XBP1, CD138, and CS1 peptides, a pharmaceutical composition containing the peptides, and a method for using the peptides and composition.

Abstract

To provide XBP1, CD138 and CS1 peptides, pharmaceutical compositions that include those peptides, and methods of using such peptides and compositions.SOLUTION: The disclosure features, inter alia, immunogenic XBP1-, CD138-, and CS1-derived peptides (and pharmaceutical compositions thereof). The peptides can be used in a variety of methods such as methods for inducing an immune response, methods for producing an antibody, and methods for treating a cancer (e.g., a breast cancer, a colon cancer, a pancreatic cancer, a blood cancer such as leukemia, or a plasma cell disorder such as multiple myeloma or Waldenstrom's macroglobulinemia). The peptides (and pharmaceutical compositions comprising the peptides) can be used, e.g., in a method of treating a precancerous condition such as smoldering multiple myeloma.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] This application claims priority to U.S. Patent Application No. 61 / 722,446, filed on November 5, 2012, and U.S. Patent Application No. 61 / 790,780, filed on March 15, 2013, the contents of which are incorporated herein by reference in their entirety.

[0002] Statement on federally sponsored research or development The research described in this application was supported by grants PO1-78378, PO1-155258, and P50-100707, respectively, awarded by the National Institutes of Health. Therefore, the Government has certain rights in this invention. [Background technology]

[0003] background Several types of vaccines, including attenuated microorganisms, recombinant proteins, and DNA vaccines, have been developed to prevent infectious diseases. In recent years, research has been conducted on the development of vaccine immunotherapy for treating cancer patients. [Overview of the project] [Means for solving the problem]

[0004] Abstract This disclosure relates to immunogenic peptides that bind to MHC class 1 molecules, such as HLA-A molecules. Peptides derived from X-Box protein 1 (XBP1), CD138, and CD2 subset 1 (CS1) have been found to be immunogenic and useful, for example, for inducing immune responses against various cancer cells. In some embodiments, these peptides possess high affinity for HLA-A molecules, high stability within the peptide binding cleft of HLA-A, and the ability to induce activation and proliferation of T cells (e.g., effector memory T cells and / or central memory T cells) when expressed in association with MHC molecules on the surface of cells (e.g., cancer cells).

[0005] Furthermore, it was found that combinations of these peptides can induce a broad immune response against target antigens, and that this broad response can overcome major therapeutic hurdles, including, in particular, heterogeneity in tumor-associated antigen expression, high frequency of mutations in specific antigens, and variability in the human T cell repertoire between individuals. Therefore, administration of various combinations of these peptides, for example, combined in pharmaceutical compositions, may provide an enhanced immune response against various cancers.

[0006] It is evident from the following description that the above peptides (and their pharmaceutical compositions) can be used in a variety of applications, such as methods for inducing immune responses, methods for activating T cells (e.g., including effector memory T cells and / or central memory T cells), methods for producing antibodies, and methods for treating cancer (e.g., breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML), multiple myeloma, Waldenström macroglobulinemia, and precancerous conditions, e.g., smoldering multiple myeloma).

[0007] In one embodiment, the disclosure features peptides, such as XBP1 peptide, CD138 peptide, and CS-1 peptide, which have affinity for multiple MHC molecules, such as HLA-A molecules (e.g., HLA-A2 and HLA-A24), high stability within the peptide bond gaps of multiple MHC molecules (e.g., HLA-A2 and HLA-A24), and the ability to induce T cell activation and proliferation, such as effector memory T cells and / or central memory T cells, when expressed in association with MHC molecules (e.g., HLA-A2 or HLA-A24) on the surface of cells (e.g., cancer cells).

[0008] It is evident from the following description that the above peptides (and their pharmaceutical compositions) can be used in a variety of applications (e.g., methods for inducing immune responses, methods for activating T cells (e.g., effector memory T cells and / or central memory T cells), methods for producing antibodies, and methods for treating cancer (e.g., lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML), multiple myeloma, and precancerous conditions, e.g., smoldering multiple myeloma).

[0009] In one embodiment, the present disclosure features an isolated peptide having at least 66% (e.g., at least 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, or 99)% identical amino acid sequence to any one of SEQ ID NOs. The peptide may bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I or class II molecule). In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs. 51-536. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids long and contains one of the amino acid sequences of SEQ ID NOs. 51-536 having three, two, or one substitutions. These substitutions may be conserved or non-conservative. In one embodiment, the peptide is used to treat subjects who have or are at risk of having cancer, e.g., cancers described herein, e.g., lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma.

[0010] In one embodiment, the peptide is used to treat a subject having a precancerous condition, such as smoldering multiple myeloma.

[0011] In one embodiment, cancer is a cancer described herein. For example, cancer is bladder cancer (including accelerated and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma and large cell carcinoma)). Cancers of the genitourinary system may include, for example, ovarian cancer (including fallopian, endometrial, and peritoneal cancers), cervical cancer, prostate cancer, and testicular cancer, lymphatic cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers (e.g., nasopharyngeal cancer). In one embodiment, cancer is breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive ductal lobular carcinoma, or invasive carcinoma). In one embodiment, the cancer is a colonic adenocarcinoma (e.g., mucinous adenocarcinoma).

