chimeric antigen receptor, and the chimeric antigen receptor binds to BCMA.

[0080] In some embodiments, the cancer expresses BCMA. In some embodiments, the human subject has multiple myeloma.

[0081] In some embodiments, the method further includes administering to the human subject a peptide comprising or consisting of a sequence set forth in either one of SEQ ID NOs: 13 or 14. In some cases, the peptide has the sequence set forth in SEQ ID NO: 13 or 14 except with one to four amino acid substitutions. In some examples, the substitutions are at one or more of positions 1, 2 or 9. In some examples, 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: (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) immune modulators including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241), (4) compounds that enhance BCMA- and TACI-specific responses, such as adjuvants, (5) therapeutics that augment BCMA- and TACI-specific responses, including vaccines, cell therapies and / or antibodies, and (6) therapeutics with a therapy-independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease.

[0082] In some embodiments, the method further comprises administering to the subject an immune agonist. 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.

[0083] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0084] In one aspect, the disclosure provides a method for identifying a T cell antigen receptor sequence of TACI, comprising: (a) generating and / or expanding TACI-specific cytotoxic T cells by a method as described herein; (b) Determine the T cell antigen receptor sequence of TACI in TACI-specific cytotoxic T cells. The present invention relates to a method comprising the steps of:

[0085] In another aspect, the disclosure relates to a method for treating a human subject having a disease or cancer that expresses TACI, the method comprising administering to the human subject a composition comprising chimeric antigen receptor T cells (CAR-T cells), where the CAR-T cells express a chimeric antigen receptor, and the chimeric antigen receptor binds to TACI.

[0086] In some embodiments, the cancer expresses TACI. In some embodiments, the human subject has multiple myeloma.

[0087] In some embodiments, the method further comprises administering to the human subject a peptide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 15-17.

[0088] In some embodiments, the method further comprises administering to the human subject an agent selected from the group consisting of: (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) immune modulators including lenalidomide, pomalidomide, HDAC inhibitors (e.g., ACY241), (4) compounds that enhance BCMA- and TACI-specific responses, such as adjuvants, (5) therapeutics that augment BCMA- and TACI-specific responses, including vaccines, cell therapies and / or antibodies, and (6) therapeutics with a therapy-independent approach targeting BCMA and TACI to broadly encompass the immune response to the disease.

[0089] In some embodiments, the method further comprises administering to the subject an immune agonist. 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.

[0090] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide.

[0091] 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 or consists of the sequence of SEQ ID NO: 13. In particular examples, the peptide has a sequence of SEQ ID NO: 13 but with one to four amino acid substitutions. In some cases, the substitutions are at one or more of positions 1, 2 or 9. The peptide can be 9 to 30 amino acids long. In other embodiments, the sequence comprises or consists of the sequence of SEQ ID NO: 16. In particular examples, the peptide has a sequence of SEQ ID NO: 16 but with one to four amino acid substitutions. In some cases, the substitutions are at one or more of positions 1, 2 or 9. The peptide can be 9 to 30 amino acids long.

[0092] In some embodiments, the peptide is encapsulated in a nanoparticle, hi some embodiments, the nanoparticle is a liposome.

[0093] 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) copolymer.

[0094] 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. In some embodiments, the amino acid sequence is SEQ ID NO: 16. In some embodiments, the amino acid sequence is SEQ ID NO: 17. In particular examples, the peptide has a sequence that is any one of SEQ ID NOs: 13-17, but with one to four amino acid substitutions. In some cases, the substitutions are at one or more of positions 1, 2 or 9. The peptide can be 9 to 30 amino acids in length.

[0095] In some embodiments, the nanoparticles comprise an adjuvant. In some embodiments, the nanoparticles comprise a Toll-like receptor agonist (e.g., R848 or an unmethylated CpG oligodeoxynucleotide).

[0096] In one aspect, the present disclosure provides a method for treating a human subject having cancer. The method comprises administering to the human subject a composition (e.g., nanoparticles) as described herein. In some embodiments, the human subject has multiple myeloma.

[0097] In some embodiments, the cancer expresses BCMA. In other embodiments, the cancer expresses TACI.

[0098] In some embodiments, the method further comprises administering to the human subject a peptide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 15-17. In some embodiments, the peptide has a sequence set forth in one of SEQ ID NOs: 15-17 except for having one to four amino acid substitutions. The substitutions can be at one or more of positions 1, 2 or 9. The peptide can be 9 to 30 amino acids in length.

[0099] In some embodiments, the method further comprises administering to the human subject a CTL specific for BCMA. In some embodiments, the method further comprises administering to the human subject a CTL specific for TACI. In certain examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of one or more of SEQ ID NO: 13 or 14. In other examples, the CTL is a CTL obtained by exposure to a peptide comprising or consisting of any one or more of SEQ ID NO: 15-17. In some examples, the CTL is a memory CD8+ CTL. In some examples, the CTL is a memory CD8+ CD45RO+ CTL. In some examples, the CTL is an effector CD8+ CTL. In some examples, the CTL is an activated CD8+ CTL. In some examples, the CTL is a tetramer-positive CD8+ CTL. In some examples, the CTL is a CD8+ CTL that has upregulated costimulatory molecule expression. In some examples, the CTL is a CD8+ CTL that has upregulated checkpoint molecule expression. In some embodiments, the method further comprises administering to the subject an immune agonist. 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.

[0100] In some embodiments, the method further comprises administering to the human subject a checkpoint inhibitor (e.g., an anti-LAG3 antibody). In certain embodiments, the method further comprises administering to the human subject lenalidomide. In some embodiments, the method further comprises administering to the human subject one or more of an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), or lenalidomide. The present invention provides, for example, the following items. (Item 1) A peptide comprising an amino acid sequence that is identical to the amino acid sequence shown in any one of SEQ ID NOs: 13 to 17 or that differs by 1 to 4 amino acid residues, wherein the amino acid at position 1 of SEQ ID NOs: 13 to 17 is not changed. (Item 2) 2. The peptide according to item 1, wherein the amino acid sequence is SEQ ID NO: 13. (Item 3) 2. The peptide according to item 1, wherein the amino acid sequence is SEQ ID NO: 14. (Item 4) 2. The peptide according to item 1, wherein the amino acid sequence is SEQ ID NO: 15. (Item 5) 2. The peptide according to item 1, wherein the amino acid sequence is SEQ ID NO: 16. (Item 6) 2. The peptide according to item 1, wherein the amino acid sequence is SEQ ID NO: 17. (Item 7) A peptide comprising a first amino acid sequence having an amino acid sequence that is at least 60% identical to any one of SEQ ID NOs: 1 to 17, and a second amino acid sequence that is heterologous to the first amino acid sequence. (Item 8) 8. The peptide according to any one of items 1 to 7, which binds to a major histocompatibility complex (MHC) molecule. (Item 9) 8. The peptide according to any one of items 1 to 7, which is recognised by an antigen-specific T cell receptor on a T cell in association with an MHC molecule. (Item 10) 10. The peptide according to item 8 or 9, wherein the MHC molecule is an MHC class I molecule. (Item 11) 10. The peptide according to item 8 or 9, wherein the MHC molecule is an MHC class II molecule. (Item 12) 10. The peptide according to item 8 or 9, wherein the MHC molecule is an HLA-A2 molecule or an HLA-A24 molecule. (Item 13) 13. A composition comprising the peptide according to any one of items 1 to 12 and a second agent. (Item 14) 14. The composition of claim 13, wherein the second agent is an immunostimulant. (Item 15) 14. The composition of claim 13, wherein the second agent is a T helper epitope. (Item 16) 16. The composition of claim 15, wherein the helper T epitope is a PADRE sequence or a universal tetanus toxoid helper T (TT Th) epitope. (Item 17) 14. The composition of claim 13, wherein the second agent is an adjuvant. (Item 18) 18. The composition according to item 17, wherein the adjuvant is selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand of the Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat-labile toxin (LT), mutant CT (MCT) and mutant E. coli heat-labile toxin (MLT). (Item 19) The composition of item 13, wherein the second agent is a toll-like receptor-3 ligand (e.g., poly-ICLC), interferon alpha (IFNα), interferon gamma (IFNγ), an anti-OX40 antibody, an anti-GITR antibody, or a granulocyte-macrophage colony-stimulating factor (GM-CSF). (Item 20) 13. A pharmaceutical composition comprising a peptide according to any one of items 1 to 12 and a pharma- ceutically acceptable carrier. (Item 21) 13. A nucleic acid encoding the peptide according to any one of items 1 to 12. (Item 22) 13. A vector comprising a nucleic acid encoding the peptide according to any one of items 1 to 12. (Item 23) 23. The vector of item 22, wherein the nucleic acid sequence is operably linked to a promoter, regulatory element or expression control sequence. (Item 24) 24. A cultured cell comprising the vector according to item 23. (Item 25) 25. The cultured cell according to item 24, which is a mammalian cell. (Item 26) 25. The cultured cell according to item 24, which is a human cell. (Item 27) 25. The cultured cell according to item 24, which is an immune cell. (Item 28) 13. A virus comprising a nucleic acid encoding the peptide according to any one of items 1 to 12. (Item 29) 29. The virus according to item 28, which is a lentivirus, an adenovirus or an adeno-associated virus. (Item 30) A combination of at least two different peptides selected from the group of peptides having the amino acid sequences shown in SEQ ID NOs:13 to 17. (Item 31) 31. The combination according to item 30, comprising at least three, four, or all five peptides having the amino acid sequences shown in SEQ ID NOs: 13 to 17. (Item 32) A combination according to item 30 or 31, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising: (Item 33) 33. The pharmaceutical composition according to item 32, further comprising an immune agonist. (Item 34) 34. The pharmaceutical composition of claim 33, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 35) A composition comprising an isolated dendritic cell, the dendritic cell presenting a peptide sequence on its surface, the peptide sequence comprising at least one major histocompatibility complex (MHC) class I peptide epitope of one or both of a BCMA antigen (SEQ ID NO: 18) and a TACI antigen (SEQ ID NO: 19). (Item 36) 36. The composition of claim 35, wherein the MHC class I peptide epitope is an HLA-A2 peptide epitope. (Item 37) 36. The composition of claim 35, wherein the dendritic cells acquire the peptide sequence in vitro by exposure to a synthetic peptide comprising the peptide sequence. (Item 38) 36. The composition according to item 35, wherein the peptide sequence is a synthetic peptide sequence. (Item 39) 36. The composition according to item 35, wherein the peptide sequence is shown in any one of SEQ ID NOs: 1 to 12 and 13 to 17. (Item 40) 10 5 ~10 8 36. The composition of claim 35, comprising dendritic cells between 1 and 2. (Item 41) 21. A method for inducing an immune response against cancer cells expressing BCMA and / or TACI in a human subject in need thereof, comprising administering to the human subject a peptide described in any one of items 1 to 12 or a composition described in any one of items 13 to 20. (Item 42) 42. The method of claim 41, wherein the subject has cancer and the immune response is directed against cancer cells. (Item 43) 43. The method of claim 42, wherein the cancer is a blood cancer. (Item 44) 43. The method of claim 42, wherein the cancer is multiple myeloma. (Item 45) 43. The method of claim 42, wherein the cancer cells are cancerous plasma cells. (Item 46) 43. The method of claim 42, wherein the cancer cells express BCMA and the level of BCMA in the cancer cells is at least 20% greater than in plasma cells in a healthy human subject. (Item 47) 43. The method of claim 42, wherein the cancer cells express TACI and the level of TACI in the cancer cells is at least 20% greater than in plasma cells in a healthy human subject. (Item 48) 42. The method of claim 41, further comprising administering to the human subject an immune agonist. (Item 49) 49. The method of claim 48, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 50) 42. The method of claim 41, further comprising determining whether an immune response against a BCMA and / or TACI expressing cancer has occurred in the human subject after administering the peptide or the composition to the human subject. (Item 51) A method for treating a human subject having cancer or a pre-malignant disease, comprising administering to said human subject a peptide according to any one of items 1 to 12 or a composition according to any one of items 13 to 20. (Item 52) 52. The method of claim 51, wherein the cancer is a blood cancer. (Item 53) 52. The method of claim 51, wherein the cancer is multiple myeloma, leukemia or lymphoma. (Item 54) 52. The method of item 51, wherein the pre-malignant disease is monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma. (Item 55) 52. The method of claim 51, further comprising detecting that one or more cancer cells express or overexpress BCMA and / or TACI in the human subject. (Item 56) 52. The method of claim 51, wherein the human subject has one or more cancer cells that overexpress BCMA and / or TACI, and the levels of BCMA and / or TACI in the cancer cells are at least 20% greater than in normal cells. (Item 57) 52. The method of claim 51, wherein the subject has one or more cancer cells that express an MHC molecule. (Item 58) 52. The method of claim 51, further comprising administering to the human subject an immune agonist. (Item 59) 59. The method of claim 58, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 60) 52. The method of claim 51, further comprising administering chemotherapy or radiation therapy to the human subject. (Item 61) 1. A method of generating and / or expanding BCMA-specific cytotoxic T cells, 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. A method comprising: (Item 62) 62. The method of claim 61, wherein the cytotoxic T cells are memory cytotoxic T cells. (Item 63) 62. The method of claim 61, wherein the cytotoxic T cells are effector cytotoxic T cells. (Item 64) 62. The method of claim 61, wherein the antigen-presenting cell is a dendritic cell. (Item 65) A method for producing 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 NOs: 15 to 17. (Item 66) 66. The method of claim 65, wherein the cytotoxic T cells are memory cytotoxic T cells. (Item 67) 66. The method of claim 65, wherein the cytotoxic T cells are effector cytotoxic T cells. (Item 68) 66. The method of claim 65, wherein the antigen-presenting cell is a dendritic cell. (Item 69) 1. A method for killing a target cell, comprising: The method comprises contacting said target cell with one or more BCMA-specific cytotoxic T cells, wherein said target cell expresses or overexpresses BCMA and expresses HLA-A. (Item 70) 70. The method of claim 69, further comprising contacting the one or more BCMA-specific cytotoxic T cells with an immune agonist. (Item 71) 70. The method of claim 69, wherein the immune agonist is an OX40 agonist or a GITR agonist. (Item 72) 71. The method of claim 70, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 73) 1. A method for killing a target cell, comprising: A method comprising 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. (Item 74) 74. The method of claim 73, further comprising contacting the one or more TACI-specific cytotoxic T cells with an immune agonist. (Item 75) 75. The method of claim 74, wherein the immune agonist is an OX40 agonist or a GITR agonist. (Item 76) 75. The method of claim 74, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 77) 1. A method of treating a human subject having cancer, comprising administering to the human subject a plurality of BCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells. (Item 78) 78. The method of claim 77, further comprising administering to the subject an immune agonist. (Item 79) 79. The method of claim 78, wherein the immune agonist is an OX40 agonist or a GITR agonist. (Item 80) 79. The method of claim 78, wherein the immune agonist is an anti-OX40 antibody or an anti-GITR antibody. (Item 81) 78. The method of claim 77, wherein the cytotoxic T cells are derived from cells of the human subject. (Item 82) 78. The method of claim 77, wherein the cytotoxic T cells are derived from induced pluripotent stem cells. (Item 83) (a) obtaining bone marrow-derived mononuclear cells from a subject; (b) culturing the mononuclear cells in vitro under conditions that allow the mononuclear cells to adhere to the culture vessel; (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; (e) contacting said antigen-presenting cells with a peptide according to any one of items 1 to 12, thereby producing antigen-presenting cells that present said peptide on a major histocompatibility complex (MHC) molecule; A method comprising: (Item 84) 84. The method of claim 83, wherein the major histocompatibility complex molecule is an MHC class I molecule. (Item 85) 84. The method of claim 83, wherein the one or more cytokines comprise granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). (Item 86) 84. The method of item 83, wherein the one or more cytokines comprises tumor necrosis factor-α (TNF-α). (Item 87) 84. The method of claim 83, wherein the bone marrow-derived cells are obtained from a subject diagnosed with multiple myeloma. (Item 88) 1. A method for identifying a T cell antigen receptor sequence for BCMA, comprising: (a) generating and / or expanding BCMA-specific cytotoxic T cells by the method described in item 61; (b) determining the T cell antigen receptor sequence of BCMA in the BCMA-specific cytotoxic T cells; A method comprising: (Item 89) 1. A method for treating a human subject having cancer, comprising: 20. The method of claim 19, further comprising administering to the human subject a composition comprising chimeric antigen receptor T cells (CAR-T cells), wherein the CAR-T cells express a chimeric antigen receptor, and the chimeric antigen receptor binds to BCMA. (Item 90) 1. A method for identifying a T cell antigen receptor sequence of TACI, comprising: (a) generating and / or expanding TACI-specific cytotoxic T cells by the method described in item 65; (b) determining the T cell antigen receptor sequence of TACI in the TACI-specific cytotoxic T cells; The method includes: (Item 91) 1. A method for treating a human subject having cancer, comprising: The method includes administering to the human subject a composition comprising chimeric antigen receptor T cells (CAR-T cells), wherein the CAR-T cells express a chimeric antigen receptor, and the chimeric antigen receptor binds to TACI. (Item 92) A composition comprising nanoparticles and a peptide comprising an amino acid sequence that is at least 60% identical to any one of SEQ ID NOs: 1-17. (Item 93) 93. The composition of claim 92, wherein the peptide is encapsulated in the nanoparticles. (Item 94) 93. The composition of claim 92, wherein the nanoparticles comprise a biodegradable polymer. (Item 95) 93. The composition of claim 92, wherein the nanoparticles comprise poly(D,L-lactide-co-glycolide) (PLGA). (Item 96) 93. The composition of claim 92, wherein the nanoparticles comprise poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) copolymer. (Item 97) 93. The composition of claim 92, wherein the nanoparticle is a liposome. (Item 98) 98. The composition of any one of items 92 to 97, wherein the amino acid sequence is SEQ ID NO: 13. (Item 99) 98. The composition of any one of items 92 to 97, wherein the amino acid sequence is SEQ ID NO: 14. (Item 100) 98. The composition of any one of items 92 to 97, wherein the amino acid sequence is SEQ ID NO: 15. (Item 101) 98. The composition of any one of items 92 to 97, wherein the amino acid sequence is SEQ ID NO: 16. (Item 102) 98. The composition of any one of items 92 to 97, wherein the amino acid sequence is SEQ ID NO: 17. (Item 103) 103. The composition of any one of items 92 to 102, wherein the nanoparticles comprise an adjuvant. (Item 104) 104. The composition of any one of items 95 to 103, wherein the nanoparticles comprise a Toll-like receptor agonist (e.g., R848 or a non-methylated CpG oligodeoxynucleotide). (Item 105) 105. The composition of any one of items 92 to 104, further comprising an adjuvant, an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody), a checkpoint inhibitor (e.g., an anti-LAG3 antibody), lenalidomide, or any combination thereof. (Item 106) 106. A method for treating a human subject having cancer, comprising administering to said human subject a composition according to any one of items 95 to 105. (Item 107) 107. The method of claim 106, wherein the human subject has multiple myeloma. (Item 108) 107. The method of claim 106, wherein the human subject has a BCMA and / or TACI expressing cancer. (Item 109) The method of any one of items 106 to 108, further comprising administering to the human subject one or more of a peptide selected from the group of SEQ ID NOs: 13 to 17, a BCMA-specific CTL, or a TACI-specific CTL. (Item 110) 109. The method of any one of items 106 to 108, further comprising administering to the human subject a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13 or 16. (Item 111) 109. The method of any one of items 106 to 108, further comprising administering to the human subject BCMA-specific CTLs generated by stimulation of CTLs with a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. (Item 112) The method of any one of items 106 to 108, further comprising administering to the human subject a TACI-specific CTL generated by stimulating the CTL with a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods and examples are merely illustrative 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 case of conflict, the present specification, including definitions, will control.

[0102] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0103] [Figure 1A-C] 1A-1I show BCMA expression in multiple myeloma cell lines. [Fig. 1D-F] 1A-1I show BCMA expression in multiple myeloma cell lines. [Figure 1G-I] 1A-1I show BCMA expression in multiple myeloma cell lines.

[0104] [Diagram 2] FIG. 2 shows the binding affinity of native BCMA peptides to HLA-A2.

[0105] [Diagram 3] FIG. 3 shows the binding affinity of native TACI peptides to HLA-A2.

[0106] [Figure 4]FIG. 4 shows the binding affinity of BCMA peptides to HLA-A2: native versus heteroclitic peptides.

[0107] [Diagram 5] FIG. 5 shows the binding affinity of TACI peptides to HLA-A2: native versus heteroclitic peptides.

[0108] [Figure 6] FIG. 6 shows HLA-A2 stability of BCMA #4 and #5 peptides: native vs. heteroclitic peptides (50ug / ml).

[0109] [Figure 7] FIG. 7 shows HLA-A2 stability of TACI #1, #3 and #4 peptides: native vs. heteroclitic peptides (50 ug / ml).

[0110] [Figure 8] 8A-8C show CD8+ cytotoxic T cells (CTLs) expanded using heteroclitic BCMA #4 peptide stimulation.

[0111] [Figure 9] Figures 9A-9C show naive CTLs depleted using heteroclitic BCMA #4 peptide stimulation.

[0112] [Figure 10] 10A-10C show memory CTL expanded using heteroclitic BCMA #4 peptide stimulation.

[0113] [Figure 11] Figures 11A-11C show CM versus effector cell kinetics with heteroclitic BCMA #4 peptide stimulation.

[0114] [Figure 12]FIG. 12 shows induction of memory CD8+ CTL by heteroclitic BCMA #4 peptide.

[0115] [Figure 13] FIG. 13 shows the induction of memory CD8+ CTLs by heteroclitic TACI no.3 peptide.

[0116] [Figure 14] FIG. 14 shows the anti-tumor activity of heteroclitic BCMA peptide 4-CTLs (N=5).

[0117] [Figure 15] FIG. 15 shows the antitumor activity of heteroclitic TACI peptide 3-CTLs (n=4).

[0118] [Figure 16] FIG. 16 shows HLA-A2-specific proliferation of heteroclitic BCMA peptide 4-CTL.

[0119] [Figure 17] FIG. 17 shows enhanced α-tumor activity by central memory cells of BCMA-specific CTLs treated with α-OX40 or α-GITR.

[0120] [Figure 18A] Figures 18A-18C show upregulation of critical T cell markers in BCMA peptide-specific CTL stimulated with heteroclitic BCMA peptides. [Figure 18B] Figures 18A-18C show upregulation of critical T cell markers in BCMA peptide-specific CTL stimulated with heteroclitic BCMA peptides. [Figure 18C] Figures 18A-18C show upregulation of critical T cell markers in BCMA peptide-specific CTL stimulated with heteroclitic BCMA peptides.