[0012] In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (e.g., at least 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, or 99) identical to any one of SEQ ID NOs. In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (e.g., at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 778, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94

[0013] In one embodiment, the peptide is a non-splicing XBP1 peptide from group C (see, for example, Table 3), for example, a non-splicing XBP-1 peptide containing any one amino acid sequence of SEQ ID NOs. 51 to 206, or a non-splicing XBP1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 51 to 206. In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 51-206. In one embodiment, the unsplicing XBP1 peptide from group C has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 3, 2, or 1 substitutions. These substitutions may be conserved or non-conserved. In one embodiment, the unsplicing XBP1 peptide from group C consists of one amino acid sequence from SEQ ID NOs. 51 to 206.

[0014] In one embodiment, the peptide is a CD138 peptide from group C, for example, a CD138 peptide containing any one amino acid sequence from sequence numbers 207 to 371, or a CD138 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence from sequence numbers 207 to 371. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence from sequence numbers 207 to 371. In one embodiment, the CD138 peptide from group C is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having three, two, or one substitutions of any one amino acid sequence from SEQ ID NOs. 207-371. These substitutions may be conserved or non-conserved. In one embodiment, the CD138 peptide from group C consists of any one amino acid sequence from SEQ ID NOs. 207-371.

[0015] In one embodiment, the peptide is a CS-1 peptide from group C, for example, a CS-1 peptide containing any one amino acid sequence of SEQ ID NOs. 372-536, or a CS-1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. 372-536. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. 372-536. In one embodiment, the CS-1 peptide from group C is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains one of the amino acid sequences of SEQ ID NOs. 372-536, having three, two, or one substitutions. These substitutions may be conserved or non-conserved. In one embodiment, the CS-1 peptide from group C consists of one of the amino acid sequences of SEQ ID NOs. 372-536.

[0016] In one embodiment, peptides from group C, e.g., XBP peptide, CD138 peptide, and / or CS-1 peptide, are used to treat subjects who have or are at risk of having cancer, e.g., cancers described herein, e.g., lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML. In one embodiment, peptides from group C are used to treat subjects who have a precancerous condition, e.g., smoldering multiple myeloma. In one embodiment, cancer includes bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). These may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, cancers of the lymphatic system, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic carcinoma), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), gastrointestinal cancer (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal cancer and glial cell cancer (e.g., glioblastoma multiforme), and head and neck cancer (e.g., nasopharyngeal cancer).

[0017] In some embodiments, any isolated peptide described herein may bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I or class II molecule). The MHC molecule may be, for example, a human MHC molecule. The MHC molecule may be, for example, an HLA-A molecule, an HLA-B molecule, and / or an HLA-C molecule. Preferably, the MHC molecule is one or more HLA-A molecules (e.g., HLA-A1, HLA-A2, HLA-A3, and HLA-A24).

[0018] In one embodiment, the disclosure features immunogenic peptides derived from X-Box protein 1 (XBP1), CD138, and CD2 subset 1 (CS1) that, for example, have high affinity for the HLA-A2 molecule, high stability within the peptide bond gap of HLA-A2, and the ability to induce activation and proliferation of T cells (e.g., effector memory T cells and / or central memory T cells) when expressed in association with MHC molecules on the surface of cells (e.g., cancer cells). For example, all or subsets of these peptides are expressed in association with MHC molecules on the surface of various cancer cells, including multiple myeloma cells, more specifically smoldering multiple myeloma cells, colon cancer cells, breast cancer cells, pancreatic cancer cells, prostate cancer cells, and leukemia cells, such as acute myeloid leukemia (AML) cells, and the presence of these peptides induces activation and proliferation of T cells against these and other cancers.

[0019] Furthermore, it was found that combinations of these peptides can induce a broad immune response against target antigens, and that this broad response can overcome major therapeutic hurdles, including, in particular, heterogeneity in tumor-associated antigen expression, high frequency of mutations in specific antigens, and variability in the human T cell repertoire between individuals. Therefore, administration of combinations of these peptides, for example, combined in pharmaceutical compositions, may provide an enhanced immune response against various cancers.

[0020] It is evident from the following description that the above peptides (and their pharmaceutical compositions) can be used in a variety of applications, such as methods for inducing immune responses, methods for activating T cells (e.g., effector memory T cells and / or central memory T cells), methods for producing antibodies, and methods for treating cancer (e.g., breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML), multiple myeloma, and precancerous conditions, e.g., smoldering multiple myeloma).

[0021] In one embodiment, the Disclosure features an isolated peptide having at least 66% (e.g., at least 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, or 99)% identical amino acid sequence to any one of Sequence IDs 1-18. The peptide may bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I or class II molecule). In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide comprises an amino acid sequence having three, two, or one substitution of any one amino acid sequence of SEQ ID NOs: 1-18. These substitutions may be conserved or non-conserved. In one embodiment, the peptide is used to treat subjects who have or are at risk of having cancer, e.g., cancers described herein, e.g., breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma.

[0022] In one embodiment, the peptide is used to treat a subject having a precancerous condition, for example, smoldering multiple myeloma. In one embodiment, cancer is one of the cancers described herein. For example, such cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0023] In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (e.g., at least 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, or 99) identical to any one of SEQ ID NOs: 1 to 18. In one embodiment, the peptide consists of any of the amino acid sequences of SEQ ID NOs: 1 to 18 having three, two, or one substitution. In one embodiment, the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1 to 18.