[0121] [Figure 19A-E] Figures 19A-19F show HLA-A2-restricted and antigen-specific immune responses by heteroclitic BCMA72-80-specific CTL against HLA-A2+ MM cell lines. [Figure 19F] Figures 19A-19F show HLA-A2-restricted and antigen-specific immune responses by heteroclitic BCMA72-80-specific CTL against HLA-A2+ MM cell lines.

[0122] [Figure 20A] Figures 20A-20H show the anti-tumor activity of heteroclitic BCMA54-62-specific CTLs or heteroclitic BCMA72-80-specific CTLs against patient MM cells. [Figure 20B-1] Figures 20A-20H show the anti-tumor activity of heteroclitic BCMA54-62-specific CTLs or heteroclitic BCMA72-80-specific CTLs against patient MM cells. [Figure 20B-2] Figures 20A-20H show the anti-tumor activity of heteroclitic BCMA54-62-specific CTLs or heteroclitic BCMA72-80-specific CTLs against patient MM cells. [Fig. 20C-H] Figures 20A-20H show the anti-tumor activity of heteroclitic BCMA54-62-specific CTLs or heteroclitic BCMA72-80-specific CTLs against patient MM cells.

[0123] [Figure 21A] Figures 21A-21C show BCMA72-80 specific tetramer+ CTLs displaying distinct phenotypes and high levels of anti-tumor activity against MM cells. [Figure 21B] Figures 21A-21C show BCMA72-80 specific tetramer+ CTLs displaying distinct phenotypes and high levels of anti-tumor activity against MM cells. [Figure 21C] Figures 21A-21C show BCMA72-80 specific tetramer+ CTLs displaying distinct phenotypes and high levels of anti-tumor activity against MM cells.

[0124] [Figure 22A] Figures 22A-22E. Differentiation of BCMA-specific CTL memory CD8+ T cells upon stimulation with heteroclitic BCMA72-80 peptide. [Fig. 22B-C] Figures 22A-22E. Differentiation of BCMA-specific CTL memory CD8+ T cells upon stimulation with heteroclitic BCMA72-80 peptide. [Fig. 22D-E] Figures 22A-22E. Differentiation of BCMA-specific CTL memory CD8+ T cells upon stimulation with heteroclitic BCMA72-80 peptide.

[0125] [Figure 23A] Figures 23A-23C characterize the high anti-tumor activity by BCMA-specific memory CTLs (Figure 23A) and the highest levels by central memory CTLs (Figures 23B, 23C). [Figure 23B] Figures 23A-23C characterize the high anti-tumor activity by BCMA-specific memory CTLs (Figure 23A) and the highest levels by central memory CTLs (Figures 23B, 23C). [Figure 23C] Figures 23A-23C characterize the high anti-tumor activity by BCMA-specific memory CTLs (Figure 23A) and the highest levels by central memory CTLs (Figures 23B, 23C).

[0126] [Figure 24A] FIG. 24A shows the results of BCMA peptide-specific CTLs co-cultured (7 days) with 25 U266 cells.

[0127] [Figure 24B] Figures 24B-24C. Enhanced anti-myeloma activity of memory CD8+ T cells of heteroclitic BCMA72-80 CTL [generated from one HLA-A2+ individual] treated with anti-LAG3 or anti-OX40. [Figure 24C]Figures 24B-24C. Enhanced anti-myeloma activity of memory CD8+ T cells of heteroclitic BCMA72-80 CTL [generated from one HLA-A2+ individual] treated with anti-LAG3 or anti-OX40.

[0128] [Figure 24D] FIG. 24D. Enhanced antitumor activity of heteroclitic BCMA72-80 CTLs [generated from HLA-A2+ donor 1, donor 2 or donor 3] treated with anti-OX40 against myeloma cells in an HLA-A2 restricted manner.

[0129] [Diagram 25] Figures 25A-25B are percentages of CD3+CD8+ T cells following peptide stimulation with heteroclitic BCMA72-80.

[0130] [Figure 26] Figures 26A-26B are percentages of CD3+CD4+ T cells following peptide stimulation with heteroclitic BCMA72-80.

[0131] [Figure 27] Figures 27A-27C show high BCMA expression in H929, MMIS, U266 and OPM1 cell lines, but not in a breast cancer cell line (MDA-MB231).

[0132] [Figure 28] Figures 28A-28B show the percentage of CD3+CD8+ T cells expressing PD-1 and LAG-3 following peptide stimulation with heteroclitic BCMA72-80.

[0133] [Figure 29A] 29A-29C show differentiation of naive cells into memory CTL upon stimulation with the heteroclitic TACI154-162 peptide. [Figure 29B]29A-29C show differentiation of naive cells into memory CTL upon stimulation with the heteroclitic TACI154-162 peptide. [Figure 29C] 29A-29C show differentiation of naive cells into memory CTL upon stimulation with the heteroclitic TACI154-162 peptide.

[0134] [Figure 30A] 30A-30F show the antitumor activity of heteroclitic TACI154-162-specific CTLs against HLA-A2+ multiple myeloma cells (McCAR). [Figure 30B] 30A-30F show the antitumor activity of heteroclitic TACI154-162-specific CTLs against HLA-A2+ multiple myeloma cells (McCAR). [Figure 30C-F] 30A-30F show the antitumor activity of heteroclitic TACI154-162-specific CTLs against HLA-A2+ multiple myeloma cells (McCAR).

[0135] [Figure 31A] 31A-31E show the antitumor activity of heteroclitic TACI154-162-specific CTLs against HLA-A2+ multiple myeloma cells. [Fig. 31B-E] 31A-31E show the antitumor activity of heteroclitic TACI154-162-specific CTLs against HLA-A2+ multiple myeloma cells.

[0136] [Fig. 31F] FIG. 31F shows induction of peptide-specific tetramer+ CTL and antitumor activity and proliferation by heteroclitic TACI154-162 peptide in HLA-A2+ multiple myeloma cells.

[0137] [Figure 32A] FIG. 32A shows the morphology of PLGA nanoparticles loaded with BCMA peptides.

[0138] [Figure 32B] FIG. 32B shows the morphology of liposomal nanoparticles loaded with BCMA peptides.

[0139] [Figure 32C] Figure 32C shows BCMA peptide quantification.

[0140] [Figure 33A] FIG. 33A shows higher loading efficiency of BCMA peptides in dendritic cells upon encapsulation in nanoparticles (PLGA, liposomes) in a time-dependent manner.

[0141] [Figure 33B] FIG. 33B shows higher loading efficiency of BCMA peptide-FITC in dendritic cells upon encapsulation in nanoparticles (PLGA, liposomes) in a time-dependent manner.

[0142] [Figure 33C] FIG. 33C shows higher PLGA / peptide uptake by dendritic cells.

[0143] [Figure 33D] FIG. 33D shows higher uptake of BCMA peptide-FITC by dendritic cells upon encapsulation in PLGA in a time-dependent manner.

[0144] [Figure 33E] FIG. 33E shows higher uptake of BCMA peptide-FITC by T2 cells upon encapsulation in PLGA in a time-dependent manner.

[0145] [Figure 34A] FIG. 34A shows the highest anti-MM activity by BCMA-CTL generated with PLGA / BCMA peptide against MM cell lines in an HLA-A2 restricted manner.

[0146] [Figure 34B]FIG. 34B shows highest IFN-γ production by BCMA-CTL generated with PLGA / BCMA peptide against MM cell lines in an HLA-A2 restricted manner.

[0147] [Figure 34C] FIG. 34C shows highest IL-2 production by BCMA-CTL generated with PLGA / BCMA peptide against MM cell lines in an HLA-A2 restricted manner.

[0148] [Fig. 34D] FIG. 34D shows highest TNF-α production by BCMA-CTL generated with PLGA / BCMA peptide against MM cell lines in an HLA-A2 restricted manner.

[0149] [Figure 34E] FIG. 34E shows the highest anti-MM activity and Th1-type cytokine (IFN-γ, IL-2, TNF-α) production by BCMA-CTL generated from different HLA-A2+ individuals (N=3) using PLGA / BCMA peptides against MM cell lines in an HLA-A2 restricted manner.

[0150] [Figure 35A] Figure 35A shows the baseline activity of BCMA-specific CTLs generated with BCMA peptide by itself, PLGA / BCMA peptide or liposome / BCMA peptide in the absence of tumor cells.

[0151] [Figure 35B] FIG. 35B shows the highest anti-MM activity by BCMA-CTLs generated with PLGA / BCMA peptides in response to primary HLA-A2+CD138+ tumor cells from HLA-A2+ myeloma patient #1.

[0152] [Figure 35C]FIG. 35C shows the highest anti-MM activity by BCMA-CTLs generated with PLGA / BCMA peptides in response to primary HLA-A2+CD138+ tumor cells from HLA-A2+ myeloma patient #2.

[0153] [Figure 35D] FIG. 35D shows the highest anti-MM activity and Th1-type cytokine (IFN-γ, IL-2, TNF-α) production by BCMA-CTLs generated with PLGA / BCMA peptide against myeloma patient tumor cells in an HLA-A2 restricted manner.

[0154] [Figure 36A-1] Figure 36A shows a higher frequency of tetramer+CD8+ T cells and costimulatory molecule expressing (CD28+)CD8+ T cells by BCMA-CTL generated using PLGA / BCMA peptide than with the BCMA peptide itself. [Figure 36A-2] Figure 36A shows a higher frequency of tetramer+CD8+ T cells and costimulatory molecule expressing (CD28+)CD8+ T cells by BCMA-CTL generated using PLGA / BCMA peptide than with the BCMA peptide itself.

[0155] [Figure 36B] FIG. 36B shows a higher increase in peptide-specific CD8+ T cell proliferation by BCMA-CTLs generated with PLGA / BCMA peptide than with the BCMA peptide itself.

[0156] [Figure 36C] Figure 36C shows a higher increase in peptide-specific IFN-γ production by BCMA-CTLs generated using PLGA / BCMA peptide than with the BCMA peptide itself.

[0157] [Figure 37A]FIG. 37A shows the highest induction of myeloma-specific CD8+ T cell proliferation by BCMA-CTLs generated with PLGA / BCMA peptide than with the BCMA peptide by itself.

[0158] [Figure 37B-1] FIG. 37B shows a higher expansion of CD45RO+ memory / CD3+CD8+ T cell subsets in BCMA-CTLs [Representative Results] upon repeated stimulation with PLGA / BCMA peptide than with the BCMA peptide itself. [Figure 37B-2] FIG. 37B shows a higher expansion of CD45RO+ memory / CD3+CD8+ T cell subsets in BCMA-CTLs [Representative Results] upon repeated stimulation with PLGA / BCMA peptide than with the BCMA peptide itself.

[0159] [Figure 37C] Figure 37C shows a higher expansion of CD45RO+ memory / CD3+CD8+ T cell subsets in BCMA-CTL [N=3 results] upon repeated stimulation with PLGA / BCMA peptide than with the BCMA peptide itself.

[0160] [Figure 37D] FIG. 37D shows higher induction and maintenance of central memory / CD3+CD8+ T cell subsets in BCMA-CTL upon repeated stimulation with PLGA / BCMA peptide than with the BCMA peptide itself.

[0161] [Figure 38A] FIG. 38A shows higher induction of central and effector memory CTLs and their anti-MM activity against myeloma cells in an HLA-A2 restricted manner by BCMA-CTLs generated with PLGA / BCMA peptide than with the BCMA peptide itself.

[0162] [Figure 38B]FIG. 38B shows higher induction of central and effector memory CTLs and their IFN-γ production against myeloma cells in an HLA-A2 restricted manner by BCMA-CTLs generated with PLGA / BCMA peptide than with the BCMA peptide itself.

[0163] [Figure 38C] FIG. 38C shows higher induction of central and effector memory CTLs against myeloma cells and their anti-MM activity and IFN-γ, IL-2, TNF-α production in an HLA-A2 restricted manner by BCMA-CTLs generated with PLGA / BCMA peptide than with the BCMA peptide itself. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0164] B-cell maturation antigen (BCMA) and transmembrane activator and interactor of CAML (TACI) are specific and critical antigens for 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, for example, to generate multiple myeloma (MM)-specific T-cell immune responses. Thus, the present disclosure relates to BCMA-derived peptides and TACI-derived peptides (and pharmaceutical compositions thereof) that can be used, for example, to induce an immune response against tumor cells (e.g., stimulate a cytotoxic T cell (CTL) response) or stimulate the production of cytokines or antibodies in a subject. The peptides can be used in a variety of applications, such as methods for inducing immune responses, producing antibodies, producing cytokines involved in anti-tumor immune responses, and treating cancers (e.g., multiple myeloma, etc.). Peptides may also be included in MHC molecule multimer compositions and used, for example, in methods of detecting T cells (eg, antigen-specific T cells) in a population of cells.

[0165] The present disclosure also provides information on different types of therapeutic applications including peptide-based vaccination, peptides constituting different approaches of vaccines (including nanoparticle-based and viral-based), adoptive transfer of ex vivo generated BCMA-specific T cells or TACI-specific T cells and antigen-specific T cells using engineered technologies (including CAR-TCR therapy-based and induced pluripotent stem cell-based) or injection of peptide-pulsed dendritic cells, as monotherapy or combination therapy as described herein in cancer patients including multiple myeloma, its pre-malignant forms or other cancers, or any disease that uniquely expresses and / or overexpresses BCMA and TACI antigens.

[0166] BCMA- and TACI-derived peptides 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 that in humans is encoded by the TNFRSF17 gene. 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 tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B / TALL-1 / BAFF), leading to NF-kappaB and MAPK8 / JNK activation. This receptor also binds to various TRAF family members, and thus may transmit signals for cell survival and proliferation. BCMA is often overexpressed in various cancer cells, for example in subjects with leukemia, lymphoma, and multiple myeloma.

[0167] 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 in humans by the TNFRSF13B gene. 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 leukemia, lymphoma, and multiple myeloma.

[0168] The amino acid sequences of human BCMA and human TACI are shown below. [ka]

[0169] The present disclosure provides peptides derived from BCMA or TACI antigens (e.g., naive peptides) and heteroclitic peptides derived from peptides (naive peptides). These peptides can be any portion or fragment of a BCMA or TACI peptide. In some embodiments, these peptides have a length 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, these peptides have a length 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, these peptides have a length of 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 peptides are 9, 10, 11, or 12 amino acid residues (e.g., 9 amino acid residues) in length.

[0170] In some embodiments, the peptide derived from BCMA or TACI comprises: [ka]

[0171] These peptides can bind to major histocompatibility complex (MHC) molecules. MHC is a large family of genes that play important roles in the immune system, autoimmunity, and reproduction. MHC molecules play a role in the presentation of peptides, including self and non-self (antigenic), on their surface to T cells. MHC class I molecules bind short peptides, whose N- and C-termini are anchored in pockets located at the ends of the peptide-binding groove. The majority of these peptides are 9 in length, but a central bulge can accommodate longer peptides, resulting in binding, for example, of peptides 8-15 in length. Peptides that bind to class II proteins are not constrained in size and can vary, for example, from 11 to 30 amino acids in length. The peptide-binding groove in MHC class II molecules is open at both ends, allowing the binding of peptides with relatively long lengths. The "core" 9-residue long segment contributes most to peptide recognition, but adjacent regions are also important for the specificity of peptides to class II alleles.

[0172] Therefore, the present disclosure also provides peptides having sequences that comprise, consist of, or essentially consist of any of the sequences described in the present disclosure.In some embodiments, peptides can bind to MHC class I molecules and / or MHC class II molecules.In some embodiments, MHC class I molecules are 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.

[0173] Various modifications can be made to the peptides to improve the stability of the peptides binding to MHC molecules (e.g., MHC class I molecules, HLA-A2) molecules, increase immunogenicity, and / or increase immune responses. For example, to increase immunogenicity, amino acid residues can be modified by enhancing affinity for the T cell receptor (TCR), for example, by altering the TCR interaction site at positions 3, 4, 5, 6, 7, and / or 8 of any of the peptides described herein (e.g., SEQ ID NOs: 1-17).

[0174] 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 peptides. 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 the T cell receptor (TCR) by modifying TCR interaction sites (e.g., positions 1, 2, and / or 9 of SEQ ID NOs: 1-17). In some embodiments, the epitopes described herein can be modified at any position (e.g., 1, 2, 3, 4, 5, or 6 positions). The peptides can also contain internal mutations that make them "superantigens" or "superagonists" for T cell stimulation. Superantigen peptides can be generated by screening T cells with positional 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(l): 79-97, 2002; US Patent 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 make the peptide more potent immunogens. In some embodiments, the first amino acid residue of the peptide derived from BCMA or TACI can be changed to tyrosine.

[0175] Some heteroclitic peptides derived from BCMA or TACI include: [ka]

[0176] As used herein, the term "heteroclitic" (e.g., a heteroclitic peptide) refers to a form of a peptide in which one or more amino acid residues have been modified from the wild-type or original sequence to produce a peptide that is more immunogenic than the corresponding peptide having the wild-type or original sequence.

[0177] The present disclosure further provides variants of peptides as described herein (e.g., SEQ ID NOs: 1-17). Variants of the peptides described herein may include forms of the peptides having 5 or less, 4 or less, 3 or less, 2 or less, 1 or less amino acid substitutions (e.g., 5, 4, 3, 2 or 1 amino acid substitutions). In some embodiments, variants of the peptides described herein may include forms of the peptides having at least 1, at least 2, at least 3 or at least 4 substitutions.

[0178] In some embodiments, the amino acids at positions 1, 2 and / or 9 of SEQ ID NO:1-17 contribute to HLA binding affinity and therefore may be substituted without affecting peptide-specific T cell responses. Thus, the immunogenicity of a peptide may still be maintained with substitutions at positions 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 acids at positions 1 and 2, 1 and 9, 2 and 9 or 1, 2 and 9 of SEQ ID NO:1-17 are substituted. In some embodiments, the disclosure provides peptides that include a sequence as described herein (e.g., one of SEQ ID NOs: 1-17, with or without substitutions as described herein) and have 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.

[0179] Substitutions can be any type of amino acid substitution, for example, conservative or non-conservative. Conservative substitutions include substitutions within 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, and (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 a member of the above polar, basic or acidic groups with another member of the same group can be considered as a conservative substitution. In contrast, a non-conservative substitution is the substitution of one amino acid with another having different characteristics, for example, the substitution of one amino acid with another amino acid in another group.

[0180] In some embodiments, one or more (e.g., one, two, three, four or all five) of positions 3, 4, 5, 6, 7 and 8 of any of the peptides are unsubstituted. In some embodiments, one or more (e.g., one, two, three, four or all five) of positions 3, 4, 5, 6, 7 and 8 of the peptides are identical to a sequence selected from SEQ ID NOs: 1-17. As used herein, the term position refers to a position starting from the N-terminus of the peptide. For example, position 3 of a peptide refers to the third amino acid residue starting from the N-terminus of the peptide. In some embodiments, residues at positions 3, 4, 5, 6, 7 and 8 of SEQ ID NOs: 1-17 contribute most to recognition of the peptide.

[0181] The present disclosure further provides amino acid or nucleotide sequences comprising, consisting of, or consisting essentially of any of the sequences as described herein, e.g., SEQ ID NOs:1-17, SEQ ID NOs:18 and 19, and sequences that are 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 of the sequences as described herein, e.g., SEQ ID NOs:1-17, SEQ ID NOs:18 and 19, and nucleotide sequences encoding SEQ ID NOs:1-17.

[0182] 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 may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may 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 corresponding amino acid or nucleotide positions are then compared. If 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, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences and the number of identical positions shared by the sequences taking into account the length of each gap. For purposes of this disclosure, comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix, which has a gap penalty of 12, a gap extension penalty of 4 and a frameshift gap penalty of 5.

[0183] Also provided herein are peptides comprising 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 that is adjacent to the first amino acid sequence, and the adjacent sequence does not occur in nature (e.g., the adjacent sequence is not naturally linked to the first amino acid sequence). The first amino acid sequence can comprise, consist essentially of, or consist of any sequence as described herein, for example, 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 with heterologous adjacent amino acid sequences generally do not (and are selected not to) adversely affect the production in cells of the immunogenic peptides of SEQ ID NO: 1-17. The cellular machinery is predicted to remove any additional sequences in the peptide to result in immunogenic peptides of SEQ ID NOs: 1-17, which are presented by class I or class II MHC molecules to stimulate an immune response against cancer cells expressing BCMA or TACI.

[0184] Heterologous flanking sequences can be, for example, sequences used in the purification of recombinant proteins (e.g., FLAG, polyhistidine (e.g., hexahistidine), hemagluttanin (HA), glutathione-S-transferase (GST), or maltose binding protein (MBP)). Heterologous sequences can also be proteins that are useful as diagnostic or detection markers, such as luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). In some embodiments, the peptide can contain all or a portion of an immunoglobulin molecule (e.g., all or a portion of an immunoglobulin heavy chain constant region).

[0185] In some embodiments, the heterologous sequence may comprise a therapeutic or immunostimulatory polypeptide sequence (e.g., a helper T epitope (e.g., the PADRE epitope or the tetanus toxoid universal helper T cell epitope) or all or a portion of a cytokine or chemokine) and / or a carrier (e.g., KLH) useful, for example, for eliciting an immune response (e.g., for antibody production). In some embodiments, the peptide may contain one or more linker peptide sequences. The peptide may also contain a targeting polypeptide. The heterologous sequence may be of varying length, 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 containing a first amino acid sequence and a second amino acid sequence that is heterologous to the first does not correspond in sequence to a naturally occurring protein.

[0186] A targeting polypeptide, as used herein, is a polypeptide that targets a moiety (or moieties) (e.g., a first amino acid sequence) to a particular tissue (e.g., lymph node) or cell (e.g., antigen-presenting cell or other immune cell) or in vitro to a particular isolated molecule or molecular complex. 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 antigen-binding fragment thereof) 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 a Fab fragment, F(ab')2 fragment, Fab' fragment, Fv fragment or scFv fragment of an antibody. Antibody fragments that include or are an Fc region (with or without an antigen-binding region) can also be used to target reagents to cells expressing Fc receptors (e.g., antigen-presenting cells such as interdigitating dendritic cells, macrophages, monocytes 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).

[0187] In some embodiments, the heterologous sequence may include, for example, a "trafficking sequence" that aids in the delivery of the peptide into a cell or to a particular compartment of the cell (e.g., the endoplasmic reticulum or Golgi apparatus). Trafficking sequences can include, for example, membrane translocation sequences, transportan sequences, antennapedia sequences, cyclic integrin-binding peptides, and Tat-mediating peptides or modified versions thereof.

[0188] A linker peptide can connect a first amino acid sequence with one or more heterologous amino acid sequences. For example, a linker peptide can connect a first amino acid sequence with a second amino acid sequence. In certain embodiments, a linker peptide can link / connect any one of the peptides of SEQ ID NOs: 1-17 with a second peptide selected from SEQ ID NOs: 1-17. A linker peptide can be or contain, for example, a stretch of amino acids in which at least 4-6 amino acids are glycines (see, for example, Mancebo et al. (1990) MoI. Cell. Biol. 10: 2492-2502). A linker can be or contain 6 or more (e.g., 7, 8, 9, 10, 11 or 12 or more) histidine residues. A linker peptide can contain or be at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 or more) protease cleavage site. The protease site can be, for example, a trypsin, chymotrypsin or factor Xa cleavage site. Such a protease site can be useful, for example, to separate the first amino acid sequence from a heterologous sequence. For example, after expression and purification of a peptide containing a first amino acid sequence linked to a polyhistidine sequence by a trypsin protease cleavage site (e.g., for purification), the polyhistidine sequence can be removed from the first amino acid sequence by contacting the peptide with trypsin.

[0189] In some embodiments, the present disclosure provides 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 119 The present invention provides a peptide having 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 (e.g., any one of SEQ ID NOs: 1-17).

[0190] 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 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 equivalent to or 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.

[0191] In some embodiments, the peptide may have additional sequences, which may be located at the amino or carboxy terminus of the peptide. In some embodiments, the additional sequence may have at least 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. In some embodiments, the additional sequence may 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.

[0192] In certain examples, the disclosure encompasses any combination of two or more peptides selected from SEQ ID NOs:13-17.

[0193] The peptides described herein can bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I or MHC class II molecules). In humans, MHC is known as HLA complex. "HLA supertype or family" as used herein refers to a set of HLA molecules grouped based on shared peptide binding specificity. HLA class I molecules that share somewhat similar binding affinity for peptides with a certain amino acid motif are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family and HLAxx-like molecule (where xx refers to a specific HLA type) are synonymous. 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 US Pat. No. 7,026,443, the entire disclosure of which is incorporated by reference in its entirety.

[0194] Peptides can bind to MHC molecules with high 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 nM (e.g., less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1 or 0.05 nM). "Moderate affinity" is the binding of a peptide to an HLA class I molecule with a KD of between about 50 nM and 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 nM (e.g., less than 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 0.05 nM). A "moderate affinity" for a peptide for an HLA class II molecule is between about 100 and about 1000 nM (e.g., 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, 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 or 1000 nM).Methods for determining the binding affinity of peptides and MHC molecules are known in the art. Suitable methods are also described, for example, in U.S. Patent No. 7,026,443.

[0195] The peptides described herein may also be recognized by antigen-specific T cell receptors on T cells in association with MHC molecules. A variety of suitable methods can be used to determine whether a peptide is recognized by a T cell receptor on a T cell in association with an MHC molecule. For example, peripheral blood lymphocytes (PBLs) from a normal subject may be cultured in vitro in the presence of antigen-presenting cells with the test peptide for a period of several weeks. The peptide-specific T cells are activated during this time and can be detected by, for example, proliferation assays (H-thymidine assay, carboxyfluorescein succinimidyl ester (CFSE) assay), limiting dilution assays, cytotoxicity assays (e.g., calcein release assays) or cytokine- (e.g., IFNγ), lymphokine- or 51Cr-release assays (e.g., Wentworth, et al., J. Immunol. 1999, 143:1311-1323, the disclosures of each of which are incorporated by reference in their entireties). PA et al., MoI. 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). A suitable in vivo method involves immunizing HLA transgenic mice by subcutaneously administering the peptide in adjuvant to the HLA transgenic mice, and several weeks after immunization, removing splenocytes and culturing them in vitro in the presence of the test peptide for approximately one week, and isolating peptide-specific T cells 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).

[0196] Additionally, direct quantification of antigen-specific T cells can be performed by staining T cells with detectably labeled MHC complexes, such as any of the MHC molecule multimeric compositions described herein, or HLA-I tetramers (see, e.g., Altman, JD et al., Proc. Natl. Acad. Sci. USA 90: 10330, 1993 and Altman, JD et al., Science 274: 94, 2002, the disclosures of each of which are incorporated by reference in their entireties). As described in 1996).

[0197] In some embodiments, the peptide may be further modified (e.g., amino acids of the peptide may be substituted) to modulate (e.g., increase or decrease) one or more properties of the peptide. For example, one or more (e.g., 2, 3, or 4) amino acids of one of the peptides described herein may be substituted to increase the affinity of the peptide to the MHC molecule. In some embodiments, one amino acid of one of the peptides described herein (e.g., an amino acid residue of the peptide that contacts the T cell receptor) may 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 often referred to as "altered peptide ligands" (see, for example, Kalergis et al. (2000) J Immunol. 165(l): 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, as well as determining the effects of the modifications, are described, for example, in Collins et al. (Immunological Reviews (1998) 163: 151-160), the disclosure of which is incorporated by reference in its entirety.

[0198] The present disclosure further provides compositions comprising any peptide as described herein. In addition, to combat the ability of tumors to evade therapies directed against them, the compositions can include various 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 seven different peptides from BCMA or TACI. Furthermore, combinations or mixtures of at least one (e.g., at least two, three, four, five) peptides from BCMA and at least one (e.g., at least two, three, four, five) peptides from TACI can also be used. Thus, in some embodiments, the present disclosure provides compositions comprising at least two, three, four, five, six, seven, or eight BCMA-derived peptides (e.g., at least two BCMA-derived peptides selected from, for example, SEQ ID NOs: 1-6 and 13-14). In some embodiments, the disclosure provides compositions 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 disclosure provides compositions 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.

[0199] In some embodiments, the composition further comprises an immunostimulatory agent (e.g., a cytokine or a helper T epitope). The helper T epitope can be a PADRE sequence or a universal tetanus toxoid helper T (TT Th) epitope. In some embodiments, the composition comprises an adjuvant, such as Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligand for the Toll receptor, QS21, RIBI, or a similar immunostimulant. Adjuvants also include, for example, cholera toxin (CT), E. coli heat-labile toxin (LT), mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185: 1203-1210), and mutant E. coli heat-labile toxin (MLT) (Di Tommaso et al. al. (1996) Infect. Immunity 64: 974-979). In some embodiments, the composition comprises a toll-like receptor-3 ligand (e.g., poly-ICLC), interferon alpha (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.

[0200] 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; Niesvizky, et al. "ACY-241, a novel, HDAC6 selective inhibitor: synergy with immunomodulatory (IMiD®) 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.

[0201] In some embodiments, the composition comprises a peptide comprising the sequence set forth 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 the sequence set forth 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.

[0202] Nucleic acids and methods for producing peptides The present disclosure also features nucleic acid sequences (as well as nucleic acid vectors containing nucleic acid sequences) encoding any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of any of the peptides described herein, as well as methods of producing any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of any of the peptides described herein. Such methods may optionally include providing a cell (or a group of cells) containing a nucleic acid vector containing a nucleic acid sequence encoding any one or more of any of the peptides described herein, the nucleic acid sequence being operably linked to an expression control sequence, and culturing the cells under conditions that allow expression of the peptide. The methods may also include isolating one or more peptides from the cells or from the medium in which the cells are cultured. Thus, in one aspect, the present disclosure provides RNA-based 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 examples, the polynucleotide encodes a peptide identical to one of SEQ ID NO: 1-17 except for having one to four amino acid substitutions. In some cases, the substitutions are at one or more of positions 1, 2, or 9. In some cases, the peptide is 2-30 amino acids in length. In some cases, the nucleic acid may include regulatory sequences (e.g., start codon, stop codon, polyA tail). In some embodiments, the RNA / DNA cancer vaccine may be formulated within a polymeric or liposomal nanocarrier (e.g., nanoparticle).

[0203] Suitable methods for constructing nucleic acid sequences and vectors (e.g., expression vectors) for recombinant expression of one or more of the peptides described herein are well known to those of skill in the art and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3, the disclosure of which is incorporated by reference in its entirety. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989. The nucleic acids and vectors can be used to express, for example, peptides in a variety of host cells, including, for example, bacteria, yeast, or mammalian cells. The nucleic acids and vectors can also be used, for example, in in vivo and ex vivo methods, as described below. The peptide-encoding sequence can be operably linked to a promoter, a regulatory element, or an expression control sequence. The promoter and / or enhancer element can direct the expression of the peptide encoded by the nucleic acid. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at variable distances from the transcription start site, provided that a promoter is present. Enhancers can also be located downstream of the transcription start site or in an exon of the associated gene. To bring a coding sequence under the control of a promoter, it is necessary to place the translation start site of the translation reading frame of the peptide 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 SV40 adenovirus early or late promoters, 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 coat protein, the promoter of 3 phosphoglycerate kinase, the promoter of acid phosphatase and the promoter of yeast alpha mating factor, the adenovirus EIb minimal promoter or the thymidine kinase minimal promoter.

[0204] A peptide-encoding sequence or a vector containing a peptide-encoding sequence may contain a leader sequence encoding a signal peptide. The leader sequence may be at the 5'-end of the sequence encoding one or more of the peptides described herein. The signal peptide may be immediately N-terminal to a given peptide or may be separated from it by one or more (e.g., 2, 3, 4, 6, 8, 10, 15 or 20) amino acids, provided that the leader sequence is in frame with the nucleic acid sequence encoding the peptide. In general, the signal peptide, which is cleaved from the peptide prior to secretion (unless, of course, the signal peptide directs the insertion of a transmembrane protein), directs the attached peptide to enter the lumen of the host cell endoplasmic reticulum (ER) during translation, and then the peptide is secreted into the host cell environment via secretory vesicles. Useful signal peptides include, for example, the natural leader sequence of a cytokine or growth factor, KDEL (Lys-Asp-Glu-Leu), or any of the signal sequences described, for example, in U.S. Pat. No. 5,827,516, the disclosure of which is incorporated herein by reference in its entirety.

[0205] In some embodiments, the 5' end of the sequence encoding the peptide may include a non-natural ATG "start sequence." That is, for example, an ATG sequence may be added to a nucleic acid encoding the peptide to ensure that the peptide is properly transcribed and translated. Leader sequences generally include an ATG start sequence, but in embodiments where this is not the case, an ATG sequence may be added to the 5' end of a nucleic acid encoding a leader sequence.

[0206] Suitable methods for constructing peptide-encoding 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 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 herein by reference in its entirety.

[0207] Recombinant vectors can be introduced into cells using a variety of methods, which may vary, at least in part, depending on the type of cell into which the nucleic acid is to be introduced. For example, bacterial cells can be transformed using methods such as electroporation or heat shock. Methods for transfecting yeast cells include, for example, the spheroplast technique or the whole-cell lithium chloride yeast transformation method (see, e.g., U.S. Pat. No. 4,929,555; Hinnen et al. (1978), the disclosures of each of which are incorporated herein by reference in their entireties). Proc. Nat. Acad. Sci. USA 75: 1929; Ito (see, e.g., Sambrook et al. (1983) J. Bacteriol. 153: 163; U.S. Patent No. 4,879,231; and Sreekrishna et al. (1987) Gene 59: 115). Transfection of animal cells can be characterized by the introduction of a vector into a cell using, for example, calcium phosphate, electroporation, heat shock, liposomes or transfection reagents such as FUGENE® or LIPOFECT AMINE®, or by contacting the naked nucleic acid vector with the cell in solution (see, e.g., Sambrook et al., supra).

[0208] 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., E. coli and B. 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 viral expression vectors (e.g., baculovirus), plant cell systems infected with recombinant viral 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) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or 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 a mammal, transfected with a plasmid vector or infected with a viral vector (e.g., viral vectors such as herpes virus, retrovirus, vaccinia virus, attenuated vaccinia virus, canary pox virus, adenovirus and adeno-associated virus, among others).

[0209] After expression of any of the peptides described herein, the peptide can be isolated from the cultured cells or from the medium in which the cells are cultured 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 chelation or immunoaffinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography, precipitation or differential solubilization.

[0210] Smaller peptides (e.g., peptides having fewer than 200 (e.g., fewer than 175, fewer than 150, fewer than 125, fewer than 100, fewer than 90, fewer than 80, fewer than 70 or fewer than 60) amino acids) can be chemically synthesized by standard chemical means such as FMOC solid phase synthesis.

[0211] The peptides described herein can be, but need not be, isolated. The term "isolated" as applied to any of the peptides described herein refers to a peptide, fragment thereof (or for compositions, macromolecular complexes) that has been separated or purified from components that naturally accompany it (e.g., proteins or other naturally occurring biological or organic molecules). It is understood that a recombinant molecule (e.g., a recombinant peptide) is always "isolated". Typically, a peptide (or fragment or macromolecular complex) is isolated when it constitutes at least 60%, 70%, 80% or 90% by weight of all molecules of the same type in a preparation, e.g., at least 60%, 70%, 80% or 90% by weight of all molecules of the same type in a sample. For example, a peptide described herein is considered isolated when it constitutes at least 60%, 70%, 80% or 90% by weight of the total proteins in a preparation or sample. In some embodiments, the molecules in a preparation consist of at least 75%, at least 90% or at least 99% by weight of all molecules of the same type in the preparation.

[0212] Similarly, the peptide-encoding sequences or vectors containing peptide-encoding sequences described herein can also be isolated. The term "isolated" as applied to any of the peptide-encoding sequences or vectors described herein refers to a peptide-encoding sequence or vector, or fragment thereof, that is separated or purified from components that naturally accompany it (e.g., nucleic acids, proteins, or other naturally occurring biological or organic molecules). It is understood that a recombinant molecule (e.g., a recombinant vector or a peptide-encoding sequence) is always "isolated". Usually, a peptide-encoding sequence or vector (or fragment thereof) is isolated when it constitutes at least 60%, 70%, 80% or 90% by weight of the total molecules of the same type in a preparation, e.g., at least 60%, 70%, 80% or 90% by weight of the total molecules of the same type in a sample. For example, a peptide-encoding sequence or vector described herein is considered to be isolated when it constitutes at least 60%, 70%, 80% or 90% by weight of the total nucleic acids in a preparation or sample. In some embodiments, the molecules in a preparation consist of at least 75%, at least 90%, or at least 99%, by weight, of all molecules of the same type in the preparation.

[0213] In some embodiments, the isolated peptide, sequence encoding the peptide, or vector can be frozen, lyophilized or fixed 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 an MHC class I molecule or an MHC class II molecule, or the ability of the vector to support expression of the peptide in a cell).

[0214] Peptide Processing After expression or synthesis of any of the peptides described herein, the peptide can be further processed. Further processing can include chemical or enzymatic modification to the peptide, or if the peptide is modified, processing can include enzymatic or chemical alteration of existing modifications or both. Further processing of the peptide can include, but is not limited to, the addition (covalent or non-covalent linkage) of a heterologous amino acid sequence, such as any of the heterologous amino acid sequences described herein. Enzymatic processing can include, for example, contacting the peptide with one or more proteases, phosphatases or kinases under conditions that allow the peptide to be modified. Enzymatic processing can include contacting the peptide with one or more enzymes (e.g., oligosaccharyltransferases or mannosidases) that can glycosylate or modify glycosylation of the peptide.

[0215] Processing can include, for example, the addition of a detectable label to the peptide. For example, the peptide can be coupled to 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, allophycocyanin (APC), or phycoerythrin), a luminescent substance (e.g., a lanthanide or a chelate thereof), a bioluminescent substance (e.g., luciferase, luciferin, or aequorin), or a radionuclide (e.g., 3 H, 32 P, 33 P, 125 I or 35 S) can be detectably labeled.

[0216] Processing may also include coupling of 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 on the peptide that is the N-terminus. In some embodiments, the peptide may contain one or more internal amino acid insertions that provide internal polymer conjugation sites to which a polymer may be conjugated.

[0217] Methods for inducing an immune response The present disclosure also provides various 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 approach). The method of inducing an immune response in a subject may, for example, include administering to the subject one or more of the compositions described herein (e.g., any of the peptides (or expression vectors containing nucleic acid sequences encoding the peptides) described herein (or any of the pharmaceutical compositions containing one or more peptides (or vectors) described herein). In some embodiments, the compositions described herein are used as vaccines.

[0218] Any of the peptides described herein can be used to stimulate immune response by using a nucleic acid expression system that encodes one or more of the peptides described herein.That is, the method of inducing immune response in a subject can include administering to the subject an expression vector (or a pharmaceutical composition that contains an expression vector) that contains a nucleic acid sequence that encodes one or more of the peptides described herein.The immune response can be CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), TH1 response, TH2 response, or a combination of both types of responses.

[0219] Any of the above methods may be, for example, a method of treating or preventing (prophylactic against) cancer in a subject (e.g., a plasma cell disorder such as multiple myeloma, or any other cancer expressing BCMA and / or TACI). When the terms "prevent", "preventing" or "prevention" are used herein in conjunction with a given treatment of a given condition, they mean that the treated subject does not develop a clinically observable level of the condition at all (e.g., the subject does not show one or more symptoms of the condition, or in the case of cancer, the subject does not develop a detectable level of cancer), or that the condition develops later and / or to a lesser extent in the subject than would be expected to have in the absence of treatment (e.g., fewer symptoms or fewer cancer cells in the subject). These terms are not limited to only those situations in which the subject does not experience any aspect of the condition at all. For example, a treatment is said to have "prevented" a condition if it is administered during a period, e.g., an early diagnosis of cancer (e.g., detection of fewer cancer cells in a sample obtained from a subject) that would be predicted to result in a given manifestation of the condition (advanced cancer), and results in the subject experiencing a condition with fewer and / or milder symptoms than would otherwise be predicted. A treatment may "prevent" a cancer (e.g., a plasma cell disorder such as multiple myeloma) if the subject exhibits only mild overt symptoms of the cancer. "Prevention" does not imply that even a single cancer cell would not have developed by the subject so treated.

[0220] Generally, the peptide to be delivered to a subject is suspended in a pharma- ceutically acceptable carrier (e.g., saline) and administered orally, rectally, or parenterally, e.g., by intravenous, subcutaneous, intramuscular, intrathecal, intraperitoneal, intrarectal, intravaginal, intranasal, intragastric, intratracheal, or intrapulmonary injection.

[0221] Administration may be by periodic injections of a bolus of the pharmaceutical composition, or may be uninterrupted or continuous by intravenous or intraperitoneal administration from a reservoir that is external (e.g., an IV bag) or internal (e.g., a bioerodable implant, a bioartificial organ, or an implanted colony of reagent-producing cells). See, e.g., U.S. Patent Nos. 4,407,957, 5,798,113, and 5,800,828, each of which is incorporated herein by reference in its entirety.

[0222] Conventional and pharma- ceutically 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 (through the nose) and other routes of effective inhalation and other parenteral routes of administration. Routes of administration may be combined and, if desired, adjusted depending on the desired effect on the nucleic acid molecule and / or immune response. Methods of administering nucleic acids to subjects may include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., viral infection / transfection or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides into subjects or cells of subjects).

[0223] Generally, the dosage of peptide or nucleic acid required will vary depending on the choice of route of administration, the nature of the formulation, the nature or severity of the subject's illness, the subject's immune status, the subject's size, weight, surface area, age, and sex, other medications being administered, and the judgment of the attending medical practitioner.

[0224] Suitable doses of peptides to induce an immune response range from 0.000001 to 10 mg of reagent or antigenic / immunogenic composition per kg of subject. Wide variations in required dosage should be expected, taking into account the diversity of reagents and the differing efficiencies of various routes of administration. For example, nasal or rectal administration may require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as well understood in the art. Administration can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-fold or more). For example, the peptide can be administered as an initial immunization, then administered one or more subsequent times as booster immunizations.

[0225] In the context of the methods described herein, the dose of nucleic acid administered to a subject must be sufficient to achieve beneficial therapeutic response or alleviate symptoms in the subject over time.However, the dosage used will vary, for example, depending on the subject or the clinical goal to be achieved.Suitable dosage ranges provide up to about 1 μg, about 1,000 μg, about 5,000 μg, about 10,000 μg, about 25,000 μg or about 50,000 μg of nucleic acid per ml of carrier in a single dose.

[0226] To optimize therapeutic efficacy (e.g., efficacy of one or more peptides or nucleic acids encoding peptides to induce immune response in a subject), compositions containing peptides or nucleic acids can be initially administered with different dosing regimens. Unit doses and regimens vary depending on factors including, for example, the species of mammal, its immune status, and the body weight of the mammal. The frequency of dosing of pharmaceutical compositions (e.g., pharmaceutical compositions containing 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 medical practitioners (e.g., doctors or nurses). Usually, dosing regimens are established by clinical trials that can establish optimal dosing parameters. However, medical practitioners can modify such dosing regimens according to the age, health, weight, sex, and medical condition of the subject.

[0227] 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 times or more). For example, the pharmaceutical composition can be administered to a subject once a month for three months, once a week for one month, once a year for three years, once a year for five years, once every five years, once every ten years, or once every three years for the rest of their lives.

[0228] As defined herein, a "therapeutically effective amount" of a peptide or a nucleic acid encoding a peptide is an amount of peptide or nucleic acid capable of producing an immune response in a treated subject. A therapeutically effective amount (i.e., an effective dosage) of a peptide includes milligram, microgram, nanogram, or picogram amounts of agent per kilogram of subject or sample weight (e.g., from about 1 nanogram per kilogram to about 500 micrograms per kilogram, from about 1 microgram per kilogram to about 500 milligrams per kilogram, from about 100 micrograms per kilogram to about 5 milligrams per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram). A therapeutically effective amount of a nucleic acid also includes microgram, nanogram or picogram amounts of the agent per kilogram of subject or sample weight (e.g., from about 1 nanogram per kilogram to about 500 micrograms per kilogram, from about 1 microgram per kilogram to about 500 micrograms per kilogram, from about 100 micrograms per kilogram to about 500 micrograms per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0229] As defined herein, a "prophylactically effective amount" of a peptide or a nucleic acid encoding a peptide is an amount of peptide or nucleic acid capable of producing an immune response against cancer cells (e.g., multiple myeloma) in a treated subject, which immune response can prevent the development of cancer in the subject or can substantially reduce the subject's chance of developing or continuing to develop cancer. A prophylactically effective amount (i.e., an effective dosage) of a peptide includes milligram, microgram, nanogram, or picogram amounts of agent per kilogram of subject or sample weight (e.g., from about 1 nanogram per kilogram to about 500 micrograms per kilogram, from about 1 microgram per kilogram to about 500 milligrams per kilogram, from about 100 micrograms per kilogram to about 5 milligrams per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram). A prophylactically effective amount of a nucleic acid also includes microgram, nanogram or picogram amounts of the agent per kilogram of subject or sample weight (e.g., from about 1 nanogram per kilogram to about 500 micrograms per kilogram, from about 1 microgram per kilogram to about 500 micrograms per kilogram, from about 100 micrograms per kilogram to about 500 micrograms per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0230] In some embodiments, the method may also include determining whether an immune response occurs in the subject after administering the composition to the subject. Suitable methods for determining whether an immune response occurs in the subject include, for example, using an immunoassay to detect the presence of peptide-specific antibodies in a biological sample obtained from the subject. For example, after administering the peptide to the subject, a biological sample (e.g., a blood sample) can be obtained from the subject and tested for the presence of peptide-specific antibodies. An immune response can also be detected by assaying for 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- or 51Cr-release assays).