[0024] In one embodiment, the peptide is a non-splicing XBP1 peptide from group A (see, for example, Table 1), for example, a non-splicing XBP-1 peptide containing any one amino acid sequence of SEQ ID NOs: 1-6, or a non-splicing XBP1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs: 1-6. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs: 1-6. In one embodiment, the unsplicing XBP1 peptide from group A is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having three, two, or one substitutions of any one of the amino acid sequences of SEQ ID NOs: 1-6. These substitutions may be conserved or non-conserved. In one embodiment, the unsplicing XBP1 peptide from group A consists of any one of the amino acid sequences of SEQ ID NOs: 1-6.

[0025] In one embodiment, the peptide is a splicing-type XBP1 peptide from group A, for example, a splicing-type XBP-1 peptide containing any one amino acid sequence of SEQ ID NOs. 7 to 10, or a splicing-type XBP1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. 7 to 10. In one embodiment, the splicing-type XBP1 peptide from group A is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having three, two, or one substitutions of any one of the amino acid sequences of SEQ ID NOs. 7-10. These substitutions may be conserved or non-conserved. In one embodiment, the splicing-type XBP1 peptide from group A consists of any one of the amino acid sequences of SEQ ID NOs. 7-10.

[0026] In one embodiment, the peptide is a CD138 peptide from group A, for example, a CD138 peptide containing any one amino acid sequence of sequence numbers 11-14, or a CD138 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of sequence numbers 11-14. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of sequence numbers 11-14. In one embodiment, the CD138 peptide from group A is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having three, two, or one substitutions of any one of the amino acid sequences of SEQ ID NOs. 11-14. These substitutions may be conserved or non-conserved. In one embodiment, the CD138 peptide from group A consists of any one of the amino acid sequences of SEQ ID NOs. 11-14.

[0027] In one embodiment, the peptide is a CS-1 peptide from group A, for example, a CS-1 peptide containing any one amino acid sequence of SEQ ID NOs. 15-18, or a CS-1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. 15-18. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of SEQ ID NOs. 15-18. In one embodiment, the CS-1 peptide from group A is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having three, two, or one substitutions of any one of the amino acid sequences of SEQ ID NOs. 15-18. These substitutions may be conserved or non-conserved. In one embodiment, the CS-1 peptide from group A consists of any one of the amino acid sequences of SEQ ID NOs. 15-18.

[0028] In one embodiment, peptides from Group A, e.g., unsplicing XBP1 peptide, splicing XBP1 peptide, CD138 peptide and / or CS-1 peptide, are used to treat subjects who have or are at risk of having cancer, e.g., cancers described herein, e.g., breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML. In one embodiment, peptides from Group A, e.g., unsplicing XBP1 peptide, splicing XBP1 peptide, CD138 peptide and / or CS-1 peptide, are used to treat subjects who have a precancerous condition, e.g., smoldering multiple myeloma. In one embodiment, cancer is one of the cancers described herein. For example, these cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0029] In some embodiments, any isolated peptide described herein may bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I or class II molecule). The MHC molecule may be, for example, an HLA-A2 molecule. The MHC molecule may be, for example, a human MHC molecule.

[0030] In another embodiment, the disclosure features immunogenic peptides derived from X-Box protein 1 (XBP1), CD138, and CD2 subset 1 (CS1) that, for example, have high affinity for the HLA-A24 molecule, high stability within the peptide bond gap of HLA-A24, and the ability to induce activation and proliferation of T cells (e.g., effector memory T cells and / or central memory T cells) when expressed in association with MHC molecules on the surface of cells (e.g., cancer cells). For example, all or subsets of these peptides are expressed in association with MHC molecules on the surface of various cancer cells, including multiple myeloma cells, colon cancer cells, breast cancer cells, pancreatic cancer cells, prostate cancer cells, and leukemia cells, such as acute myeloid leukemia (AML) cells, and the presence of these peptides induces activation and proliferation of T cells against these and other cancers.

[0031] It is evident from the following description that the above peptides (and their pharmaceutical compositions) can be used in a variety of applications (e.g., methods for inducing immune responses, methods for activating T cells (e.g., effector memory T cells and / or central memory T cells), methods for producing antibodies, and methods for treating cancer (e.g., lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML), multiple myeloma, and precancerous conditions, e.g., smoldering multiple myeloma).

[0032] In one embodiment, the present disclosure features an isolated peptide having at least 66% (e.g., at least 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, or 99)% identical amino acid sequence to any one of SEQ ID NOs. The peptide may bind to a major histocompatibility complex (MHC) molecule (e.g., an MHC class I or class II molecule). In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide comprises an amino acid sequence having three, two, or one substitution of any one of the amino acid sequences of SEQ ID NOs. 29-50. These substitutions may be conserved or non-conserved. In one embodiment, the peptide is used to treat subjects who have or are at risk of having cancer, for example, cancers described herein, such as lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., colon adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma.