[0231] In some embodiments, the methods described herein (e.g., therapeutics, vaccines, cell therapies, antibodies that augment BCMA- and TACI-specific responses and / or therapeutic approaches that target some target other than BCMA and TACI) 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), immune modulators (e.g., lenalidomide, pomalidomide, HDAC inhibitors such as ACY241) and / or adjuvants.

[0232] The method also includes administering to the subject one or more chemotherapeutic agents, one or more forms of ionizing radiation, or one or more immunomodulatory agents. The one or more forms of ionizing radiation can be gamma radiation, X-ray radiation, or beta radiation. The one or more chemotherapeutic agents may be selected from the group consisting of cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, thalidomide, lenalidomide, proteosome inhibitors (e.g., bortezomib), hsp90 inhibitors (e.g., tenespinmycin), transplatinum, 5-flurouracil, vincristine, vinblastine, methotrexate, or an analog of any of the above. Immunomodulatory agents 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 a nucleic acid encoding the peptide can be administered with an immune modulator, such as a Toll receptor ligand or an adjuvant.

[0233] In some embodiments, the additional therapeutic agent is a histone deacetylase 6 (HDAC6) inhibitor (e.g., ACY241).

[0234] In some embodiments, the one or more additional therapeutic agents may be an immunomodulatory agent, a checkpoint inhibitor (e.g., an anti-LAG3 antibody) or an immune agonist (e.g., an anti-OX40 antibody, an anti-GITR antibody).

[0235] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, 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).

[0236] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0237] In some embodiments, the combination therapy includes one or more of the following: (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) immune modulators, including, for example, lenalidomide, pomalidomide, HDAC inhibitors, such as ACY241, (4) adjuvants, (B) Independent approaches to target therapeutics or other targets (e.g., non-BCMA or non-TACI targeted therapies) that can augment BCMA and TACI specific responses, including any therapeutics (e.g., vaccines, cell therapies and / or antibodies) as described herein; and (C) Additional compounds such as one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, 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 isocitrate dehydrogenase 2 (IDH2) and / or inhibitors of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0238] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinfluridine ... The therapeutic agent may include one or more therapeutic agents selected from the group consisting of Lunin, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, Vinblastine, Thalomid, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacitidine, Lenalidomide, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate and Enzastaurin.

[0239] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) alpha, 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 treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0240] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0241] 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.

[0242] The present disclosure also provides a virus comprising a nucleic acid encoding one or more peptides as described herein or a viral particle comprising one or more peptides as described herein.Various viruses can be used, such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, alphaviruses, etc. These viruses can be used to deliver peptides or the nucleic acid encoding the peptides to subjects to induce immune responses.Similarly, liposomes comprising one or more peptides and / or the nucleic acid encoding the peptides as described herein can be used to deliver peptides or nucleic acids to subjects in need thereof to induce immune responses.

[0243] In some embodiments, the methods described herein can increase immune response, activity (e.g., producing cytokines, IFN-γ, IL-2 or TNF-α) or number of 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 as described herein can increase the number of CD8+ effector T cells (e.g., the total number of CD8+ effector T cells or, e.g., the percentage of CD8+ among CD45+ cells) that specifically target cancer cells or recognize a peptide described herein by at least 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold or 50-fold.

[0244] Nanoparticles The present disclosure further provides nanoparticles or nanocarriers comprising one or more peptides as described herein (e.g., SEQ ID NOs: 1-17) or one or more polynucleotides as described herein (e.g., sequences encoding SEQ ID NOs: 1-17). In some cases, the nanocarriers comprise peptides identical to the amino acid sequences of SEQ ID NOs: 1-17 but with one to four amino acid substitutions. In some examples, the substitutions are at one or more of positions 1, 2, and 9. Polynucleotides (e.g., mRNA, DNA) encoding such peptides can also be encapsulated in the nanocarriers. The nanoparticles or nanocarriers can be administered to a subject in need thereof to induce an immune response.

[0245] Peptides can be attached to nanoparticles or nanocarriers through various attachment mechanisms. This attachment mechanism can be electrostatic attraction, covalent coupling or hydrophobic interaction. In some embodiments, nanoparticles can be loaded with adjuvants. The adjuvant can be a dendritic cell targeting molecule, for example, a Toll-like receptor agonist, for example, R-848, which is recognized as a strong synthetic agonist of TLR7 / TLR8, or a non-methylated CpG oligodeoxynucleotide, which is an immunostimulatory agonist of TLR-9, or a monophosphoryl lipid A, which is an immunostimulatory agonist of TLR-4, or an endosomal membrane targeting agent, for example, Endo-Porter peptide.

[0246] 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), poly(glycolic acid) (PGA) or poly(lactic-co-glycolic acid) (PLGA). These polymers have an established safety record 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 polymers can be used alone, as physical mixtures, or by forming copolymers. In certain embodiments, the nanoparticles are formed by mixtures of poly(lactic acid-co-glycolic acid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG) triblock copolymers, PLGA-PEG diblock copolymers 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.

[0247] As used herein, a "nanoparticle" is a particle having a diameter in the range between 500 nm and 0.5 nm, e.g., having a diameter between 50 and 500 nm, having a diameter that is between 100 and 400 nm, or having a diameter that is between 200 and 400 nm.

[0248] Nanoparticles and methods of making and using nanoparticles are known in the art and are described, for example, in US2016 / 0008451, US2010 / 0129439, US2018 / 0021258, each of which is incorporated by reference in its entirety. In some embodiments, the nanoparticles are liposomes. In some embodiments, the nanoparticles are polymeric particles.

[0249] 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), poly(glycolic acid) (PGA) or poly(lactic-co-glycolic acid) (PLGA). These polymers have an established safety record 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 compositions disclosed herein (see Akagi et al., Adv Polym Sci. 247: 31-64, 2012).

[0250] The polymers can be used alone, as physical mixtures, or by forming copolymers. In some embodiments, the nanoparticles are formed by mixtures of poly(lactic acid-co-glycolic acid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG) triblock copolymers, PLGA-PEG diblock copolymers, 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. The linear structure (e.g., PLGA-PLH-PEG) can provide the nanoparticles with some characteristics that are compatible with extended circulation and charge-mediated targeting.

[0251] 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 prolamines and hydrophobic proteins, copolymers and mixtures thereof. Generally, these materials degrade by surface or bulk erosion, either by enzymatic hydrolysis in vivo or exposure to water.

[0252] Other suitable biodegradable polymers include, but are not limited to, poly(hydroxy acids), such as polymers and copolymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co-caprolactone).

[0253] The polymer can be a bioadhesive polymer that is hydrophilic or hydrophobic. Hydrophilic polymers include CARBOPOL™ (a high molecular weight, cross-linked, acrylic acid-based polymer manufactured by Noveon), polycarbophil, cellulose esters, and dextran.

[0254] These polymers can be obtained 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.

[0255] Various polymers and methods for forming polymer matrices therefrom are known in the art. Generally, a 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 a copolymer comprising two or more monomers. In terms of sequence, the copolymer may be random, block, or contain a combination of random and block sequences. Typically, the polymer according to the present invention is an organic polymer.

[0256] Examples of polymers suitable for use in the compositions described herein include, but are not limited to, polyethylene, polycarbonates (e.g., poly(1,3-dioxan-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactides, polyglycolides, polylactide-co-glycolides, polycaprolactones, polyhydroxy acids (e.g., poly(-hydroxyalkanoates)), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0257] In some embodiments, polymers in accordance with the present invention include polymers approved for human use by the U.S. Food and Drug Administration (FDA) under 21 C.FR §177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0258] In some embodiments, the polymer can be hydrophilic. For example, the polymer can include 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 hydrophilic or hydrophobic nature of the polymer can have an impact on the nature of the material incorporated (e.g., coupled) into the synthetic nanoparticles.

[0259] In some embodiments, the polymer can be modified with one or more moieties and / or functional groups. A variety of moieties or functional groups can be used according to the present invention. In some embodiments, the polymer can be modified with polyethylene glycol (PEG), with carbohydrates, and / or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certain embodiments can be made using the general teachings of U.S. Patent No. 5,543,158 to Gref et al. or International Publication WO2009 / 051837 to Von Andrian et al.

[0260] In some embodiments, the polymer can be modified with lipid or fatty acid groups. In some embodiments, the fatty acid groups can be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid groups can be one or more of palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha linoleic acid, gamma linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid.

[0261] In some embodiments, the polymer can be a copolymer comprising lactic acid and glycolic acid units, e.g., poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA", and a polyester comprising homopolymers comprising glycolic acid units, collectively referred to herein as "PGA", and lactic acid units, e.g., 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[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof. The degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio. In some embodiments, PLGA to be used according to the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0262] In some embodiments, the polymer may be one or more acrylic polymers.In certain embodiments, the acrylic polymer may 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, polyacrylamides, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the above polymers.The acrylic polymer may include fully polymerized copolymers of acrylic and methacrylic acid esters with low content of quaternary ammonium groups.

[0263] In some embodiments, the polymer may be a cationic polymer. In general, cationic polymers can condense and / or protect the negatively charged strands of nucleic acids (e.g., DNA or its derivatives). Polymers containing amines, 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) are positively charged at physiological pH and form ion pairs with nucleic acids to mediate transfection in a variety of cell systems.

[0264] 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; Kwon et 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 esters) (Zhou et al, 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline esters) (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc, 121 :5633) and poly(4-hydroxy-L-proline esters) (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc, 121 :5633).

[0265] The properties of these and other polymers as well as methods for preparing them are well known in the art (e.g., U.S. Pat. Nos. 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., 2003, pp. 111-115 ... 123:9480;Lim et 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 (See, e.g., et al, 1999, Chem. Rev., 99:3181.) More generally, various methods for synthesizing specific suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al, Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386, and in U.S. Patent Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732, each of which is incorporated herein by reference in its entirety.

[0266] In some embodiments, the polymers may be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers may be substantially crosslinked to one another. In some embodiments, the polymers may be substantially free of crosslinks. In some embodiments, the polymers may be used in accordance with the present invention without a crosslinking step. It should further be understood that the synthetic nanoparticles of the present invention may include block copolymers, graft copolymers, blends, mixtures and / or adducts of any of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, but not comprehensive, list of polymers that may be used in accordance with the present invention.

[0267] In some embodiments, the synthetic nanoparticles may optionally include one or more amphiphilic entities. In some embodiments, the amphiphilic entities may facilitate the production of synthetic nanoparticles with increased stability, improved uniformity, or increased viscosity. In some embodiments, the amphiphilic entities may be associated with the inner surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in the production of synthetic nanoparticles according to the present invention.Such amphiphilic entities include, but are not limited to, phosphoglycerides, phosphatidylcholines, dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylethanolamine (DOPE), dioleyloxypropyltriethylammonium (DOTMA), dioleoylphosphatidylcholine, cholesterol, cholesterol esters, diacylglycerol, diacylglycerol succinate, diphosphatidylglycerol (DPPG), hexanedecanol, polyethyleneglycol, and the like. fatty alcohols such as polyoxyethylene (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, sorbitan trioleate (Span® 85) glycocholate, sorbitan monolaurate (Span® 20), polysorbate 20 (Tween® 20), polysorbate 60 (Tween® 60), polysorbate 65 (Tween® 65), Polysorbate 80 (Tween® 80), Polysorbate 85 (Tween® 85), polyoxyethylene monostearate, surfactin, poloxamer, sorbitan fatty acid esters such as sorbitan trioleate, lecithin, lysolecithin, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, dipalmitoylphosphatidylglycerol, ... These include cerol, stearylamine, dodecylamine, hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, 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, cyclodextrins, chaotropic salts, ion pairing agents and combinations thereof. The amphiphilic entity component can be a mixture of various amphiphilic entities.Those skilled in the art will recognize that this is an exemplary, but not comprehensive, list of substances with surfactant activity. Any amphiphilic entity can be used in the manufacture of synthetic nanoparticles to be used according to the present invention.

[0268] 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, glucoronic acid, galacturonic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, the carbohydrate is a polysaccharide, including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, the synthetic nanoparticles of the present invention do not include (or specifically 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.

[0269] In some embodiments, the nanoparticles comprise a peptide (e.g., SEQ ID NO: 13) comprising a sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16. In certain examples, the nanoparticles disclosed herein can be administered in combination with another therapy (e.g., APC-based therapy, CTL-based therapy, peptide vaccine therapy) as described below. In some examples, the nanoparticles disclosed herein can be administered to a human subject in combination with an immune agonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpoint inhibitor (e.g., anti-LAG3 antibody), and / or lenalidomide.

[0270] Antigen-presenting cell (APC)-based immunotherapy Ex vivo strategies for inducing an immune response in a subject may include contacting suitable antigen-presenting cells (e.g., dendritic cells, monocytes or macrophages) obtained from the subject with any of the peptides described herein. Alternatively, cells can be transfected with a nucleic acid (e.g., an expression vector) encoding one or more of the peptides, and optionally cultured under conditions that allow expression of the peptide for a period of time. Transfection methods vary depending on the type of cell and the nucleic acid that is transfected into the cell. After contacting and transfection, the cells are returned to the subject.

[0271] The cells can be of any of a variety of types that express MHC class I or II molecules. For example, the cells can include myeloid cells, macrophages, monocytes, dendritic cells, T cells (e.g., helper T cells, CD4+ cells, CD8+ cells, or cytotoxic T cells), or B cells.

[0272] Thus, the present disclosure provides compositions comprising APCs (e.g., dendritic cells), the APCs presenting a peptide sequence on their surface, the peptide sequence comprising 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 APCs are dendritic cells. 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 APCs acquire the peptide sequence in vitro by exposure to a synthetic peptide comprising the peptide sequence.

[0273] 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 a reagent (or immunogenic / antigenic composition) and administered to the subject.

[0274] In some embodiments, the composition comprises at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 In some embodiments, the composition comprises 10 APCs (e.g., dendritic cells). 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 The antibody comprises less than one APC (e.g., dendritic cell).

[0275] A. Preparation of antigen-presenting cells Antigen-presenting cells (APCs), e.g., 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 otherwise cultured and provided. APCs (e.g., DCs) can be found, for example, in mammalian bone marrow or peripheral blood mononuclear cells (PBMCs), in mammalian spleen, or in mammalian skin (i.e., Langerhans cells, which have certain qualities similar to those of DCs, can be found in the skin). For example, bone marrow can be collected from a mammal and cultured in a medium that promotes the growth of DCs. This medium may include GM-CSF, IL-4 and / or other cytokines (e.g., TNF-α), growth factors and supplements. After culturing in medium containing the appropriate cytokines for a suitable amount of time (e.g., 4, 6, 8, 10, 12 or 14 days suitable for expanding and differentiating DCs into mature DCs), clusters of DCs 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 a cancer vaccine using standard techniques. Antigens (e.g., isolated or purified peptides, or synthetic peptides) can be added to the cultures at a concentration of 1 μg / ml to 50 μg / ml per antigen, e.g., 2, 5, 10, 20, 30 or 40 μg / ml per antigen.

[0276] In some embodiments, APCs are isolated from a subject (e.g., a human). Mononuclear cells are isolated from blood using leukapheresis (e.g., using a COBE Spectra apheresis system). Mononuclear cells are allowed to become adherent by incubation at 37° C. for 2 hours in tissue culture flasks. Non-adherent cells are removed by washing. Adherent cells are cultured for 5 days in 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.). On day 5, TNF-α is added to the culture medium for an additional 3-4 days. On day 8 or 9, cells are harvested, washed, and incubated with peptide antigen for 16-20 hours on a tissue rotator. Peptide antigen is added to the culture at a concentration of approximately 10 μg / ml (per antigen).

[0277] As will be appreciated by those skilled in the art, various other methods can be used to isolate APC.DC occurs in small numbers in all tissues where they exist, which makes it necessary to isolate and enrich DC.Any of several procedures that require 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 DC. Guidance regarding 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., J. Immunol., 151: 6840-6852, 1993. One method for isolating DCs from human peripheral blood is described in US Patent No. 5,643,786.

[0278] The dendritic cells prepared according to the method described herein present the epitopes corresponding to the antigens at a higher average density than the epitopes present on the dendritic cells exposed to tumor lysate (e.g., neural tumor lysate). The relative density of one or more antigens on the antigen-presenting cells can be determined by both indirect and direct means. The primary immune response of naive animals is roughly proportional to the antigen density of the antigen-presenting cells (Bullock et al., J. Immunol., 170: 1822-1829, 2003). Thus, the relative antigen density between two populations of antigen-presenting cells can be estimated by immunizing animals with each population, isolating B or T cells, and monitoring the specific immune response against specific antigens, for example, by tetramer assay, ELISPOT or quantitative PCR.

[0279] Relative antigen density can also be measured directly. In one method, antigen-presenting cells are stained with an antibody that specifically binds to MHC-antigen complexes, and then the cells are analyzed to determine the relative amount of antibody binding to each cell (see, for example, 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, for example, as the percentage of cells that are positive for staining for individual MHC-antigen complexes or the average relative amount of staining per cell. In some embodiments, a histogram of the relative amount of staining per cell can be generated.

[0280] In some embodiments, antigen density can be measured directly, for example, by direct analysis of MHC-bound peptides by mass spectrometry (see, e.g., Purcell and Gorman, Mol. Cell. Proteomics, 3: 193-208, 2004). Typically, MHC-bound peptides are isolated by one of several methods. One method involves analyzing cell lysates of antigen-presenting cells, often after ultrafiltration to enrich for small peptides (see, e.g., Falk et al., J. Exp. Med., 174: 425-434, 1991; Rotzxhke et al., Nature, 348: 252-254, 1990). In another method, MHC-bound peptides are directly isolated from the cell surface, for example, 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 MHC-bound peptides are eluted by acid treatment (see, e.g., Falk et al., Nature, 351: 290-296). After isolation of MHC-bound peptides, the peptides are often analyzed by mass spectrometry after a separation step (e.g., liquid chromatography, capillary gel electrophoresis, or two-dimensional gel electrophoresis). Individual peptide antigens can be identified and quantified using mass spectrometry, and the relative average proportion of each antigen in a population of antigen-presenting cells can be determined. In some methods, the relative amounts of peptides in two populations of antigen-presenting cells are compared using stable isotope labeling of one population followed by mass spectrometry (Lemmel et al. al., Nat. Biotechnol., 22: 450-454, 2004).

[0281] b. Administration of antigen-presenting cells APC-based cancer vaccine can be delivered to patient or test animal by any suitable delivery route, which can include injection, infusion, inoculation, direct surgical delivery or any combination thereof.In some embodiments, cancer vaccine is administered to human in deltoid region or axillary region.For example, vaccine is administered as intradermal injection in axillary region.In some embodiments, vaccine is administered intravenously.

[0282] A suitable carrier for administering cells can be selected by one of skill in the art by routine techniques. For example, the pharmaceutical carrier can be a buffered saline solution, such as cell culture medium, and can contain DMSO to preserve cell viability.

[0283] The amount of APC suitable for administration to a patient as a cancer vaccine to accomplish the method of the present invention and the most convenient route of such administration may be based on a variety of factors, as well as the formulation of the vaccine itself. Some of these factors include the physical characteristics of the patient (e.g., age, weight and sex), the physical characteristics of the tumor (e.g., location, size, rate of growth and accessibility), and the extent to which other therapeutic methodologies (e.g., chemotherapy and beam radiation therapy) are being implemented in conjunction with the overall treatment regimen. Notwithstanding the various factors that must be considered in carrying out the method of the present invention to treat a disease state, a mammal may be administered approximately 10 mL of APC in about 0.05 mL to about 2 mL of solution (e.g., saline) in a single dose. 5 ~about 10 8 APCs (e.g., 10 7 Depending on the above and other factors, e.g., the severity of the tumor pathology, further administrations can be performed. In one embodiment, about 10 6 About 1 to about 5 administrations of the APCs are administered at 2-week intervals.

[0284] DC vaccination may be accompanied by other treatments. For example, the patient who receives DC vaccination may also be undergoing chemotherapy, radiation and / or surgical therapy at the same time. Methods of using DC vaccination with chemotherapy to treat cancer are described in Wheeler et al., US Patent Publication No. 2007 / 0020297. In some embodiments, the patient who receives DC vaccination has already undergone chemotherapy, radiation and / or surgical treatment for cancer. In one embodiment, the patient who receives DC vaccination is treated with COX-2 inhibitors, as described in Yu and Akasaki, WO2005 / 037995.

[0285] T cell-based immunotherapy Ex vivo methods for stimulating an immune response may also include contacting in vitro T cells (e.g., in a population of lymphocytes obtained from a subject) with an antigen-presenting cell (APC) expressing an MHC molecule that is bound to one of the peptides described herein for an amount of time (and under 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 generating and / or expanding BCMA-specific and / or TACI-specific T cells (e.g., cytotoxic T cells and / or CD4+ helper T cells). The methods include 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 may be cytotoxic T cells, e.g., memory cytotoxic T cells, effector cytotoxic T cells, or CD4+ helper T cells.

[0286] Activated T cells can be used to kill target cells. In some embodiments, the method comprises contacting a target cell with one or more BCMA-specific cytotoxic T cells, where the target cell expresses or overexpresses BCMA and expresses HLA-A. In some embodiments, the method comprises contacting a target cell with one or more TACI-specific cytotoxic T cells, where the target cell expresses or overexpresses TACI and expresses HLA-A.

[0287] 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 a peptide disclosed herein (e.g., one or more of SEQ ID NOs: 13-17), APCs presenting a BCMA (e.g., SEQ ID NO: 13 or 14) or TACI (SEQ ID NO: 16) peptide, lenalidomide, an immunomodulatory agent, a checkpoint inhibitor (e.g., anti-LAG3 antibody), or an immune agonist (e.g., anti-OX40, anti-GITR). In some embodiments, the additional therapeutic agent administered with the BCMA or TACI specific CTLT cells 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. In some embodiments, the T cells are administered in combination with an immune agonist, such as an anti-OX40 or anti-GITR antibody.

[0288] Activated T cells can also be reintroduced into the subject from which they were obtained, in some embodiments, T cells can be obtained from a subject of the same species other than the subject (allogeneic), contacted with a reagent (or immunogenic / antigenic composition), and administered to the subject.

[0289] In some embodiments, T cells are derived from in vitro induction in patient-derived peripheral blood mononuclear cells (PBMCs). Antigen-specific CTLs can be produced in vitro from patient-derived PBMCs using the following protocol. To generate dendritic cells, plastic-adherent cells obtained from PBMCs were cultured in AIM-V medium supplemented with recombinant human GM-CSF and recombinant human IL-4 in a humidified CO2-conditioned room temperature. 2 The cells are cultured in a 5% CO incubator at 37°C. After 6 days, the 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 diluted in PBS at 1 x 10 per mL with peptide (2 µg / mL). 6 The PBMCs are resuspended in 100 ml of PBS and incubated for 2 hours at 37° C. Autologous CD8+ T cells are enriched from the PBMCs using magnetic microbeads (Miltenyi Biotech, Auburn, Calif.). CD8+ T cells (2×10 per well) are 6 ) in each well of a 24-well tissue culture plate at 2 × 10 per well in 2 mL / well of AIM-V medium supplemented with 5% human AB serum and 10 units / mL of rhIL-7 (Cell Sciences). 5 The lymphocytes are co-cultured with autologous dendritic cells pulsed with peptides of 1000 to 10 ...

[0290] Chimeric antigen receptor (CAR) T cell-based immunotherapy The present disclosure further provides a method for adoptive transfer of T cells expressing chimeric antigen receptors to treat cancer. CAR-modified T cells can be engineered to target virtually any tumor-associated antigen (e.g., BCMA and / or TACI). Usually, T cells are engineered to express a CAR specifically directed against an antigen on the patient's tumor cells, and then infused back into the patient.

[0291] A common form of CAR is a fusion of a single chain variable fragment (scFv) fused to the CD3-zeta transmembrane and endodomain. The scFv can be derived from an antibody that specifically binds to an antigen-specific receptor or antigen on a T cell (e.g., a BCMA-specific cytotoxic T cell or a TACI-specific cytotoxic T cell).

[0292] In some embodiments, the sequence of the T cell receptor in the BCMA-specific cytotoxic T cells or the 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 the TACI-specific cytotoxic T cells can be used to generate a CAR.

[0293] In some embodiments, these T cells are collected from a patient. In some embodiments, these T cells are obtained from induced pluripotent stem cells (iPSCs).

[0294] A viral vector such as a retrovirus, lentivirus, or transposon is often used to integrate the transgene (e.g., CAR) into the host cell genome. Alternatively, a non-integrating vector such as a plasmid or mRNA can be used to transfer the CAR gene to T cells, causing the T cells to express the CAR under appropriate conditions.

[0295] Induced pluripotent stem cell approach Adoptive T cell therapy, using the administration of large numbers of ex vivo expanded activated antigen-specific cytotoxic T lymphocytes (CTLs) targeting tumor-specific antigens, has induced durable remissions in selected malignancies. Utilizing TCRs that primarily recognize intracellular antigens that have already been processed and presented as peptide complexes with MHC molecules (Johnson et al. 2009; Morgan et al. 2006) can further enhance tumor selectivity, but the introduction of exogenous TCR genes can result in mismatches of the transferred and endogenous α and β chains, resulting in severe autoimmune adverse events (Bendle et al. 2010, Hinrichs et al. 2013). In contrast, CAR-T recognizes antigens expressed on the cell surface in an MHC-unrestricted manner. To date, the most successful CAR-T therapy targeting the B cell antigen CD19 has achieved minimal residual disease-negative complete responses in patients with relapsed and chemotherapy-resistant B cell malignancies (Kochenderfer et al. 2010, Grupp et al. 2013). Nevertheless, ongoing efforts are directed to minimize adverse effects, including cytokine release syndrome, and improve the durability of responses (Brentjens et al. 2011, Kalos et al. 2011, Kochenderfer et al. 2012, Porter et al. 2011). Importantly, CTLs continuously exposed to tumor antigens during long-term expansion used for TCR-based or CAR-based therapy may lose their proliferative capacity (become "exhausted") and lose their functional activity upon terminal differentiation.

[0296] To overcome these limitations, a technology currently being developed is the use of fully rejuvenated CTLs from "induced pluripotent stem cells (iPSCs)". These iPSCs are a special type of pluripotent cells derived from adult somatic cells upon ectopic expression of a defined set of transcription factors. Importantly, tumor antigen-specific CTLs can be reprogrammed by iPSC technology from antigen-specific CTLs (Vizcardo et al. 2013, Ando et al. 2014). al. 2015, Timmermans et al. 2009, Kennedy et al. 2012). These iPSC-CTLs are functionally rejuvenated and demonstrate longer telomeres (1.5-fold increase) and higher proliferative capacity (5-50-fold increase) than the original CTLs from which they were derived (Nishimura et al. 2013). This powerful reprogramming therapeutic approach has the potential to significantly increase the efficacy and durability of antigen-specific cancer immunotherapy. Thus, the present disclosure provides a method of rejuvenating cytotoxic T cells. In some embodiments, the method can increase proliferative capacity by at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100-fold.

[0297] Activation of tumor-specific CTLs is the main 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 of adaptive immunotherapy using T cells. iPSC technology can be used to improve the efficacy of adoptive cell transfer immunotherapy (ACT).

[0298] iPSCs can be obtained from differentiated cells (e.g., fibroblasts, immune cells, T cells, B cells) induced by retroviral transfection of Yamanaka factors (a combination of Oct3 / 4, Sox2, Klf4 and c-Myc) and differentiated into the T cell lineage by culturing them in monolayer OP9-DL1 cell line in addition to Flt-3 ligand and IL-7.

[0299] In some embodiments, iPSCs can be generated from T cells. These cells are redifferentiated into T cells after expansion. Human T lymphocytes can serve as a cell source for iPSC generation. Peripheral blood mononuclear cells (PBMCs) can be separated from whole blood by leukapheresis or venipuncture, and then CD3+ T cells can be expanded by stimulation with IL-2 and anti-CD3 antibodies. T cell-derived iPSCs (TiPS) can be generated from activated T cells when exposed to retroviral transduction of reprogramming factors. These T-iPSCs preserve their original T cell receptor (TCR) gene rearrangements, so they can be used as an unlimited source of hematopoietic stem cells with endogenous tumor-specific TCR genes for cancer ACT therapy.

[0300] Thus, in some embodiments, iPSCs are generated from antigen-specific cytotoxic T cells. These antigen-specific T cells are generated by methods as described herein, for example, by contacting one or more T cells with antigen-presenting cells pulsed with a peptide comprising one or more amino acid sequences as described herein (e.g., SEQ ID NOs: 1-17). T-iPSCs preserve their original T cell receptor (TCR) gene rearrangements, so that after these T-iPSCs differentiate into T cells, these T cells can recognize BCMA and / or TACI on cancer cells.

[0301] In some embodiments, a nucleic acid encoding a CAR that specifically recognizes BCMA and / or TACI can be introduced into T-iPSCs. After these T-iPSCs differentiate into T cells, these T cells can recognize BCMA and / or TACI on cancer cells.

[0302] In some embodiments, the differentiated T cells are administered to the subject. In some embodiments, T-iPSCs are administered to the subject, and these cells then differentiate into cytotoxic T cells in the subject's body.

[0303] Subject A subject may be any animal capable of immune response to an antigen. The terms "subject" and "patient" are used interchangeably throughout the present specification to describe an animal, human or non-human, to which treatment according to the method of the present disclosure is provided. Veterinary and non-veterinary applications are contemplated by the present invention. A human patient may be an adult human or a juvenile human (e.g., a human under the age of 18 years). In addition to humans, subjects include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, mini pigs), horses, dogs, cats, cows, and other domestic, livestock, and zoo animals.

[0304] The subject may have cancer, be suspected of having cancer, or be at risk of developing cancer. As used herein, the term "cancer" refers to cells capable of autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. The term "tumor" as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that may be treated or diagnosed using the methods described herein include, for example, malignancies of various organ systems affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer, and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. In some embodiments, the agents described herein are designed to treat or diagnose carcinoma in a subject. The term "carcinoma" is art-recognized and refers to malignant diseases of epithelial or endocrine tissues, including respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, testicular carcinoma, breast cancer, prostate carcinoma, endocrine system carcinoma, and melanoma. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues, for example. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal derivation. In some embodiments, the subject has a hematological cancer, for example, multiple myeloma, leukemia, non-Hodgkin's lymphoma, or Hodgkin's lymphoma.

[0305] In some embodiments, the subject has a BCMA expressing / overexpressing disease or a TACI expressing / overexpressing disease, including, for example, multiple myeloma, a B cell associated malignancy, a plasma cell associated malignancy, a pre-malignant disease (e.g., a pre-malignant disease of MM, e.g., SMM or MGUS).

[0306] In some embodiments, the subject may have, be suspected of having, or be 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 clonal plasma cell (PC) proliferation and hypersecretion of paraproteins (e.g., monoclonal immunoglobulins and / or free light chains (FLC)).

[0307] Non-limiting examples of plasma cell disorders include Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple Myeloma (MM), Waldenstrom's Macroglobulinemia (WM), Light Chain Amyloidosis (AL), Monocentric Plasmacytoma (e.g., Monocentric Plasmacytoma of the bone or Extramedullary Plasmacytoma), Polyneuropathy, Organomegaly, Endocrinopathy Monoclonal Gammopathy and Cutaneous Changes Syndrome (POEMS), and Heavy Chain Disease. Other plasma cell disorders include, for example, Monoclonal Gammopathy of Renal Significance (MGRS), MGUS-associated neuropathy, and other proteinogenic neuropathy.

[0308] MGUS, smoldering MM (SMM) and symptomatic MM represent a spectrum of the same disease. Symptomatic or active multiple myeloma is defined as any level of monoclonal protein and the presence of end-organ damage consisting of any form of myeloma-defyning events in the CRAB criteria (hypercalcemia, renal failure, anemia or bone lesions likely associated with a plasma cell clone) or any of the novel biomarkers of malignancy (BM involvement by clonal plasma cells equal to or greater than 60%; ratio of involved to uninvolved FLCs equal to or greater than 100 and / or presence of two or more bone lesions on MRI (Kyle RA et al., Leukemia, 23: 3-9 (2009); Rajkumar VS et al, Lancet Oncology, 15: 12, MM is characterized by >10% BM infiltration by clonal plasma cells and / or biopsy-proven plasmacytoma in addition to MM-associated clonal plasma cells (Kyle RA et al., N. Engl. J. Med., 351: 1860-73 (2014)). MM is a plasma cell malignancy that characteristically involves excessive infiltration of the bone marrow (BM) and occasionally formation of plasmacytomas as discrete clusters of malignant plasma cells inside or outside the BM space (Kyle RA et al., N. Engl. J. Med., 351: 1860-73 (2004)). The consequences of the 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 variably results in fatigue, increased susceptibility to infections, and less commonly, an increased tendency to bleeding. Disease involvement in bone produces osteolytic lesions, resulting in bone pain and may be associated with hypercalcemia (Kyle RA et al., Blood, 111: 2962-72 (2008).

[0309] 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 defynying biomarkers of malignancy (Rajkumar et al. al, Lancet, 2014). Natural history studies of SMM suggest that there are two distinct 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 progressive increase in M ​​protein and a shorter median time to progression (TTP) to active multiple myeloma of 1.3 years. Non-evolving SMM has a more stable M protein that can change rapidly with time to progression to active multiple myeloma with a median TTP of 3.9 years.

[0310] Monoclonal gammopathy of undetermined significance (MGUS) is a condition in which abnormal immunoglobulin proteins (known as paraproteins) are found in the blood during standard laboratory blood tests. MGUS resembles multiple myeloma and similar diseases, but has lower levels of antibodies, fewer plasma cells (antibody-secreting white blood cells) in the bone marrow, and no symptoms or major problems.

[0311] In some embodiments, the subject has multiple myeloma, SMM, or MGUS. In some embodiments, the subject may be in remission from multiple myeloma. In some embodiments, the subject has a pre-malignant disease (e.g., MM, e.g., SMM, or MGUS).

[0312] In some embodiments, the subject may have a type of cancer that expresses or overexpresses BCMA or TACI. Thus, the method may include determining whether one or more cancer cells of the subject's cancer (e.g., multiple myeloma) express or overexpress BCMA or TACI before administering one or more peptides (or nucleic acids) to the subject. 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, and include, for example, enzyme-linked immunosorbent assay (ELISA), Western and dot blotting techniques or immunohistochemistry techniques for detecting protein, and reverse transcription polymerase chain reaction (RT-PCR) or Northern blotting techniques for detecting mRNA. In some embodiments, the average level of BCMA or TACI expression in cancer cells is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher than the average level of BCMA or TACI expression in normal cells (e.g., normal tissue cells in the same subject, normal plasma cells in the same subject, or tissue or plasma cells in a healthy subject). In some embodiments, the average level of BCMA or TACI expression in cancer cells is at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or 50-fold higher than the average level of BCMA or TACI expression in normal cells (e.g., normal tissue cells in the same subject, normal plasma cells in the same subject, or tissue or plasma cells in a healthy subject).

[0313] A 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 one who has one or more symptoms of cancer. 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 shortness of breath, hemoptysis, blood in urine, blood in stool, nausea, vomiting, abdominal fullness, bloating, fluid in the abdominal cavity, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, excessive sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, difficulty swallowing, etc. Symptoms of multiple myeloma specifically include, for example, bone pain (e.g., in the back or ribs), high levels of calcium in the blood, excessive thirst or urination, constipation, nausea, loss of appetite, confusion, weakness or numbness in the legs, weight loss, or recurring infections.

[0314] As used herein, a subject "at risk of developing cancer" is a subject who 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 BRCAl, p53, RB, or APC), has been exposed to a condition that may lead to cancer, or is currently affected by a condition. Thus, a subject may also be "at risk of developing cancer" if the subject is exposed to mutagenic or carcinogenic levels of a particular compound (e.g., carcinogenic compounds in tobacco smoke, such as acrolein, 4-aminobiphenyl, aromatic amines, benzene, benzo[a]anthracene, benzo[a]pyrene, formaldehyde, hydrazine, polonium-210 (radon), urethane, or vinyl chloride). A subject may be "at risk of developing cancer" if the subject is, for example, exposed to high doses of ultraviolet or X-ray radiation, or exposed to (e.g., infected with) a tumor-causing / tumor-associated virus, such as papilloma virus, Epstein-Barr virus, Hepatitis B virus, or human T-cell leukemia-lymphoma virus. Additionally, a subject may be "at risk of developing cancer" if the subject suffers from inflammation (e.g., chronic inflammation). A subject may be at risk of developing multiple myeloma, for example, if the subject has monoclonal gammopathy of undetermined significance (MGUS). Thus, it is understood that subjects "suspected of having cancer" or "at risk of developing cancer" are not all subjects within a species of interest.

[0315] In some embodiments, the method may also include a step of determining whether the subject has cancer. Suitable methods for such determination vary depending 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, a medical professional may diagnose a subject as having multiple myeloma if the subject shows two or more (e.g., three, four, five, or six or more) symptoms of multiple myeloma, such as any of those described herein. A subject may also be determined to have multiple myeloma by measuring blood calcium levels, white or red blood cell counts, or the amount of protein in the subject's urine.

[0316] MHC molecule multimer The present disclosure also features a composition comprising (i) one or more of any of the peptides described herein and (ii) a major histocompatibility complex (MHC) molecule multimer. The multimer contains two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) whole 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.

[0317] The MHC molecules of the multimer can be MHC class I molecules (e.g., HLA-A2 molecules) or MHC class II molecules. The MHC molecules can be mammalian (e.g., rodent, non-human primate, human, or any other mammalian molecule described herein) MHC molecules.

[0318] The two or more MHC molecules (or peptide binding regions of the MHC molecules) in the multimer may be derived from the same MHC molecule or from different MHC molecules. For example, an MHC molecule multimer may contain five MHC molecules, three of which are the same MHC molecule 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 at least one of the peptides.

[0319] In some embodiments, the composition may contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 15 or more) of any of the peptides described herein.

[0320] The composition may also be associated with a detectable label. For example, one or more of the MHC molecules of the multimer may be covalently or non-covalently linked to a detectable label. Suitable detectable labels (e.g., enzymes, fluorescent substances, luminescent substances, bioluminescent substances or radionuclides) as well as methods for linking detectable labels to peptides or MHC molecules are also provided.

[0321] MHC multimer compositions can be made using the peptides described herein as follows: peptides that bind to HLA molecules are refolded in the presence of the corresponding HLA heavy chain and β2 microglobulin to make a trimolecular complex. The complex is then biotinylated at the carboxyl terminus of the heavy chain at a site previously engineered into the heavy chain. Multimer formation is then induced by the addition of streptavidin.

[0322] Because T cell receptors can recognize specific peptide-MHC complexes on target cells among a variety of other peptide-MHC complexes, the MHC multimer compositions described herein can be used, for example, to detect antigen-specific T cells in a population of unrelated T cells. For such assays, the multimers are generally detectably labeled.

[0323] For example, the multimeric MHC molecule / peptide complex can be used in an assay to evaluate peripheral blood mononuclear cells for the presence of antigen-specific CTLs after exposure to an immunogen. The MHC multimeric complex 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 determine the frequency of antigen-specific CTL populations in a sample of peripheral blood mononuclear cells. In some embodiments, detectably labeled streptavidin, which is used to multimerize the MHC multimer, can be used to label T cells that bind to the multimeric MHC molecule / peptide complex. To do this, the cells treated with the multimer are exposed to, for example, a label (e.g., a fluorophore conjugated to biotin). The cells can then be easily isolated or detected, for example, using flow cytometry.

[0324] The peptides (and pharmaceutical compositions thereof), MHC multimers containing compositions, kits and articles of manufacture described herein can be used in various ways.For example, the peptides can be used to (i) induce immune responses in subjects (e.g., subjects with cancer), (ii) activate T cells in culture, and / or (iii) treat or prevent events in cancers such as multiple myeloma.As described herein, the MHC multimers containing compositions can be used, for example, to detect antigen-specific T cells in unrelated T cell populations.

[0325] Methods for Producing Antibodies in a Subject Methods for producing antibodies specific to immunogens (e.g., any one or more of the 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 animals 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.

[0326] The peptides can be used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other mammal, such as a human) with the peptide. An appropriate immunogenic preparation can contain, for example, any of the compositions described herein. The preparation can further 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), E. coli heat-labile toxin (LT), mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185: 1203-1210), and mutant E. coli heat-labile toxin (MLT) (Di Tommaso et al. (1996) Infect. Immunity 64: 974-979). MCT and MLT contain point mutations that substantially reduce toxicity without substantially impairing adjuvant activity relative to those of parent molecules. Immunization of a suitable subject with an immunogenic peptide preparation (e.g., any of the compositions described herein) induces a polyclonal anti-peptide antibody response. In some embodiments, toll-like receptor-3 ligand (e.g., poly-ICLC), interferon alpha (IFNα), interferon gamma (IFNγ) or granulocyte macrophage colony-stimulating factor (GM-CSF) can be administered to the subject, for example, to boost immune response.

[0327] The term antibody, as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen-binding site that specifically binds to a peptide, such as a peptide described herein). Antibodies that specifically bind to the peptides described herein are antibodies that bind to the peptide but do not substantially bind to other molecules in a sample. Examples of immunologically active portions of immunoglobulin molecules include, for example, F(ab) fragments, F(ab')2 fragments, or any other antibody fragments described herein.

[0328] The isolated antibody or antigen-binding fragment thereof produced by the methods described herein can selectively bind to an epitope within or overlapping with any of the amino acid sequences of SEQ ID NOs: 1-17. The antibody can also cross-block binding of antibodies that bind to an epitope within or overlapping with any of the amino acid sequences of SEQ ID NOs: 1-17. Typically, binding of an antibody to an epitope occurs within 10 -6 An antibody is considered selective if it binds with a KD less than M. If necessary, the binding conditions can be modified to reduce non-specific binding without substantially affecting selective binding. A "cross-blocking" antibody or a "cross-blocking antibody" refers to a first antibody that, when bound to an epitope (e.g., contained within or overlapping with any of SEQ ID NOs: 1-17), 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). It is understood that the antibodies produced by the methods described herein (e.g., antibodies specific for one or more of the peptides described herein) can be used to detect, for example, cancer cells expressing BCMA or TACI, and are therefore useful in a number of exemplary methods described herein.

[0329] Immunologic testing The antigen-specific cellular immune response of vaccinated subjects can be monitored by several different assays, such as tetramer assays, ELISPOT and quantitative PCR. These methods and protocols are described, for example, in Current Protocols in Immunology, Coligan, J. et al., Eds., (John Wiley & Sons, Inc.; New York, NY).

[0330] Tetramer assays can be used to detect and quantitate T cells that are specific for a given antigen in blood samples. Tetramers composed of recombinant MHC molecules complexed with peptides can be used to identify populations of antigen-specific T cells. To detect T cells specific for antigens, fluorescently labeled specific peptide tetramer complexes (e.g., phycoerythrin (PE)-tHLA) containing peptides from these antigens have been synthesized and provided by Beckman Coulter (San Diego, Calif.). Specific CTL clone CD8 cells were cultured at 10 5 Resuspend cells / 50 μl FACS buffer (phosphate buffer and 1% inactivated FCS buffer). Incubate cells with 1 μl tHLA for 30 min at room temperature and continue incubation with 10 μl anti-CD8 mAb (Becton Dickinson, San Jose, Calif.) for 30 min at 4° C. Wash cells twice with 2 ml cold FACS buffer and then analyze by FACS (Becton Dickinson).