[0033] In one embodiment, the peptide is used to treat a subject having a precancerous condition, for example, smoldering multiple myeloma. In one embodiment, the cancer is one of the cancers described herein. For example, the cancer is one of the cancers described herein, including bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (including small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). ), genitourinary cancers, for example, ovarian cancer (including fallopian tube cancer, endometrial cancer and peritoneal cancer), cervical cancer, prostate cancer and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic carcinoma), gastric cancer (for example, gastroesophageal cancer, upper gastric cancer or lower gastric cancer), gastrointestinal cancers (for example, anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (for example, acute myeloid leukemia), neuronal and glial cell cancers (for example, glioblastoma multiforme), and head and neck cancers (for example, nasopharyngeal cancer).

[0034] In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (for example, at least 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, or 99) identical to any one of SEQ ID NOs. In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (for example, at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 98, or 99) identical to any one of SEQ ID NOs. In one embodiment, the peptide consists of an amino acid sequence that is at least 66% (for example, at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 7 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96

[0035] In one embodiment, the peptide is a non-splicing XBP1 peptide from group B (see Table 2), for example, a non-splicing XBP-1 peptide containing any one of the amino acid sequences of SEQ ID NOs. 29 and 33-37, or a non-splicing XBP1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 29 and 33-37. In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 29 and 33-37. In one embodiment, the unsplicing XBP1 peptide from group B has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 3, 2, or 1 substitutions with any one of SEQ ID NOs. 29 and 33-37. These substitutions may be conserved or non-conserved. In one embodiment, the unsplicing XBP1 peptide is derived from the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 29, and includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or more (e.g., 1, 2, 3) amino acids at the C-terminal end of SEQ ID NO: 29 in SEQ ID NO: 19. In one embodiment, the unsplicing XBP1 peptide is derived from the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 33, and includes 1, 2, 3, 4, 5 or more (e.g., 1 or 2) amino acids at the N-terminal end of SEQ ID NO: 33 in SEQ ID NO: 19 and / or 1, 2, 3 or more (or It contains (e.g., 1) amino acids. In one embodiment, the unsplicing XBP1 peptide is derived from the amino acid sequence of SEQ ID NO: 19 and includes the amino acid sequence of SEQ ID NO: 36, as well as 1, 2, 3, 4, 5, 6, 7, 8, 9 or more (e.g., 1, 2, 3, or 4) amino acids at the N-terminal side of SEQ ID NO: 36 in SEQ ID NO: 19 and / or 1, 2, 3, 5, 6 or more (e.g., 1, 2, 3, 4, 5, or 6) amino acids at the C-terminal side of SEQ ID NO: 36 in SEQ ID NO: 19. In one embodiment, the unsplicing XBP1 peptide is derived from the amino acid sequence of SEQ ID NO: 19 and includes the amino acid sequence of any one of SEQ ID NO: 34, 35, or 37, as well as 1, 2, 3, 4, 5, 6, 7, 8, 9 or more (e.g., 1, 2, 3) amino acids at the N-terminal and / or C-terminal side of any one of SEQ ID NO: 34, 35, or 37 in SEQ ID NO: 19. In one embodiment, the unsplicing XBP1 peptide from group B consists of one of the amino acid sequences of SEQ ID NOs. 29 and 33-37.

[0036] In one embodiment, the peptide is a splicing-type XBP1 peptide from group B, for example, a splicing-type XBP-1 peptide containing any one of the amino acid sequences of SEQ ID NOs. 30, 38, and 39, or a splicing-type XBP1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 30, 38, and 39. In one embodiment, the peptide has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 30, 38, and 39. In one embodiment, the splicing-type XBP1 peptide from group B has an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids and contains an amino acid sequence having 3, 2, or 1 substitutions with any one of SEQ ID NOs. 30, 38, and 39. These substitutions may be conserved or non-conserved. In one embodiment, the splicing-type XBP1 peptide is derived from the amino acid sequence of SEQ ID NO: 20, and includes one of the amino acid sequences of SEQ ID NOs: 30, 38, and 39, as well as 1, 2, 3, 4, 5, 6, 7, 8, 9 or more (e.g., 1, 2, 3) amino acids at the N-terminal and / or C-terminal end of one of SEQ ID NOs: 30, 38, and 39 in SEQ ID NO: 20. In one embodiment, the splicing-type XBP1 peptide from group B consists of one of the amino acid sequences of SEQ ID NOs: 30, 38, and 39.

[0037] In one embodiment, the peptide is a CD138 peptide from group B, for example, a CD138 peptide containing any one amino acid sequence of SEQ ID NO: 31, or a CD138 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 31 and 40-45. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 31 and 40-45. In one embodiment, the CD138 peptide from group B is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains one of the amino acid sequences of SEQ ID NOs. 31 and 40-45, having three, two, or one substitutions. These substitutions may be conserved or non-conserved. In one embodiment, the CD138 peptide is derived from the amino acid sequence of SEQ ID NOs. 21, and contains the amino acid sequence of SEQ ID NOs. 31 and one, two, three, four, five, six, seven, eight or more (e.g., one, two, three, four) amino acids at the N-terminal end of SEQ ID NOs. 31 in SEQ ID NOs. In one embodiment, the CD138 peptide is derived from the amino acid sequence of SEQ ID NO: 21, and includes the amino acid sequence of SEQ ID NO: 42 or 44, as well as 1, 2, 3, 4, 5 or more (e.g., 1, 2, 3, 4, 4) amino acids at the N-terminus of SEQ ID NO: 42 or 44 in SEQ ID NO: 21 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9 or more (e.g., 1, 2, 3, 4, 5, 6) amino acids at the C-terminus of SEQ ID NO: 42 or 44 in SEQ ID NO: 21.In one embodiment, the CD138 peptide is derived from the amino acid sequence of SEQ ID NO: 21, and includes the amino acid sequence of SEQ ID NO: 45, as well as 1, 2, 3, 4, 5, 6, 7, 8 or more (e.g., 1, 2, 3, or 4) amino acids at the N-terminus of SEQ ID NO: 45 in SEQ ID NO: 21 and / or 1, 2, 3, 5, 6, 7, 8, or 9 (e.g., 1, 2, 3, 4, 5, or 6) amino acids at the C-terminus of SEQ ID NO: 45 in SEQ ID NO: 21. In one embodiment, the CD138 peptide is derived from the amino acid sequence of SEQ ID NO: 21, and includes the amino acid sequence of any one of SEQ ID NOs: 40, 41, or 43, as well as 1, 2, 3, 4, 5, 6, 7, 8, or 9 (e.g., 1, 2, 3) amino acids at the N-terminus and / or C-terminus of any one of SEQ ID NOs: 40, 41, or 43 in SEQ ID NO: 21. In one embodiment, the CD138 peptide from group B consists of one of the amino acid sequences from SEQ ID NOs. 31 and 40-45.

[0038] In one embodiment, the peptide is a CS-1 peptide from group B, for example, a CS-1 peptide containing any one amino acid sequence of SEQ ID NOs. 32 and 46-50, or a CS-1 peptide containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 32 and 46-50. In one embodiment, the peptide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths and contains an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs. 32 and 46-50. In one embodiment, the CS-1 peptide from group B is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acid lengths, and the peptide contains an amino acid sequence having one, three, two, or one substitution from any of SEQ ID NOs. 32 and 46-50. These substitutions may be conserved or non-conserved. In one embodiment, the CS-1 peptide is from the amino acid sequence of SEQ ID NOs. 22, and contains the amino acid sequence of SEQ ID NOs. 32 and one, two, three, or more (e.g., one, two, or three) amino acids at the C-terminal end of SEQ ID NOs. 22. In one embodiment, the CS-1 peptide is derived from the amino acid sequence of SEQ ID NO: 22 and includes one amino acid sequence from any one of SEQ ID NOs. 46-50 and one, two, three, four, five, six, seven, eight, nine or more (e.g., one, two, three) amino acids at the N-terminal and / or C-terminal end of one of SEQ ID NOs. 46-50 in SEQ ID NO: 22. In one embodiment, the CS-1 peptide from group B consists of SEQ ID NOs. 32 and one of the amino acid sequences from 46-50.

[0039] In one embodiment, peptides from Group B, e.g., unsplicing XBP1 peptide, splicing XBP1 peptide, CD138 peptide and / or CS-1 peptide, are used to treat subjects who have or are at risk of having cancer, e.g., cancers described herein, e.g., lung cancer, liver cancer, bile duct cancer, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML. In one embodiment, peptides from Group B, e.g., unsplicing XBP1 peptide, splicing XBP1 peptide, CD138 peptide and / or CS-1 peptide, are used to treat subjects who have a precancerous condition, e.g., smoldering multiple myeloma. In one embodiment, cancer is one of the cancers described herein. For example, these cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (including small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). ), genitourinary cancers, for example, ovarian cancer (including fallopian tube cancer, endometrial cancer and peritoneal cancer), cervical cancer, prostate cancer and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic carcinoma), gastric cancer (for example, gastroesophageal cancer, upper gastric cancer or lower gastric cancer), gastrointestinal cancers (for example, anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (for example, acute myeloid leukemia), neuronal and glial cell cancers (for example, glioblastoma multiforme), and head and neck cancers (for example, nasopharyngeal cancer).

[0040] In some embodiments, any isolated peptide described herein may be recognized by antigen-specific T cell receptors on T cells while associated with major histocompatibility complex (MHC) molecules.

[0041] In another embodiment, the Disclosure features a first amino acid sequence (e.g., as described herein) comprising peptides described herein, such as a non-splicing XBP1 peptide from group A, group B, or group C, a splicing XBP1 peptide from group A or group B, a CD138 peptide from group A, group B, or group C, and / or a CS-1 peptide from group A, group B, or group C; and a fusion protein comprising a second amino acid sequence heterogeneous to the first amino acid sequence.

[0042] In some embodiments, the second amino acid sequence may or may include a targeted polypeptide, an immunostimulatory molecule, an immunoglobulin or its antigen-binding fragment, an Fc receptor-binding region of an immunoglobulin molecule, or a carrier polypeptide. The targeted polypeptide may, for example, be a polypeptide that targets an isolated peptide against antigen-presenting cells (e.g., dendritic cells, macrophages, monocytes, or B cells). The immunostimulatory molecule may, for example, be a cytokine or a T helper epitope. The immunoglobulin may, for example, be a single-chain Fv immunoglobulin fragment or an entire immunoglobulin molecule. The carrier polypeptide may or may include a KLH (keyhole limpet hemocyanin) polypeptide or an albumin polypeptide.