[0331] For example, ELISPOT assays can be used to detect cytokine-secreting cells to determine whether cells in a vaccinated patient secrete cytokines in response to an antigen, thereby demonstrating whether an antigen-specific response has been elicited. ELISPOT assay kits are supplied by R & D Systems (Minneapolis, Minn.) and are performed as described in the manufacturer's instructions. 1 x 10 responders (R) obtained before and after vaccination were5 PBMC cells of patients are plated in 96-well plates with nitrocellulose membrane inserts coated with capture Ab. Stimulator (S) cells (antigen-pulsed TAP-deficient T2 cells) are added at a 1:1 R:S ratio. After 24 h incubation, cells are removed by washing the plate four times. Detection Ab is added to each well. Plates are incubated overnight at 4° C. and the washing step is repeated. After 2 h incubation with streptavidin-AP, plates are washed. An aliquot (100 μl) of BCIP / NBT chromogen is added to each well to develop the spots. After 60 min, the reaction is stopped by washing with water. Spots are scanned and counted using computer-assisted image analysis (Cellular Technology Ltd, Cleveland, Ohio). If the experimental value is significantly different from the mean number of spots for non-pulsed T2 cells (background value) as determined by a two-tailed Wilcoxon rank sum test, the background value is subtracted from the experimental value.

[0332] Quantitative PCR is another means to assess immune responses. To examine IFN-γ production in patients by quantitative PCR, cryopreserved PBMCs and autologous dendritic cells from the patient's pre- and post-vaccination samples are thawed in RPMI DC culture medium with 10% patient serum, washed, and counted. PBMCs are plated at 3×10 in 2 ml of medium in 24-well plates. 6 Plate 1 x 10 PBMCs and 1 x 10 dendritic cells. 6 Dendritic cells are plated at 1x10 cells / ml and pulsed with 10 μg / ml tumor peptide in 2 ml per well in a 24-well plate for 24 hours. Dendritic cells are harvested, washed, counted and diluted to 1x10 6 Dilute to 3 × 10 cells / ml and inject 3 × 10 cells into wells with PBMCs. 5100 μl of solution (i.e., 300 μl of solution) (DC:PBMC=1:10) is added. Every 3-4 days, 2.3 μl of IL-2 (300 IU / mL) is added, and the cells are harvested between the 10th and 13th days after the start of the culture. The harvested cells are then stimulated for 4 hours at 37°C with tumor cells or autologous PBMC pulsed with 10 μg / ml of tumor peptide. On days 11-13, the cultures are harvested, washed twice, and then split into 4 different wells, with two wells used for control (without target) and another two wells of CTLs co-cultured (1:1) with tumor cells if tumor cells are available. If tumor cells are not available, 10 μg / ml of tumor lysate is added to the CTLs. After 4 hours of stimulation, the cells are collected, RNA is extracted, and IFN-γ and CD8 mRNA expression is evaluated using a thermocycler / fluorescence camera system. PCR amplification efficiency follows a natural logarithmic progression, and linear regression analysis demonstrates a correlation coefficient of >0.99. Based on experimental analysis, a difference of one cycle is interpreted as a 2-fold difference in mRNA abundance, and CD8-normalized IFN-γ abundance is determined. A >1.5-fold increase in post-vaccine over pre-vaccine IFN-γ is the established standard for a positive type I vaccine response.

[0333] How to Choose a Therapy A method of selecting a therapy for a subject having cancer (e.g., a plasma cell disorder such as multiple myeloma or any cancer in which BCMA or TACI is expressed or overexpressed) optionally includes determining whether one or more cancer cells of the subject express or overexpress BCMA or TACI, and if 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 has no more than four substitutions of the amino acid sequence of any of SEQ ID NOs: 1-17) as a therapy for the subject, provided that the amino acid sequence (i) is capable of inducing an immune response in the subject, (ii) is capable of binding to an MHC molecule, and (iii) is capable of being recognized in the context of an MHC molecule by a T cell receptor on a T cell.

[0334] In some embodiments, the method further includes determining whether one or more cancer cells of the subject express an MHC molecule, e.g., an MHC class I molecule (e.g., HLA-A2) or an MHC class II molecule.

[0335] It is understood that a suitable combination of peptides can be delivered to a subject when one or more cells (e.g., plasma cells) of a cancer of a subject express or overexpress both BCMA and TACI. For example, when one or more cells (e.g., plasma cells) of a cancer of a subject are determined to express or overexpress both BCMA and TACI, a method of selecting a therapy can include selecting a combination of a composition containing at least one peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1-6 and 13-14 and a composition containing at least one peptide comprising any one of the amino acid sequences of SEQ ID NOs: 7-12 and 15-17 as a therapy for the subject.

[0336] Methods for determining whether one or more cells express BCMA, TACI and / or MHC molecules are known in the art. For example, a biological sample (e.g., a blood sample or a lymph node tissue sample) obtained from a subject can be tested using a BCMA and / or TACI specific antibody produced by the methods described herein to detect the presence or amount of BCMA and TACI polypeptides expressed by a cell (or cell lysate). Methods for assaying a biological sample for the presence or amount of a polypeptide include, for example, ELISA, immunohistochemistry, flow cytometry, Western blotting or dot blotting assays. In some embodiments, any of the methods described herein can also include a step of providing a biological sample obtained from a subject and / or a step of obtaining a biological sample from a subject. Biological samples suitable for the methods described herein include any biological fluid, cell, tissue or fraction thereof that contains the analyte protein of interest. A biological sample can be, for example, a specimen obtained from a subject or can be derived from such a subject. For example, the sample may be a tissue section obtained by biopsy, or cells placed in or adapted to tissue culture. A biological sample may also be a biological fluid containing cells, such as urine, blood, plasma, serum, saliva, semen, sputum, cerebrospinal fluid, tears, mucus, or aspirates (e.g., lung or breast nipple aspirates) or such samples absorbed onto paper or polymeric substrates. A biological sample may be further fractionated into fractions containing specific cell types, if desired. For example, a blood sample may be fractionated into serum, or into fractions containing specific types of blood cells, such as red blood cells or white blood cells (white blood cells). If desired, a sample may be a combination of sample types obtained from a subject, such as a combination of tissue and biological fluid.

[0337] Biological samples can be obtained from subjects, for example, subjects who have, are suspected of having, or are at risk of developing cancer (e.g., multiple myeloma). Any suitable method for obtaining biological samples can be used, but exemplary methods include, for example, venisection, swab (e.g., buccal swab), aspirate, or fine needle aspiration biopsy procedures. Non-limiting examples of tissues susceptible to fine needle aspiration include lymph nodes, lung, thyroid, breast, and liver. Samples can also be collected, for example, by microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder washing, smear (PAP smear) or ductal lavage.

[0338] The medical practitioner can also select, prescribe and / or administer one or more additional therapeutic agents for treating cancer or one or more medicines for treating side effects of anti-cancer drugs. 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 anti-cancer drugs include, for example, anemia, gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea), leukopenia (a decrease in white blood cell count that can lead to infection), temporary hair loss or thrombocytopenia (a decrease in platelet count that can lead to bleeding). Thus, the medical practitioner can prescribe or administer a chemotherapeutic agent such as vincristine to a subject together with an anti-anemia drug such as epoetin alpha (e.g., Procrit® or Epogen®).

[0339] Nucleic Acid Vaccines The present disclosure provides a nucleic acid vaccine (NAV) comprising one or more polynucleotides, e.g., polynucleotide constructs, encoding one or more polypeptides as described herein. Exemplary polynucleotides include, e.g., DNA, RNA, polynucleotide constructs comprising RNA polynucleotides encoding antigens, e.g., mRNA. In some embodiments, the polynucleotide, e.g., RNA polynucleotides encoding antigens, may comprise at least one chemical modification. In some embodiments, the nucleic acid vaccine may be formulated in a polymer or liposomal nanocarrier (e.g., nanoparticle).

[0340] In some embodiments, an adjuvant or immune enhancer can also be administered with or in combination with one or more NAVs. In some embodiments, the adjuvant acts as a co-signal to prime T cells and / or B cells and / or NK cells.

[0341] NAVs can vary in their valency. Valency refers to the number of antigenic components in the NAV or NAV polynucleotide (e.g., RNA polynucleotide) or polypeptide. In some embodiments, the NAV is monovalent. In some embodiments, the NAV is bivalent. In some embodiments, the NAV is trivalent. In some embodiments, the NAV is multivalent. Multivalent vaccines can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic components of the NAV can be on a single polynucleotide or on separate polynucleotides.

[0342] NAVs can be used as therapeutic or prophylactic agents. They are provided for use in medicine and / or for priming immune effector cells, e.g., stimulating / transfecting peripheral blood mononuclear cells (PBMCs) ex vivo and reinfusing activated cells. For example, the NAVs described herein can be administered to a subject, where the polynucleotide is translated in vivo to produce an antigen. Compositions, methods, kits and reagents are provided for the diagnosis, treatment or prevention of diseases or conditions in humans and other mammals. Active therapeutic agents can include NAVs, cells containing NAVs or polypeptides translated from polynucleotides contained in said NAVs.

[0343] Provided herein is a method 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. A cell, tissue, or organism is contacted with an effective amount of a composition containing a NAV that contains a polynucleotide having at least one translatable region that encodes a polypeptide (e.g., an antigen or immunogen) of interest.

[0344] An "effective amount" of a NAV composition is provided based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides) and other components of the NAV, as well as other determinants. In general, an effective amount of a NAV composition preferably provides an induced or boosted immune response as a function of antigen production in cells that is more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen. Increased antigen production may 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 (e.g., as demonstrated by an increased duration of protein translation from a modified polynucleotide), or altered innate immune response of the host cell.

[0345] The present disclosure also provides a method for inducing in vivo translation of a polypeptide antigen in a mammalian subject in need thereof. In it, an effective amount of a NAV composition containing a polynucleotide having at least one structural or chemical modification and a translatable region that encodes a polypeptide (e.g., an antigen or immunogen) is administered to the subject using the delivery method described herein. The polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell. The cell in which the polynucleotide is localized or the tissue in which the cell resides can be targeted using one or more rounds of NAV administration.

[0346] The proteins described herein can be engineered for localization within a cell, potentially within a specific compartment such as the cytoplasm or nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.

[0347] In some embodiments, nucleic acids (e.g., DNA, RNA) may have one or more modifications. In some embodiments, nucleic acid molecules (e.g., RNA molecules) as described herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. Backbone modifications in the context of the present invention are modifications in which the backbone phosphate of the nucleotide contained in the nucleic acid molecule as defined herein is chemically modified. Sugar modifications in the context of the present invention are chemical modifications of the sugar of the nucleotide of the nucleic acid molecule as defined herein. Furthermore, base modifications in the context of the present invention are chemical modifications of the base portion of the nucleotide of the nucleic acid molecule of the nucleic acid molecule. In this context, it is preferred that the nucleotide analogs or modifications are selected from nucleotide analogs that are applicable to transcription and / or translation.

[0348] Modified nucleosides and nucleotides that may be incorporated into nucleic acid molecules can be modified at the sugar moiety. For example, the 2' hydroxyl group (OH) of an RNA molecule can be modified and replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH 2 CH 2 O)nCH 2 CH 2 OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, for example, by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, where the amino group (e.g., NRR) can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0349] The sugar group can also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acid molecules can include, for example, nucleotides that contain arabinose as the sugar.

[0350] In modified nucleosides and nucleotides that can be incorporated into nucleic acid molecules (e.g., RNA) as described herein, the phosphate backbone can be further modified. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can include the complete replacement of unmodified phosphate moieties with modified phosphates as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by replacing linking oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0351] The modified nucleosides and nucleotides that can be incorporated into nucleic acid molecules (e.g., RNA molecules) as described herein can be further modified at the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified at the major groove surface. In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0352] In some embodiments, the nucleotide analog / modification may be, 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'-amino-2'-deoxycytidine ... phosphate, 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, Cytidine-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-aza The base modifications may be selected from adenosine-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.Particularly preferred are base-modified nucleotides selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate.

[0353] In some embodiments, the nucleic acid molecule can be modified by the addition of a so-called "5' cap" structure. The 5'-cap is generally an entity, usually a modified nucleotide entity, that "caps" the 5' end of a mature mRNA. The 5'-cap can usually be formed by a modified nucleotide, in particular a derivative of a guanine nucleotide. Preferably, the 5'-cap is linked to the 5' end via a 5'-5'-triphosphate linkage. The 5'-cap can be methylated, for example m7GpppN, where N is the terminal 5' nucleotide of the nucleic acid bearing the 5'-cap, usually the 5' end of the RNA. m7GpppN is a 5' cap structure that is naturally present in mRNA transcribed by polymerase II, and is therefore not considered a modification to be included in the modified RNA according to the present invention.

[0354] Methods of making and using nucleic acid vaccines are described, for example, in US20070269451, US20160317647, US9872900, and US2017002984, each of which is incorporated by reference in its entirety.

[0355] Pharmaceutical Compositions Any of the peptides, peptide-encoding nucleic acids, nanoparticles and cells described herein can be incorporated into pharmaceutical compositions. The compositions can include one or more of the peptides (and / or peptide-encoding nucleic acids) and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration. One or more peptides can be formulated as pharmaceutical compositions in the form of syrup, elixir, suspension, powder, granules, tablets, capsules, lozenges, troches, aqueous solutions, creams, ointments, lotions, gels, emulsions and the like. Complementary active compounds (e.g., one or more chemotherapeutic agents) can also be incorporated into the composition.

[0356] Pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include oral, rectal and parenteral, such as intravenous, intramuscular, intradermal, subcutaneous, inhalation, transdermal or transmucosal. Solutions or suspensions used for parenteral application may contain the following components: sterile diluents, such as water for injection, saline solution, hardened oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate buffer, citrate buffer or phosphate buffer, and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. The composition can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0357] 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™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the pharmaceutical composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against any contamination by microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial contamination can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be expected that it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be facilitated by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0358] Sterile injectable solutions can be prepared by incorporating one or more of the peptides (or one or more nucleic acids encoding the peptides) in the required amount in a suitable solvent with one or a combination of ingredients as required, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the peptides (or nucleic acids encoding the peptides) into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method can include vacuum drying or freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0359] Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, one or more peptides can be incorporated with excipients and used in the form of tablets, troches or capsules, for example, gelatin capsules. For use as mouthwash, oral compositions can also be prepared using fluid carriers. Pharmaceutically compatible binding agents 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 a similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate or orange flavoring.

[0360] Powders and tablets may contain 1%-95% (w / w) of individual peptides or mixtures of two or more peptides. In certain embodiments, the peptides may range from about 5%-70% (w / w). Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term "preparation" is intended to include formulations of peptides (or nucleic acids) with encapsulating material as a carrier, in which the peptide, with or without other carriers, is surrounded by the carrier, thus providing a capsule associated therewith. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges may be used as solid dosage forms suitable for oral administration.

[0361] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavorings, 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, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.

[0362] For administration by inhalation, the peptides or nucleic acids can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0363] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate for the barrier to be permeated by the formulation is used. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the peptide or nucleic acid can be formulated into ointments, salves, gels or creams, as generally known in the art.

[0364] The peptides or nucleic acids can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0365] In some embodiments, peptides or nucleic acids can be prepared with carriers that protect peptides from rapid elimination from the body, such as controlled release formulations, 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. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including, for example, liposomes targeted to APCs using monoclonal antibodies against APC-specific antigens) can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Pat. No. 4,522,811.

[0366] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically separate unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of peptide (or nucleic acid) calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.Dosage unit may also be accompanied by instructions for use.

[0367] The nucleic acid molecule encoding the peptide can be inserted into a vector and used as a gene therapy vector. The gene therapy vector can be delivered to a subject, for example, by 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 vehicle is embedded. Alternatively, if the complete gene delivery vector, for example, a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0368] Additional examples of gene delivery vehicles include, but are not limited to, liposomes, biocompatible polymers including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, bacteria, viruses such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vehicles commonly used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as for simple protein expression.

[0369] Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, etc. Liposomes containing a targeting moiety, e.g., an antibody or fragment thereof, can also be used to prepare a pharmaceutical composition of the nucleic acid for delivery to a subject.

[0370] Any of the pharmaceutical compositions described herein can be included in a container, pack, or dispenser together with instructions for administration, as described below.

[0371] Kits and Manufactured Products The present disclosure also features various kits. The kit may, for example, comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of any of the peptides described herein (or expression vectors containing nucleic acid sequences encoding one or more peptides) and instructions for administering the peptides to a subject. The kit may comprise one or more pharma- ceutically acceptable carriers and / or one or more immune stimulants. The immune stimulant may, for example, be a helper T epitope, a modified peptide ligand, or an adjuvant. The kit may also comprise one or more therapeutic, diagnostic, or prophylactic agents. The one or more therapeutic, diagnostic or prophylactic agents may include, but are not limited to, (i) agents that modulate inflammatory responses (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, cromolyn sodium 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, (v) anticancer drugs (e.g., cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, or decarbazine); (vi) immunomodulatory agents (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 acting on blood and / or blood-forming organs (e.g., interleukins, G-CSF, GM-CSF, 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, drugs affecting calcium deposition and bone turnover (e.g., calcium, phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride).

[0372] In some embodiments, the kit may contain one or more (e.g., 1, 2, or 3 or more) of any of the BCMA and / or TACI antibodies described herein. In some embodiments, the kit may contain two antibodies, each specific for a different protein. For example, the kit may contain one BCMA-specific antibody (described herein) and one TACI-specific antibody (described herein). The kit may optionally include instructions for assaying a biological sample for the presence or amount of one or more of the BCMA and / or TACI proteins. Also featured is an article of manufacture that includes a container and a composition contained within the container, the composition includes an active ingredient for inducing an immune response in a mammal (e.g., a human), the active ingredient includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of any of the peptides described herein, and the container has a label indicating that the composition is for use in inducing an immune response in a mammal (e.g., any of the mammals described herein). The label may further indicate that the composition is to be administered to a mammal having, suspected of having, or at risk for developing multiple myeloma. The composition of the article of manufacture may be dried or lyophilized and may include, for example, one or more solutions (and / or instructions) for solubilizing the dried or lyophilized composition.

[0373] The article of manufacture may also include instructions for administering the composition to a mammal. EXAMPLES

[0374] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0375] Example 1 BCMA Expression in Multiple Myeloma Cell Lines A total of 12 cancer cell lines, including 11 MM cell lines and one breast cancer cell line (MDA-MB231), were assessed for their expression levels of BCMA antigen by staining with antibodies specific for each of the following clones: no. 1. ANC3B1 (LifeSpan Biosciences, Catalog No. LS-C357630), no. 2. VICKY1 (LifeSpan Biosciences, Catalog No. LS-C18662), and no. 3. 19F2 (BioLegend, Catalog No. 357506). Among the cell lines, H929 (MM cell line) showed the highest level of BCMA expression, and MDA-MB231 (breast cancer cell line; BCMA negative) showed the lowest level of BCMA expression. (Figures 1A-1I)

[0376] Example 2 Selection of BCMA and TACI native peptides specific for HLA-A2 Six native peptides derived from the BCMA or TACI antigens, respectively, were identified as follows: [ka]

[0377] Example 3 Binding affinity of BCMA or TACI native peptides to HLA-A2 molecules The listed BCMA peptides were assessed for HLA-A2 specific binding capacity using the T2 cell line. For the assay, T2 cells were washed and resuspended in serum-free AIM-V medium at 1×10 6 The cells were resuspended to a final concentration of 10 cells / ml and transferred to wells of a 24-well tissue culture plate. The cells were pulsed with various concentrations of the respective BCMA peptide (0-200 μg / ml) and 3 μg / ml human β2-microglobulin (Sigma) and incubated at 37 °C in 5% CO in humidified air. 2 After overnight incubation, cells were washed and stained with mouse anti-human HLA-A2-FITC mAb for 15 min at 4° C., washed, and analyzed using CellQuest™ v2.1 software (Becton Dickinson, San Jose, CA). Analysis was performed using a FACSort™ flow cytometer equipped with a 24-well plate (Joseph, Calif.). Peptide binding to HLA-A2 was determined by upregulation of HLA-A2 molecules in T2 cells caused by HLA-A2-specific peptide binding and demonstrated by measuring mean fluorescence intensity (MFI) by flow cytometric analysis. Among the BCMA peptides evaluated, “no. 4.BCMA 72~80 (VLMFLLRKI (SEQ ID NO: 4))" showed the highest level of HLA-A2 specificity, and "5.BCMA 54~62 (AILWTCLGL (SEQ ID NO: 5))" showed the second highest level of specificity (Figure 2). Among the TACI peptides evaluated, all peptides except TACI no. 5 showed HLA-A2 specificity, with the highest level being TACI no. 4. 166~174 (TLGLCLCAV (SEQ ID NO: 10)) (Figure 3).

[0378] Example 4 Stability of BCMA or TACI native peptides to HLA-A2 molecules To improve the stability of peptide binding to the HLA-A2 molecule, the following heteroclitic BCMA or TACI peptides were designed: [ka]

[0379] Native and heteroclitic BCMA and TACI peptides were examined for HLA-A2 binding stability using the T2 cell line. T2 cells were pulsed with each peptide. After overnight incubation, cells were washed to remove unbound peptides and they were assessed for binding affinity as indicated above and for stability as follows. Cells were incubated with 10 μg / ml Brefeldin A (Sigma) at 37° C. and 5% CO. 2The cells were incubated with Brefeldin A for 1 h to block cell surface expression of newly synthesized HLA-A2 molecules. Peptide / HLA-A2 binding stability was assessed 0, 2, 4, 6 and 18 h after Brefeldin A treatment. After the incubation period, cells were harvested, washed, stained with mouse anti-human HLA-A2-FITC mAb and analyzed by flow cytometry. 72~80 (YLMFLLRKI (SEQ ID NO: 13))" and "Heteroclitic 5 BCMA 54~62 (YILWTCLGL (SEQ ID NO: 14))" and "heteroclitic 3 TACI 154~162 The HLA-A2 binding affinity of (YLSADQVAL (SEQ ID NO: 16)) was increased from its native peptide (Figures 4 and 5). In terms of binding stability, the heteroclitic BCMA 72~80 (YLMFLLRKI (SEQ ID NO: 13))" and "heteroclitic 3 TACI 154~162 The (YLSADQVAL (SEQ ID NO: 16)) peptide showed a significant improvement in its HLA-A2 affinity at all time points evaluated, including 0, 2, 4, 6 and 18 hours, compared to the native peptide (Figures 6 and 7). Therefore, heteroclitic 4-BCMA mAb was used for further evaluation of its immunogenic potential to generate MM-specific cytotoxic T cells (CTLs). 72~80 (YLMFLLRKI (SEQ ID NO: 13)) and heteroclitic no. 3.TACI 154~162 (YLSADQVAL (SEQ ID NO: 16) peptide was selected.