[0043] In some embodiments, any isolated peptide described herein may include a linker sequence that directly or indirectly connects a first amino acid sequence to a second amino acid sequence. The linker sequence may include, or consist of, one or more amino acids, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the linker may include, or consist of, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) protease cleavage sites.

[0044] In some embodiments, the second amino acid sequence may be at the amino-terminal or carboxy-terminal end of the first amino acid sequence.

[0045] In some embodiments, any isolated peptide or fusion protein described herein may be detectably labeled. The detectable labeling may be selected from the group consisting of luminescence labeling, fluorescence labeling, radioactive labeling, and enzymatic labeling.

[0046] In yet another embodiment, the disclosure features (i) an isolated nucleic acid encoding any isolated peptide described herein; (ii) a vector comprising the isolated nucleic acid of (i); or (iii) a cultured cell comprising the vector of (ii). The vector may be operably ligated to an expression regulatory sequence. The cultured cell may be a prokaryotic or eukaryotic cell. The cultured cell may be, for example, a fungal cell, a plant cell, or an animal cell (e.g., a nematode cell, an insect cell, a bird cell, a fish cell, or a mammalian cell (e.g., a human cell)). The cultured cell may be an immune cell, such as any immune cell described herein.

[0047] In another embodiment, the present disclosure features a method for producing a peptide. The method includes culturing any cultured cells described herein under conditions that enable the expression of the peptide. The method may also include isolating the peptide from the cells or from the culture medium in which the cells were cultured.

[0048] In another embodiment, the Disclosure features a pharmaceutical composition comprising one or more of the isolated peptides (or fusion proteins) described herein and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises at least two peptides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more peptides described herein. For example, in one embodiment, the composition comprises at least 2, 3, or 4 peptides described herein.

[0049] In one embodiment, the composition comprises at least two peptides. For example, the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group A, and a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group A; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group A, and a CD138 peptide, e.g., a CD138 peptide from group A; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group A, and a CS-1 peptide, e.g., The composition comprises a CS-1 peptide from group A; the composition comprises a splicing-type XBP1 peptide, for example, a splicing-type XBP1 peptide from group A, and a CD138 peptide, for example, a CD138 peptide from group A; the composition comprises a splicing-type XBP1 peptide, for example, a splicing-type XBP1 peptide from group A, and a CS-1 peptide, for example, a CS-1 peptide from group A; the composition comprises a CD138 peptide, for example, a CD138 peptide from group A, and a CS-1 peptide, for example, a CS-1 peptide from group A.

[0050] In one embodiment, the composition comprises at least three peptides. For example, the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide described from group A, a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group A, and a CD138 peptide, e.g., a CD138 peptide from group A; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group A, a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group A, and a CS-1 peptide, e.g., The composition comprises a CS-1 peptide from group A; the composition comprises a non-splicing type XBP1 peptide, e.g., a non-splicing type XBP1 peptide from group A, a CD138 peptide, e.g., a CD138 peptide from group A, and a CS-1 peptide, e.g., a CS-1 peptide from group A; the composition comprises a splicing type XBP1 peptide, e.g., a splicing type XBP1 peptide from group A, a CD138 peptide, e.g., a CD138 peptide from group A, and a CS-1 peptide, e.g., a CS-1 peptide from group A. In one embodiment, the composition comprises at least three peptides, e.g., a non-splicing type XBP1 peptide (e.g., a non-splicing type XBP1 peptide from group A), a splicing type XBP1 peptide (e.g., a splicing type XBP-1 peptide from group A), and a CD138 peptide (e.g., a CD138 peptide from group A).

[0051] In one embodiment, the composition comprises four peptides, for example, an unsplicing XBP1 peptide, e.g., an unsplicing XBP1 peptide from group A; a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group A; a CD138 peptide, e.g., a CD138 peptide from group A; and a CS-1 peptide, e.g., a CS-1 peptide from group A.

[0052] In one embodiment, the composition comprises a non-splicing type XBP1 peptide from group A having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing the amino acid sequence of any of SEQ ID NOs. 1 to 6, for example, SEQ ID NO. 6. In one embodiment, the composition comprises a splicing type XBP1 peptide from group A having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing the amino acid sequence of any of SEQ ID NOs. 7 to 10, for example, SEQ ID NO. 10. In one embodiment, the composition comprises a CD138 peptide from group A, having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing the amino acid sequence of any of SEQ ID NOs. 11-14, for example, SEQ ID NO. 12. In one embodiment, the composition comprises a CS-1 peptide from group A, having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing the amino acid sequence of any of SEQ ID NOs. 15-18, for example, SEQ ID NO. 16.

[0053] In one embodiment, the composition comprises four peptides, the four peptides being a peptide from group A comprising (e.g., consisting thereof) the amino acid sequence of SEQ ID NO: 6, a peptide from group A comprising (e.g., consisting thereof) the amino acid sequence of SEQ ID NO: 10, a peptide from group A comprising (e.g., consisting thereof) the amino acid sequence of SEQ ID NO: 12, and a peptide from group A comprising (e.g., consisting thereof) the amino acid sequence of SEQ ID NO: 16.