[0380] Example 5 BCMA or TACI peptide-specific CD3 + CD8 + Induction of CTLs For evaluation of functional activity in targeting MM cell lines, various HLA-A2 + Peptide-specific CTLs were generated from normal donors. To generate peptide-specific CTLs, mature dendritic cells (mDCs) generated from the same donor were resuspended in serum-free AIM-V medium and incubated with 50 μg / ml of heteroclitic BCMA no. 4. 72~80(YLMFLLRKI (SEQ ID NO: 13)) peptide or "heteroclitic 3 TACI 154~162 (YLSADQVAL (SEQ ID NO: 16)) peptide in humidified air at 37°C and 5% CO 2 Peptide-pulsed mDCs were washed, counted, irradiated at 10 Gy, and used to culture 1:20 antigen-presenting cells / peptide to CD3 + CD3 T cell ratio + T cells were primed. Cultures were restimulated with peptide-pulsed irradiated T2 cells every 7 days for a total of 4 cycles. IL-2 (50 U / ml) was added to the cultures 2 days after the second stimulation to maintain the T cells ex vivo. Control T cell cultures were maintained under identical culture conditions in the presence of IL-2 (50 U / ml) but without peptide stimulation. The phenotype of the resulting CTLs was assessed 1 week after each cycle of peptide stimulation. Heteroclitic BCMA number 4 CTLs were identified by flow cytometric analysis. 72~80 CD3 stimulated with (YLMFLLRKI (SEQ ID NO: 13)) + CD8 + There was a clear change in the phenotype of the T cell subsets, which gradually expanded in the population. + CD8 + T cell expansion was mediated by the immunogenic peptide CD138, previously identified as an immunogenic peptide. 260~268 (GLVGLIFAV (SEQ ID NO: 20)), suggesting potential immunogenicity of the BCMA peptide. BCMA peptide-specific CTL cultures expressed a higher percentage of CD8 T cells upon four cycles of peptide stimulation compared to non-peptide stimulated control T cells (approximately 20%). + It contained T cells (approximately 80%) (Figures 8A to 8C).

[0381] Example 6 Heteroclitic BCMA 72~80 Peptide-stimulated naive CD3 + CD8 + Decrease in CTL and memory CD3 +CD8 + Increase in CTL Antigen-specific CTLs can be phenotypically identified as activated / memory T cells from naive T cells by their expression of distinct cell surface antigens. The phenotype of BCMA-CTLs was examined as potential effector cells by analyzing the phenotype of naive and memory cells. BCMA peptide-specific CTLs were generated by inducing activation of BCMA peptide-specific CTLs with 50 μg / ml of heteroclitic BCMA 72~80 Weekly HLA-A2 using antigen-presenting cells pulsed with (YLMFLLRKI (SEQ ID NO: 13)) + Normal donor CD3 + CTLs were generated by repeated stimulation of T cells. One week after each peptide stimulation, the resulting CTLs were evaluated for their phenotypic profile by flow cytometry. BCMA-CTLs were significantly more potent than naive CD3 T cells compared to control T cells. + CD8 + They showed a decreased frequency of T cells (Donor 1: 80% unstimulated vs. 2% after 4 cycles of stimulation, Donor 2: 83% unstimulated vs. 2% after 4 cycles of stimulation). Heteroclitic BCMA 72~80 When the (YLMFLLRKI (SEQ ID NO: 13)) peptide was used, memory CD3 + CD8 + A corresponding increase in the frequency of T cells was observed (donor 1: 18% naive vs. 86% after 4 cycles of stimulation; donor 2: 10% naive vs. 92% after 4 cycles of stimulation). These phenotypic changes were consistent with heteroclitic BCMA 72~80 CD3 using (YLMFLLRKI (SEQ ID NO: 13)) + Repeated stimulation of T cells induces the expression of CD8 + We demonstrated that it led to CTL expansion, demonstrating the immunogenicity of the BCMA peptide (Figures 9 and 10).

[0382] Example 7 Heteroclitic BCMA 72~80 Peptide-stimulated central memory and effector CD3 + CD8 + Changes in CTL frequency Heteroclitic BCMA 72~80Further evaluation of central memory and effector cells upon stimulation of T cells with (YLMFLLRKI (SEQ ID NO: 13)) peptide was performed. After three cycles of stimulation, expansion of central memory CTLs by BCMA peptide was detected, which coincided with a decrease in effector CTLs. Upon four cycles of peptide stimulation, a decrease in central memory CTLs and an increase in effector CTLs, including effector memory cells, were also detected. Heteroclitic BCMA 72~80 This pattern of phenotypic change in CD8+ T cells with the (YLMFLLRKI (SEQ ID NO: 13)) peptide was consistent with that observed in CD138 260~268 (GLVGLIFAV (SEQ ID NO: 20)) (FIGS. 11A-11C).

[0383] Example 8 Heteroclitic BCMA 72~80 (YLMFLLRKI-SEQ ID NO:13) peptide or heteroclitic TACI 154~162 (YLSADQVAL-SEQ ID NO:16) Specific CTL stimulated with peptides exhibit distinct phenotypes representing specific T cell subtypes)

[0384] The present inventors also investigated the effect of peptide-stimulated CD3 + CD8 in T cell cultures + Naïve (CD45RO - / CCR7 + ), Central Memory (CD45RO + / CCR7 + ), Effector Memory (CD45RO + / CCR7 - ) and terminal effectors (CD45RO - / CCR7 - ) frequency of heteroclitic BCMA cells 72~80 (YLMFLLRKI (SEQ ID NO: 13)) peptide or heteroclitic TACI 154~162 CD3 in CTL stimulated with (YLSADQVAL (SEQ ID NO: 16)) peptide + CD8 +We observed distinct phenotypic changes in T cell subsets. After four cycles of peptide stimulation, effector memory CD3 + CD8 + The frequency of T cells was increased, and naive T cells (CD45RO - CCR7 + / CD3 + CD8 + ) and central memory T cells (CD45RO + CCR7 + / CD3 + CD8 + ) with a corresponding decrease in CD3+ / -100%. Thus, these results support the conclusion that CD3+ / -100% with selected heteroclitic BCMA or TACI peptides + Repeated stimulation of T cells induces the expression of CD3 + CD8 + We demonstrate that this results in distinct phenotypic changes and expansion of T cell subsets (Figures 12 and 13).

[0385] Example 9: BCMA-specific CTLs and TACI-specific CTLs induce cytotoxic activity, produce Th1-type cytokines (IFN-γ, IL-2, TNF-α), and upregulate 41BB expression on MM cells in an HLA-A2-restricted manner.

[0386] Heteroclitic BCMA 72~80 (YLMFLLRKI (SEQ ID NO: 13)) peptide or heteroclitic TACI 154~162 Peptide-specific CTLs stimulated with (YLSADQVAL (SEQ ID NO: 16)) were analyzed by flow cytometry for their ability to lyse myeloma cells and produce critical cytokines involved in antitumor activity. BCMA-CTLs and TACI-specific CTLs were analyzed by flow cytometry for their ability to lyse myeloma cells and produce critical cytokines involved in antitumor activity. + Upon recognition of U266 cells, we demonstrated a significant increase in the frequency of cells expressing the CD107a degranulation marker, a measure of cytotoxic activity, which was consistent with the recognition of HLA-A2 - It was higher than that of OPM2 cells. HLA-A2 +Increased levels of IFN-γ, IL-2 and TNF-α production were detected in BCMA- and TACI-specific CTLs against MM cells, whereas HLA-A2 - No signal was detected against MM cells, demonstrating that the immune response was in an HLA-A2 restricted manner (FIGS. 14 and 15).

[0387] Example 10 BCMA-specific CTLs proliferate in response to MM cells in an HLA-A2-restricted and antigen-specific manner

[0388] Heteroclitic BCMA 72~80 The functional activity of peptide-specific CTL stimulated with (YLMFLLRKI (SEQ ID NO: 13)) was further analyzed using a CFSE-proliferation assay. + MM(U266), HLA-A2 + Breast cancer (MDA-MB231) or HLA-A2 - CD8 in BCMA peptide-specific CTLs after stimulation with MM (MM1S) cells + T cell proliferation was measured on day 4 as evidenced by a decrease in fluorescence of CFSE-labeled CTLs (CFSE low gated). BCMA-CTLs express HLA-A2 + Significant CD8 expression in response to the U266 MM cell line + Induced T cell proliferation (proliferating cells: 46%). However, CD8 + T cell proliferation was not induced in response to MDA-MB231 or MM1S and remained at low levels (11%-14%) similar to cells cultured in medium alone (10%). Taken together, these results suggest that BCMA-CTL respond specifically to myeloma cells and that CD8+ T cell proliferation is HLA-A2 restricted and antigen-specific (Figure 16).

[0389] Example 11 Higher levels of cytotoxicity by BCMA-specific CTLs in combination with immune agonists Heteroclitic BCMA 72~80The activity of peptide-specific CTL stimulated with (YLMFLLRKI (SEQ ID NO: 13)) was measured upon treatment of the cells with anti-OX40 or anti-GITR for 48 hours. The level of cytotoxicity was measured using gated CD3 + CD8 + As measured by CD107a degranulation in T cells, increased CD107a degranulation was observed upon treatment of BCMA peptide-specific CTLs with anti-OX40 (92%) or anti-GITR (55%) compared to the untreated group (43%), suggesting that combined treatment with immune agonists helps induce the antitumor activity of BCMA-CTLs (Figure 17).

[0390] Example 12 Selective targeting of multiple myeloma by BCMA-specific central memory CD8+ cytotoxic T lymphocytes Despite recent advances in the treatment of multiple myeloma (MM) incorporating novel therapies into the stem cell transplantation paradigm, ongoing DNA damage and genomic evolution underlie relapse in a large number of patients. Thus, novel therapeutic approaches with distinct mechanisms of action are needed. Constitutive or evolving genetic complexity, coupled with the immune responsiveness of B-cell malignancies, have stimulated the development of immunotherapeutic options in MM, including monoclonal antibodies, bispecific antibodies, immunotoxins, and CART cells. Although MM patient-specific CART cell therapies have achieved remarkably deep responses, durability of responses has not been established, and they are labor-intensive, time-consuming, and expensive. To overcome these limitations, this example provides an immunogenic peptide-based cancer vaccine as an off-the-shelf immunotherapy to treat patients more broadly and efficiently. Peptide-based therapeutic approaches do not have the limitations of recombinant protein, mRNA, or DNA-based vaccines, which require a process of protein internalization, degradation into optimally immunogenic peptides for HLA, along with the additional steps required for suitable translation (for mRNA) or transcription (for DNA). To overcome MHC restriction and treat a more diverse patient population using an immunogenic epitope vaccine approach, peptide cocktails were pooled to include major HLA subtypes. Furthermore, lenalidomide can enhance peptide vaccine-specific immune responses and memory cytotoxic T cell (CTL) activity, setting the stage for combinatorial approaches with checkpoint inhibitors and / or immune agonists. Furthermore, the antitumor efficacy elicited by immunogenic peptides can be enhanced by its ability to induce "epitope spreading" upon the generation of effector cells, whereby targeted lysed cancer cells release novel antigenic epitopes that are subsequently taken up, processed, and presented by antigen-presenting cells to a new repertoire of CTLs.

[0391] B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily 17 (TNFRSF17), has been characterized as a type III transmembrane protein containing a cysteine-rich extracellular domain with a central role in regulating B-cell maturation and differentiation into plasma cells. As a receptor for the MM cell growth and survival factors B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), BCMA is required for MM cell survival, making it a promising therapeutic target. Nearly all MM tumor cells express BCMA and it has been proposed as a marker for tumor cell identification. Its selective expression on subsets of mature B and long-term surviving plasma cells further suggests a favorable therapeutic index for BCMA-directed treatment approaches. Currently, BCMA is targeted by several immunotherapeutic strategies, including antibodies (naked antibodies, antibody-drug conjugates and bispecific antibodies) and cell therapy (chimeric antigen receptor T cells), with promising clinical results even in relapsed and refractory MM. Furthermore, serum soluble BCMA is elevated in patients with MM and chronic lymphocytic leukemia and may serve as a prognostic marker and response monitor. Finally, recent studies indicate that BCMA is expressed in non-hematopoietic tissues, and that BCMA is aberrantly expressed in non-small cell lung cancer cell lines and may play a role in tumors via the ERK1 / 2 signaling pathway. These data support targeting BCMA in immunotherapy strategies in MM and potentially in solid tumors that express BCMA as well.

[0392] This example demonstrates that antigen-specific CD8 + We provide a peptide-based immunotherapy approach targeting BCMA by generating CTL. We identified novel immunogenic natural and heteroclitic HLA-A2-specific BCMA peptides that are capable of inducing MM-specific responses with highly effective antitumor activity. Importantly, heteroclitic BCMA 72~80 ( YThe LMFLLRKI (SEQ ID NO: 13) peptide exhibited the highest level of immunogenicity with the greatest affinity / stability for the HLA-A2 molecule, as well as the highest level of immunogenicity with respect to HLA-A2. + We demonstrated robust induction of BCMA-specific memory CTLs with polyfunctional activity against patient MM cells and MM cell lines. Experiments support the use of this novel engineered immunogenic BCMA in cancer vaccine and adoptive immunotherapy protocols. 72~80 We present a framework for clinical application of peptides to provide long-lasting memory anti-tumor immunity in patients with MM or BCMA-expressing cancers.

[0393] In particular, the results were consistent with those from CD138 + We demonstrate that tumor-associated antigens on tumor cells can be used to expand the breadth and extent of current multiple myeloma (MM)-specific immunotherapies. These experiments demonstrate that BCMA-specific memory CD8 T cells mediate effective, long-lasting immune responses against MM cells. + The study was designed to target B cell maturation antigen (BCMA), which promotes MM cell growth and survival, by generating CTL. 72~80 ( Y LMFLLRKI (SEQ ID NO: 13) and BCMA 54~62 ( Y We show that a novel engineered peptide specific for ILWTCLGL (SEQ ID NO: 14) shows improved affinity / stability for HLA-A2 compared to the native peptide and induces BCMA-specific CTLs with increased activation (CD38, CD69) and costimulatory (CD40L, OX40, GITR) molecule expression. Importantly, heteroclitic BCMA 72~80 Specific CTLs demonstrated polyfunctional Th1-specific immune activity against MM [IFN-γ / IL-2 / TNF-α production, proliferation, and cytotoxicity], which was mediated by the tetramer + and Memory CD8 + When combined with anti-OX40 or anti-LAG3, heteroclitic BCMA 72~80Specific CTLs showed enhanced cytotoxicity against MM, especially by central memory CTLs. These results support the use of heteroclitic BCMA alone and in combination with anti-LAG3 and / or anti-OX40 in vaccination and adoptive immunotherapy strategies. 72~80 It provides a framework for clinical application of the peptide to generate long-lasting autologous anti-tumor immunity in patients with MM and other BCMA-expressing tumors.

[0394] In this example, the following materials and methods were used:

[0395] material and method cell line 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). The T2 cell line, a human B and T cell hybrid expressing HLA-A2 molecules, was kindly provided by Dr. J. Molldrem (University of Texas MD Anderson). Cell lines were provided by the National 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) medium supplemented with 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Gibco-Life Technologies).

[0396] reagent Fluorochrome-conjugated anti-human BCMA, HLA-A2, CD3, CD8, CD38, CD40L, CD69, 41BB, CCR7, CD45RO, CD107a, IFN-γ, IL-2, TNF-α, 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 kits were purchased from Molecular Probes (Grand Island, NY). Recombinant human GM-CSF was obtained from Immunex (Seattle, WA), and human IL-2, IL-4, IFN-α, and TNF-α 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).

[0397] Synthetic peptides Native BCMA peptide [BCMA 64~72 (LIISLAVFV (SEQ ID NO: 1)), BCMA 69~77 (AVFVLMFLL (SEQ ID NO: 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))], heteroclitic BCMA peptide [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-Gag77~85 (SLYNTVATL (SEQ ID NO: 21)) was synthesized by standard fmoc (9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >95% using reverse phase chromatography, and verified by mass spectrometry for molecular weight (Biosynthesis, Lewisville, TX).

[0398] HLA-A2 affinity and stability assay T2 cells were pulsed overnight with various doses of peptide and β2-microglobulin (3 μg / ml) (Sigma, St Louis, MO). After overnight incubation, cells were stained with HLA-A2-PE mAb and analyzed using a FACSCanto™ flow cytometer (BD). Peptide / HLA-A2 complex stability was measured in peptide-loaded T2 cells by staining with HLA-A2-PE mAb and flow cytometric analysis at 0, 2, 4, 6, and 14 hours after Brefeldin A treatment.

[0399] Generation of dendritic cells Monocytes isolated from peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in the presence of 1,000 units / ml GM-CSF and 1,000 units / ml IL-4 in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS. Fresh medium and GM-CSF and IL-4 were added to the cultures every other day. Mature DCs (mDCs) were obtained on day 7 and continued to be incubated with 1,000 units / ml IFN-α and 10 ng / ml TNF-α for an additional 3 days.

[0400] Induction of BCMA peptide-specific CTLs BCMA peptide-specific CTLs (BCMA-CTLs) were generated using peptide-pulsed antigen-presenting cells (APCs) and expressed in the presence of HLA-A2 + CD3 obtained from donor +They were generated ex vivo by repeated stimulation of T cells. Briefly, APCs pulsed with peptide (50 μg / ml) were irradiated (10 Gy) 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).

[0401] Phenotypic analysis of BCMA peptide-specific CTLs or tumor cells Phenotypic characterization was performed on BCMA-CTLs after staining with a Live / Dead Aqua staining kit and fluorochrome-conjugated anti-human mAb and tetramer-PE. Alternatively, MM and breast cancer cell lines were stained with fluorochrome-conjugated BCMA or HLA-A2 mAb. After staining, cells were washed, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0402] Cell proliferation tracked by carboxyfluorescein succinimidyl ester (CFSE) BCMA-CTLs were labeled with CFSE (Molecular Probes) and co-incubated with irradiated (10 Gy) tumor cells or peptide-pulsed APCs in the presence of IL-2 (10 units / ml). On days 4, 5, 6 or 8 of co-culture, cells were harvested and stained with the Live / Dead Aqua staining kit and CD3 / CD8 / CD45RO / CCR7 mAbs. CD3 + CD8 + The level of CTL proliferation was determined as the reduction in CFSE fluorescence intensity as measured by flow cytometry.

[0403] CD107a degranulation and intracellular IFN-γ / IL-2 / TNF-α cytokine production Functional cytolytic activity of BCMA-CTLs was measured by CD107a degranulation and Th1 cytokine production by flow cytometry. Briefly, BCMA-CTLs were co-incubated with tumor cells or T2 / peptide in the presence of CD107a mAb. After 1 h of incubation, CD28 / CD49d mAb, Brefeldin A and Monensin (BD) were added for another 5 h. Cells were harvested, washed with 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 mAb, washed with Perm / Wash solution (BD), fixed with 2% paraformaldehyde and analyzed by flow cytometry.

[0404] statistical analysis Results are presented as mean ± SE. Groups were compared using unpaired Student's t-test. Differences were considered significant when p<0.05.

[0405] BCMA peptide binding affinity and stability to HLA-A2 molecules.

[0406] Various search software programs, including BIMAS and NetCTL, were used to evaluate the full-length BCMA protein sequence to predict epitopes with HLA-A2 affinity, extended half-time dissociation rate, proteasomal C-terminal cleavage and TAP transport. Six selected native peptides [BCMA 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 Among the designed heteroclitic peptides, the heteroclitic BCMA peptide (AILWTCLGL) showed the highest HLA-A2 binding affinity in a dose-dependent manner.72~80 ( Y LMFLLRKI (SEQ ID NO: 13)) and heteroclitic hBCMA 54~62 ( Y ILWTCLGL (SEQ ID NO: 14) showed the highest increase in HLA-A2 binding affinity compared to the native peptide (n=3, p<0.05). In contrast, non-HLA-A2 specific BCMA 9~17 The anchor motif in (SQNEYFDSL) is heteroclite BCMA 9~17 ( Y Substituting QNEYFDSL (SEQ ID NO: 22) did not change its HLA-A2 affinity status, indicating improved HLA-A2 affinity due solely to modifications within the HLA-A2-specific peptide.

[0407] BCMA after Brefeldin A treatment of peptide-pulsed T2 cells 72~80 and BCMA 54~62 The HLA-A2 stability of HLA-A2 specific peptides was evaluated. 72~80 and BCMA 54~62 The peptides demonstrated extended HLA-A2 stability for >6 hours, demonstrating their potential to inhibit heteroclitic BCMA 72~80 ( Y LMFLLRKI (SEQ ID NO: 13) and BCMA 54~62 ( Y This was further enhanced by engineering BCMA ILWTCLGL (SEQ ID NO: 14) into 72~80 ( Y The highest levels of HLA-A2 affinity and stability were detected using LMFLLRKI (SEQ ID NO: 13), which was significantly higher than the HLA-A2 positive control HIV-Gag 77~85 was higher than that of the peptide.

[0408] Heteroclitic BCMA 72~80 ( Y LMFLLRKI (SEQ ID NO: 13) or BCMA 54~62 ( YBCMA-specific CTLs generated with ILWTCLGL (SEQ ID NO: 14) show increased T cell activation and costimulatory molecule expression.

[0409] Heteroclitic BCMA using flow cytometry 72~80 Peptide-specific CTL (hBCMA 72~80 CTL) or heteroclitic hBCMA 54~62 Peptide-specific CTL (hBCMA 54~62 Phenotypic characterization of hBCMA CTLs was performed after the fourth round of peptide stimulation. Both CTL populations showed increased expression of activation markers (CD69, CD38) and were associated with increased expression of hBCMA CTLs. 72~80 The highest upregulation was detected in CTLs: CD38 increased from baseline 23% to 80% and CD69 increased from baseline 7% to 38% (Figure 18A). 72~80 CTLs are 54~62 They showed higher expression of 41BB, CD40L, OX30 and GITR costimulatory molecules than CTLs (FIGS. 18B and 18C).

[0410] In Figures 18A-18C, CD3+ T cells obtained from HLA-A2+ individuals were stimulated weekly with irradiated APCs pulsed with either the respective heteroclitic BCMA peptide, BCMA72-80 (YLMFLLRKI (SEQ ID NO: 13)) or BCMA54-62 (YILWTCLGL (SEQ ID NO: 14)). One week after the fourth cycle of stimulation, CD3+CD8+ T cells were analyzed by flow cytometry. Expression of T cell activation markers (CD69, CD38) and costimulatory molecules (41BB, CD40L, OX30, GITR) was assessed in CD8+ T cells. Results are demonstrated as representative (Figures 18A and 18B) or as a summary (Figure 18C) of three independent experiments using BCMA-CTLs generated from different individuals (N=3).