[0054] In one embodiment, the composition comprises at least two peptides. For example, the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group B, and a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group B; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group B, and a CD138 peptide, e.g., a CD138 peptide from group B; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group B, and a CS-1 peptide, e.g., The composition comprises a CS-1 peptide from group B; the composition comprises a splicing-type XBP1 peptide, for example, a splicing-type XBP1 peptide from group B, and a CD138 peptide, for example, a CD138 peptide from group B; the composition comprises a splicing-type XBP1 peptide, for example, a splicing-type XBP1 peptide from group B, and a CS-1 peptide, for example, a CS-1 peptide from group B; the composition comprises a CD138 peptide, for example, a CD138 peptide from group B, and a CS-1 peptide, for example, a CS-1 peptide from group B.

[0055] In one embodiment, the composition comprises at least three peptides. For example, the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide described from group B, a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group B, and a CD138 peptide, e.g., a CD138 peptide from group B; the composition comprises a non-splicing XBP1 peptide, e.g., a non-splicing XBP1 peptide from group B, a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group B, and a CS-1 peptide, e.g., The composition comprises a CS-1 peptide from group B; the composition comprises a non-splicing type XBP1 peptide, e.g., a non-splicing type XBP1 peptide from group B, a CD138 peptide, e.g., a CD138 peptide from group B, and a CS-1 peptide, e.g., a CS-1 peptide from group B; the composition comprises a splicing type XBP1 peptide, e.g., a splicing type XBP1 peptide from group B, a CD138 peptide, e.g., a CD138 peptide from group B, and a CS-1 peptide, e.g., a CS-1 peptide from group B. In one embodiment, the composition comprises at least three peptides, e.g., a non-splicing type XBP1 peptide (e.g., a non-splicing type XBP1 peptide from group B), a splicing type XBP1 peptide (e.g., a splicing type XBP-1 peptide from group B), and a CD138 peptide (e.g., a CD138 peptide from group B).

[0056] In one embodiment, the composition comprises four peptides, for example, an unsplicing XBP1 peptide, e.g., an unsplicing XBP1 peptide from group B; a splicing XBP1 peptide, e.g., a splicing XBP1 peptide from group B; a CD138 peptide, e.g., a CD138 peptide from group B; and a CS-1 peptide, e.g., a CS-1 peptide from group B.

[0057] In one embodiment, the composition comprises a non-splicing XBP1 peptide from group B having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing one of the amino acid sequences of SEQ ID NOs. 29 and 33-37. In one embodiment, the composition comprises a splicing XBP1 peptide from group B having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing one of the amino acid sequences of SEQ ID NOs. 30, 38, and 39. In one embodiment, the composition comprises a CD138 peptide from group B, having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing one of the amino acid sequences of SEQ ID NOs. 31 and 40-45. In one embodiment, the composition comprises a CS-1 peptide from group B, having an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 25, 30, or 35 amino acids, and containing one of the amino acid sequences of SEQ ID NOs. 32 and 46-50.

[0058] In one embodiment, the composition comprises four peptides, the four peptides being a peptide from Group B comprising (e.g., consisting of) any one amino acid sequence of SEQ ID NOs. 29 and 33-37, a peptide from Group B comprising (e.g., consisting of) any one amino acid sequence of SEQ ID NOs. 30, 38 and 39, a peptide from Group B comprising (e.g., consisting of) any one amino acid sequence of SEQ ID NOs. 31 and 40-45, and a peptide from Group B comprising (e.g., consisting of) any one amino acid sequence of SEQ ID NOs. 32 and 46-50.

[0059] In one embodiment, the composition comprises peptides from group A and group B. For example, the composition comprises one, two, three, or four or more peptides from group A and one, two, three, or four or more peptides from group B.

[0060] The above composition may also include, for example, one or more further agents, such as one or more therapeutic agents, diagnostic or prophylactic agents, or immunostimulants or immunomodulating agents. Examples of immunostimulants include, but are not limited to, T helper epitopes, modified peptide ligands, adjuvants, or any other immunostimulants described herein. T helper epitopes may be, for example, PADRE sequences or universal tetanus toxoid T helper (TT Th) epitopes. The adjuvant may be selected from the group consisting of complete Freund's adjuvant, incomplete Freund's adjuvant, alum, ligands for Toll receptors, saponins (e.g., QS21), RIBI, cholera toxin (CT), Escherichia coli thermolabile toxin (LT), mutant CT (MCT), mutant Escherichia coli thermolabile toxin (MLT), carboxymethylcellulose, adjuvants containing polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., hiltonol), adjuvants containing water-and-oil emulsions (e.g., montanaid), and adjuvants containing proteins (e.g., cytokines, complement, GCSF, GM-CSF). In one embodiment, the immunostimulant is an adjuvant comprising carboxymethylcellulose, polyinosinate-polycytidylic acid, and poly-L-lysine double-stranded RNA, e.g., polyIC-LC, e.g., Hiltonol. In one embodiment, the adjuvant is a water-oil emulsion, e.g., montanaide. In one embodiment, the adjuvant is a protein, e.g., cytokine, complement, GCSF, GM-CSF. In one embodiment, the immunomodulator may be a protein, e.g., an antibody that modulates the immune system. For example, an antibody that modulates the immune system may be an anti-CTLA4 antibody, e.g., ipilimumab or tremelimumab, or an anti-PD-1 antibody, or an anti-PDL-1 antibody. In one embodiment, the immunomodulator may be a small molecule adjuvant, e.g., thalidomide or a thalidomide derivative, e.g., lenalidomide.