[0411] Heteroclitic BCMA 72~80 Specific CTLs exhibit antigen-specific antitumor activity depending on the MM cell line.

[0412] hBCMA after each round of peptide stimulation 72~80 The phenotype and activity of CTLs were evaluated. Among the generated specific CTLs (n=3), heteroclitic BCMA 72~80 ( Y Upon stimulation with LMFLLRKI (SEQ ID NO: 13), CD3 + CD8 + Gradual increase in T cell % (Figures 25A-25B) and CD3 + CD4 + A corresponding decrease in % T cells was observed (Figures 26A-26B). In parallel, phenotypic analysis of target cells stained with BCMA mAb clones (ANC3B1, VICKY1, 19F2) showed high BCMA expression in H929, MMIS, U266 and OPM1 cell lines, but not in a breast cancer cell line (MDA-MB231) (Figures 27A-27C). Assessment of functional activity showed that hBCMA 72~80 CTLs are HLA-A2 - BCMA + MM1S (7%), HLA-A2 + BCMA - HLA-A2 compared with MDA-MB231 (9%) or medium alone (6%) + BCMA + Significantly, according to U266 (49%) * p<0.05) higher CD3 + CD8 + This HLA-A2-restricted and MM-specific CD8 T cell proliferation was observed (Figures 19A-19D, histograms). + CTL proliferation was determined by the three HLA-A2 + hBCMA generated from individuals 72~80 This was consistently observed in hBCMA CTLs (Figure 19E; bar graph). 72~80 CTLs are HLA-A2 + CD8 expressing the CD107a degranulation marker (47.1%) and producing Granzyme B (32.6%) and Perforin (29.9%) in response to U266 + We demonstrated an expansion of T cells, but HLA-A2 +MDA-MB231 cells did not respond (FIG. 19F). Consistent with the antitumor activity, BCMA + In an HLA-A2-restricted manner, as measured by IFN-γ / IL-2 / TNF-α production, 41BB upregulation, and CD107a degranulation on MM cells, other HLA-A2 + hBCMA generated from individuals (N=5) 72~80 These data were observed in heteroclitic BCMA 72~80 The induction of MM-specific immune responses by peptides is further demonstrated.

[0413] In Figures 19A-9F, heteroclitic BCMA 72~80 ( Y BCMA-specific CTLs generated by repeated stimulation with LMFLLRKI (SEQ ID NO: 13) peptide were + MM cells or BCMA - Antigen-specific, HLA-A2-restricted CD8 expression in response to breast cancer cells is mediated by proliferation, CD107a degranulation, Granzyme B / perforin production, IFN-γ / IL-2 / TNF-α production, and 41BB upregulation. + T cell responses were examined and results are shown as representative (Figures 19A-19F) or a summary of three independent experiments using BCMA-CTL generated from different individuals (N=3).

[0414] HLA-A2 + Heteroclitic BCMA on patient MM cells 72~80 CTL functional immune response

[0415] CD138 in MM patients + Tumor cells were used as target cells to evaluate BCMA-specific CTLs generated with each heteroclitic peptide. 54~62 Compared to BCMA 72~80 The peptide inhibits CD107a degranulation (hBCMA 54~62 CTL 13.8% vs hBCMA 72~80hBCMA elicited more robust antigen-specific CTL and antitumor activity against patient MM cells as measured by CTL production (21.5%) and IL-2 production (4.4% vs. 16.3%, respectively) (Figure 20A). 72~80 CTLs consistently demonstrated higher anti-MM activity against patient cells, including CD107a degranulation, Granzyme B / IFN-γ / TNF-α upregulation (Figure 20B) and perforin / IL-2 production (n=3) (Figures 20C-20H) in an HLA-A2 restricted manner. 72~80 The anti-MM responses detected in CTLs were consistent with higher activation (CD69, CD38) and costimulatory molecule expression (41BB, CD40L, OX40, GITR) (Figures 18A-18C). These data suggest that heteroclitic BCMA 72~80 This study provides further evidence regarding the immunogenicity of and supports its potential clinical application in novel MM treatments.

[0416] In Figures 20A-20H, heteroclitic BCMA peptide-specific CTLs were evaluated for their functional activity against patient MM cells. CD138 CTLs obtained from HLA-A2 negative or HLA-A2 positive MM patients versus baseline responses (not stimulated with tumor cells) + The specific activity of BCMA-CTL in response to tumor cells was measured. Results are shown as representative (Figure 20A, Figure 20B) or a summary (Figure 20C-20H) of three independent experiments using BCMA-specific CTL generated from different individuals (N=3).

[0417] Heteroclitic BCMA 72~80 Specific CTLs are CD8 with robust anti-MM activity + tetramer + Enriched for T cells.

[0418] hBCMA 72~80 CTLs were further characterized for their phenotype and antitumor activity within the tetramer-positive population. Tetramer-positive CTLs showed significantly expanded CD8 + Indicating T cells activation (CD38 +:tetramer + vs. tetramer - 49.4% vs. 3.2%) and costimulatory 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%) (Figure 21A). + hBCMA generated from individuals (n=3) 72~80 CTLs consistently demonstrated higher levels of anti-MM activity against U266 MM cells by tetramer-positive cells (83%, 97%, 97%; donors A, B, or C BCMA-CTL) compared to tetramer-negative cells (6%, 18%, 13%; donors A, B, or C BCMA-CTL) (Figure 21B). + We further assessed the frequency of tetramer-positive cells within either CTL. Tetramers within degranulated CD107a-positive CTLs compared to CD107a-negative CTLs (1%, 2%, 1%; donor A, B or C BCMA-CTLs). + A significantly higher frequency of hBCMA cells (82%, 98%, 98%; donors A, B, or C) was observed (Figure 21C). These results therefore support the conclusion that 72~80 The specific anti-MM activity of CTL is confirmed to be contained within BCMA peptide-specific tetramer-positive cells, indicative of CTL activation and upregulation of costimulatory molecules.

[0419] In Figures 21A-21C, heteroclitic BCMA 72~80 Recognizing tetramer-positive CTLs or non-recognizing tetramer-negative CTLs were cultured in a CD8 + Expression of CD38, CD40L, 41BB, OX40 and GITR on T cells was analyzed (Figure 21A). Heteroclitic BCMA 72~80 The antitumor activity of specific CTLs (N=3) was further characterized by measuring CD107 upregulation within tetramer-positive or tetramer-negative CTL subsets (Figure 21B) and by assessing the tetramer-positive status within CD107a-positive or CD107a-negative CTLs (Figure 21C).

[0420] Heteroclitic BCMA 72~80 The peptide targets MM-specific memory CD8 + Induce CTLs.

[0421] To characterize BCMA-specific CTL activity, experiments were performed to evaluate the composition of naive:memory CTL subsets after two cycles of peptide stimulation and after four cycles of peptide stimulation compared to baseline. - CCR7 + [Donor 1-naive: 83.0%, CM: 0.4%, Donor 2-naive: 84.1%]. After two cycles of peptide stimulation, central memory (CM; CD45RO + CCR7 + ) [donor 1-naive: 37.4%, CM: 32.1%, donor 2-naive: 19.0%, CM: 49.6%], and then effector memory (EM; CD45RO + CCR7 - ) CTL (Donor 1-CM: 44.2%, EM: 54.6%, Donor 2-CM: 18.3%, EM: 77.6%), which gradually progressed to a phenotypic change in CD8 + In general, memory CD8 + CTL development was assessed by heteroclitic BCMA 72~80 There was a gradual increase after peptide stimulation (Figures 22B-22C), accompanied by a corresponding decrease in naive T cells (Figures 22D-22E). Thus, these results support the notion that heteroclitic BCMA 72~80 The induction and progressive development of memory CTLs upon stimulation of T cells with peptides is demonstrated.

[0422] In Figures 22A-22E, heteroclitic BCMA 72~80 The naïve:memory phenotype of CTLs (donor 1, donor 2) was analyzed at baseline (no peptide stimulation) or 1 week after each cycle of peptide stimulation. Phenotypic change, naïve to memory CD8 +The patterns of T cell differentiation and memory CTL expansion were demonstrated in dot plots (Figure 22A) and bar graphs (Figures 22B-22E) after each cycle of BCMA peptide stimulation.

[0423] Central Memory hBCMA 72~80 CTLs demonstrate the greatest anti-MM activity Eight (N=8) different HLA-A2 + Specific memory T cell subsets within the BCMA-specific CTLs generated from the individuals were then characterized for their anti-MM activity. - Compared with non-memory CTLs, CD45RO + Memory CTL is HLA-A2 + Increased CD107a degranulation in response to U266 MM cells (non-memory vs. memory: 7.25% vs. 28.2%) and HLA-A2 + McCAR MM cells (non-memory vs. memory: 4.14% vs. 13.2%) (Figure 23A; Donor A BCMA-CTL). 72~80 specific tetramer + Cells predominantly and consistently displayed the CM phenotype in BCMA-CTL generated from different individuals (tetramers + Intracellular CM% - Donor B: 88.2%, Donor C: 97.4%, Donor D: 100%) (Figure 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%) (Figure 23C). Furthermore, high responders (Donor E, Donor F) exhibiting higher anti-MM activity showed increased frequencies of BCMA peptide-specific CM CTL compared to intermediate (Donor G) or low (Donor H) responders. Thus, these results support the notion that heteroclitic BCMA 72~80 We further demonstrate the distinct peptide specificity and anti-MM activity induced by peptide-generated CM subsets.

[0424] In Figures 23A-23C, heteroclitic BCMA 72~80 The anti-MM activity of CTLs was determined by HLA-A2 + Naïve:memory CD3 in response to MM cells (U266, McCAR; Figure 23A) + CD8 + T cell subsets were assessed: central memory CD8 + The frequency of T cells was compared with that of heteroclitic BCMA 72~80 Different CTL subsets of CTL (N=3) were analyzed within tetramer-positive or tetramer-negative CTL subsets (FIG. 23B) and within CD107a-positive or CD107a-negative CTL subsets (FIG. 23C).

[0425] Inhibition of LAG3 or stimulation of OX40 inhibits hBCMA 72~80 Enhancement of CTL proliferation and anti-MM activity Finally, experiments were performed to characterize the specific T cell subsets of BCMA-CTLs that are highly responsive to MM cells. + T cell subsets were gated to demonstrate HLA-A2-restricted and MM-specific CTL expansion and characterized the naïve:memory subsets with the most robust responsiveness to U266 MM cells and the most proliferative hBCMA 72~80 CTLs were predominantly within the CM subset (donor 1: 97.4%, donor 2: 100%) (Figure 24A), confirming the major role of the CM subset within the BCMA antigen-specific CTLs in anti-MM activity. Experiments were then performed to examine the effect of checkpoint inhibitors (anti-LAG3) or immune agonists (anti-OX40) on these memory T cells. hBCMA treated with either anti-LAG3 or anti-OX40 72~80 CTLs are particularly sensitive to HLA-A2 + against U266 MM cells (untreated 28.2% vs. anti-LAG3 treated 35.8% vs. anti-OX40 treated 39.5%), and HLA-A2 +We demonstrated enhanced cytotoxic activity by memory CTLs against McCAR MM cells (untreated 13.2% vs. anti-LAG3 treated 14.5% vs. anti-OX40 treated 20.0%) (Figure 24B). Interestingly, checkpoint inhibitors and immune agonists did not induce an enhanced anti-MM response in non-memory cells in BCMA-CTLs. Finally, the beneficial effects of anti-LAG3 and anti-OX40 were confirmed by the use of hBCMA 72~80 Further investigation was performed within the CM and EM subsets of CTLs. Both treatments induced a greater effect on BCMA-specific CM cells, as evidenced by higher CD107a degranulation in response to anti-LAG3 or anti-OX40 treatment compared to EM cells (Figure 24C). Thus, these results support the notion that hBCMA, which further enhances anti-MM activity within the BCMA-specific CM subset, is a potentiator of MM activity in BCMA-specific CMs. 72~80 This supports the utility of anti-LAG3 or anti-OX40 antibodies in combination with peptide-induced CTLs.

[0426] In Figures 24A-24C, heteroclitic BCMA in response to U266 cells 72~80 MM-specific CD8 in specific CTL + We identified a specific subset that induced T cell proliferation (Figure 24A). Furthermore, heteroclitic BCMA 72~80 CTLs were evaluated in combination with anti-LAG3 or anti-OX40 for their modulation of anti-myeloma activity by memory T cells (FIG. 24B) or central memory T cell subsets (FIG. 24C).

[0427] Consideration Even in patients with refractory MM who have relapsed after allogeneic transplantation, long-lasting responses have been achieved with infusion of donor lymphocytes (DLI). These early promising results of DLI have provided a framework for the evaluation of active specific immunotherapy approaches to treat MM. Cancer-targeting vaccines, such as those of active specific immunotherapy approaches, have been shown to be highly effective CD8+ / -cell-specific inhibitors with antitumor activity. +The successful vaccination will depend on the selection of appropriate patient populations, targeting antigens selectively expressed in tumors, and utilizing a combinatorial approach to effectively induce and maintain antigen-specific memory antitumor immune responses. The present disclosure provides immunogenic HLA-A2 and HLA-A24 specific peptides derived from XBP1, CD138 and CS1 antigens, which are highly overexpressed in MM and solid tumors, including breast, pancreatic and colon cancer, and has demonstrated the ability to induce HLA-A24 in both preclinical and clinical studies. + or HLA-A24 + Peptide-specific CD8 with antitumor activity against tumor cells + Furthermore, peptide stimulation / vaccination combination studies with immunomodulatory drugs such as lenalidomide or with the histone deacetylase 6 inhibitor ACY241 enhanced peptide-specific CTL activity against tumor cells. The experiments showed that the combination of peptide stimulation with either lenalidomide or ACY241 enhanced antigen-specific CD8 + We demonstrated that it enhances T cell activity and involves activation of AKT, which upregulates transcriptional regulators such as T-bet / Eomes and antigen-specific CTL differentiation with the FOXO, mTOR and Wnt / β-catenin signaling pathways. Importantly, these effects are primarily mediated by antigen-specific CD45RO + IFN-γ and granzyme B production and CD8 expression in response to tumor cells are restricted to memory CTLs. + The most robust increases in T cell proliferation were observed primarily within specific CM subsets.

[0428] Due to promising preclinical results, the XBP1 / CD138 / CS1 multi-peptide vaccine has been evaluated alone and in combination with lenalidomide in clinical trials treating patients with smoldering MM (SMM), as well as in combination with anti-PD1 in clinical trials treating patients with triple-negative breast cancer. In patients with SMM, the multi-peptide vaccine was well tolerated and immunogenic as monotherapy, and increased tetramer production along with IFN-γ production. + CD8+ Furthermore, the combination with lenalidomide increased the frequency of CD8+ CTLs with tetramer positivity and IFN-γ production. + Importantly, the peptide vaccine induced a higher mean fold increase in CD45RO T cells specific for the XBP1 / CD138 / CS1 peptide. + Memory CTLs were induced and further enhanced in combination with lenalidomide. Stable disease and responses have been observed in SMM, but randomized trials are needed to evaluate whether peptide vaccination can extend the progression-free interval from SMM to active disease.

[0429] To expand MM-specific immunotherapy beyond the XBP1 / CD138 / CS1 antigen, the present disclosure also provides a method for identifying CD138 antigens from newly diagnosed MM patients (N=616). + Additional tumor-associated antigens on tumor cells have been identified. Here, the present disclosure provides the identification and characterization of BCMA selectively expressed on normal and malignant plasma cells and immunotherapeutic strategies that target several current immune treatment targets in MM. Examples include highly immunogenic engineered BCMA-specific nanopeptides, heteroclitic BCMA with highly improved HLA-A2 affinity / stability from the native BCMA peptide. 72~80 ( Y LMFLLRKI (SEQ ID NO: 13) and BCMA 54~62 ( Y ILWTCLGL (SEQ ID NO: 14). These peptides are useful for the identification of BCMA-specific CTLs, BCMA-specific tetramers, and the like. + It induces cell expansion, enhanced CD107 degranulation, Th1-type cytokine (IFN-γ / IL-2 / TNF-α) production, and proliferation into MM cells in an HLA-A2-restricted manner. Most importantly, it induces BCMA-specific memory CD8 + The expansion of both CTL, CM and EM cells, together with their capacity for self-renewal and response to MM cells, strongly supports the potential of heteroclitic BCMA peptides in novel vaccination and / or immunotherapy approaches in MM. Indeed, the present disclosure provides a novel approach to the use of heteroclitic BCMA peptides in MM.72~80 A clinical protocol is provided that involves peptide vaccination, ex vivo recovery and expansion of BCMA-specific CM cells, re-infusion of these CM cells as adoptive immunotherapy, and then subsequent vaccination with BCMA peptides as required to ensure their durability to effectively treat MM patients.

[0430] BCMA-specific memory CD8 + CTLs have been observed to express key molecules that regulate T cell function for both costimulation and immunosuppression. The highest induction of costimulatory and immune checkpoint molecules was observed in hBCMA, a population that demonstrated highly efficacious polyfunctional activity against MM. 72~80 It was detected in the CM subset within the peptide-specific CTL. Importantly, these findings indicated the possibility of combination therapy of BCMA-CTL with checkpoint inhibitors or immune agonists to enhance its functional anti-MM activity. This may be particularly relevant given the recent concern of combining PD-1 checkpoint inhibitors with the immunomodulatory drugs lenalidomide or pomalidomide, or with Ab daratumumab, whose toxicity has curtailed studies. Here, the examples attempted to target alternative inhibitory receptors and suppressive mechanisms within the MM tumor microenvironment. In particular, LAG3 (CD223), the third inhibitory receptor to be targeted in the clinic after CTLA and PD1 / PD-L1, was expressed in BCMA-specific CM CTL. In parallel, immune agonists, in particular costimulatory tumor necrosis factor receptor targeting OX40 (CD134), 41BB (CD137) and GITR (CD357), have attracted considerable attention for their therapeutic utility in enhancing antitumor immune responses, and among these, anti-OX40 mAb has recently demonstrated promising efficacy in inducing tumor regression by boosting 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 inhibit the expression of heteroclitic BCMA against MM cells. 72~80The functional activity of specific CTLs was evaluated. Ex vivo experiments demonstrated that both anti-LAG3 and anti-OX40 specifically enhanced the functional activity of memory CTLs in BCMA-CTLs against MM cells without affecting the activity of non-memory CTLs. + The impact on BCMA-CTLs generated from individual T cells was greater following treatment with anti-OX40 than anti-LAG3, and was greater in the CM versus EM subsets 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.

[0431] In summary, these experiments demonstrate that antigen-specific CD8 + We identified and validated novel immunogenic HLA-A2-specific engineered BCMA peptides capable of inducing CTLs. These results provide a framework for the therapeutic application of these highly immunogenic heteroclitic BCMA peptides in MM patients as vaccines and / or stimuli for the expansion and proliferation of autoantigen-specific memory CTLs. They further support the potential utility of combinations incorporating BCMA peptide vaccines or BCMA-specific adoptive T cell immunotherapy with anti-OX40 and / or anti-LAG3 to enhance BCMA-induced anti-MM responses.

[0432] Example 13 HLA-A2-specific immunogenic TACI peptides for eliciting TACI-specific CD8+ cytotoxic T lymphocytes Experiments were carried out to identify novel immunogenic engineered heteroclitic TACI 154~162 ( Y LSADQVAL (SEQ ID NO: 16) peptide inhibits antigen-specific memory CD8 with robust polyfunctional immune responses against MM +We demonstrated that heteroclitic TACI peptides can induce CTLs in MM patients. These results in this example provide a framework for the therapeutic application of heteroclitic TACI peptides in MM patients and support the therapeutic application of TACI peptide-specific vaccines or TACI peptide-specific adoptive T cell immunotherapy to treat patients with myeloma or other diseases that express TACI.

[0433] The following materials and methods were used in the examples.

[0434] material and method cell line HLA-A2 + (U266 and McCAR) and HLA-A2 - MM cell lines (OPM2 and RPMI) were obtained from ATCC (Manassas, VA). The T2 cell line, a human B and T cell hybrid expressing HLA-A2, was kindly provided by Dr. J. Molldrem (University of Texas MD Anderson). Cell lines were provided by the National 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).

[0435] reagent Fluorochrome-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 kits were purchased from Molecular Probes (Grand Island, NY). Recombinant human GM-CSF was obtained 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).

[0436] Synthetic peptides Natural TACI peptide [TACI 178~186 (FLVAVACFL (SEQ ID NO: 7)), TACI 174~182 (VLCCFLVAV (SEQ ID 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))], heteroclitic 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 (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 verified by mass spectrometry for molecular weight (Biosynthesis, Lewisville, TX).

[0437] HLA-A2 affinity and stability assay T2 cells were pulsed overnight with various concentrations of peptides and β2-microglobulin (3 μg / ml) (Sigma, St Louis, MO). After overnight incubation, cells were stained with HLA-A2-PE mAb and analyzed using a FACSCanto™ flow cytometer (BD). The stability of peptide / HLA-A2 complex binding was measured in peptide-loaded T2 cells 0, 2, 4, 6, and 14 hours after Brefeldin A treatment, followed by staining with HLA-A2-PE mAb and flow cytometric analysis.

[0438] Generation of dendritic cells Monocytes isolated from peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in the presence of 1,000 units / ml GM-CSF and 1,000 units / ml IL-4 in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS. Fresh medium and GM-CSF and IL-4 were added to the cultures every other day. Mature DCs (mDCs) were obtained on day 7 and continued to be incubated with 1,000 units / ml IFN-α and 10 ng / ml TNF-α for an additional 3 days.

[0439] Induction of TACI peptide-specific CTL ...

Claims

1. A peptide comprising an amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 15-17, wherein said peptide enhances a response specific for Transmembrane Activator and CAML Interactor (TACI), and said peptide is 9-30 amino acids in length.

2. A peptide comprising a first amino acid sequence consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 15-17, and a second amino acid sequence heterologous to the first amino acid sequence, wherein the peptide enhances a response specific for Transmembrane Activator and CAML Interactor (TACI).

3. 3. The peptide of claim 1 or 2, wherein the peptide binds to a major histocompatibility complex (MHC) molecule.

4. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 3 and a pharma- ceutically acceptable carrier.

5. A nucleic acid encoding the peptide according to any one of claims 1 to 3.

6. A vector comprising a nucleic acid encoding the peptide according to any one of claims 1 to 3.

7. A cultured cell comprising the vector of claim 6.

8. A virus comprising a nucleic acid encoding the peptide according to any one of claims 1 to 3.

9. A composition comprising nanoparticles and a peptide according to any one of claims 1 to 3.

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