[0061] The above composition may also include immunogenic peptides other than those disclosed above, such as immunogenic peptides derived from WT1 or derivatives thereof. Exemplary WT1 peptides are described in U.S. Patent No. 7,598,221, which is incorporated herein by reference. In one embodiment, the above composition may include, for example, a WT1 class 1 epitope; a peptide comprising (or consisting thereof) RMFPNAPYL (SEQ ID NO: 538) (WT1 126-134); a peptide comprising (or consisting thereof) YMFPNAPYL (SEQ ID NO: 539); a peptide comprising (or consisting thereof) RSDELVRHHNMHQRNMTKL (SEQ ID NO: 540) (WT1 427-445); a peptide comprising (or consisting thereof) PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 541) (WT1 331-352); SGQARMFPNAPYLPSCLES (SEQ ID NO: 542) (WT1 The peptide comprises one or more immunogenic peptides or derivatives derived from WT1, selected from one or more peptides containing (or consisting of) 122-140; and one or more peptides containing (or consisting of) SGQAYMFPNAPYLPSCLES (SEQ ID NO: 543). Other immunogenic peptides include, but are not limited to, immunogenic peptides derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0062] In one embodiment, the compositions described herein are used to treat subjects who have or are at risk of having cancer, for example, cancers described herein, such as breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., colon adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma. In one embodiment, the compositions described herein are used to treat subjects who have a precancerous condition, for example, smoldering multiple myeloma. In one embodiment, cancer is cancers described herein. For example, these cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0063] In another embodiment, the Disclosure provides (i) one or more isolated peptides from Group A, Group B and / or Group C; and instructions for administering such peptides to a subject, for example, a subject with cancer, for example, cancers described herein, for example, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma, or a precancerous condition, for example, a subject with smoldering multiple myeloma; (ii) compositions described herein, and instructions for administering such peptides to a subject, for example (iii) Instructions for administration to a subject having cancer, for example, cancers described herein, for example, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma, or a subject having a precancerous condition, for example, smoldering multiple myeloma; and / or (iii) a kit comprising one or more isolated nucleic acids encoding an isolated peptide, one or more vectors containing the isolated nucleic acids, or one or more cultured cells containing those vectors and instructions for generating the isolated peptide. In one embodiment, cancer is a cancer described herein. For example, these cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0064] In some embodiments, the kit may also include, for example, one or more pharmaceutically acceptable carriers, one or more immunostimulants or immunomodulators, or one or more therapeutic agents, diagnostic agents or prophylactic agents. In one embodiment, the immunostimulant is an immunostimulant described herein. One or more immunostimulants may be selected from the group consisting of T helper epitopes, modified peptide ligands, and adjuvants. In one embodiment, the immunostimulant is an adjuvant containing carboxymethylcellulose, polyinosinate-polycytidylic acid, and poly-L-lysine double-stranded RNA (e.g., polyIC-LCt, e.g., Hiltonol); an adjuvant containing a water-oil emulsion (e.g., Montanaide); or an adjuvant containing a protein (e.g., cytokines, complement, GCSF, GM-CSF). In one embodiment, the immunomodulator is an immunomodulator described herein, e.g., a protein, e.g., an antibody that modulates the immune system (e.g., anti-CTLA4 antibody, e.g., ipilimumab or tremelimumab, anti-PD-1 antibody, anti-PDL-1 antibody), or a small molecule adjuvant (e.g., thalidomide or a thalidomide derivative, e.g., lenalidomide). In one embodiment, the kit further includes instructions for administering the immunostimulator and / or immunomodulator together with one or more peptides or compositions described herein.

[0065] In one embodiment, the kit further comprises a further immunogenic peptide, for example, an immunogenic peptide derived from Wt1 or a derivative thereof, for example, the immunogenic WT1 peptide described herein. Other immunogenic peptides include, but are not limited to, immunogenic peptides derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2. In one embodiment, the kit further comprises instructions for administering a further immunogenic peptide, for example, the WT1 peptide, together with one or more peptides described herein or compositions described herein.

[0066] In another embodiment, the Disclosure features a container; and a product comprising a composition contained within the container, wherein the composition is one of the compositions described herein. The container may have a marking indicating that the composition is intended for use in inducing an immune response in a mammal, e.g., a human. The marking may further indicate that the composition is to be administered to a subject having cancer, e.g., cancers described herein, e.g., breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma, or a precancerous condition, e.g., smoldering multiple myeloma. The product may also include instructions for administering the composition to a mammal, e.g., a human. The composition may be, for example, in solution, dried, or lyophilized form.

[0067] In one embodiment, cancer is a cancer described herein. For example, such cancer is bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0068] In another embodiment, the Disclosure features a method for inducing an immune response in a subject, the method comprising the steps of delivering, for example, administering, one or more of the isolated peptides and / or compositions described herein to the subject. In one embodiment, the subject is administered at least two, for example, two, three, or four peptides from Group A. For example, the subject may be administered two or more of the unsplicing XBP1 peptide from Group A, the splicing XBP1 peptide from Group A, the CD138 peptide from Group A, the CS-1 peptide from Group A, and combinations thereof. In one embodiment, the subject is administered a non-splicing XBP1 peptide from group A (e.g., a non-splicing XBP1 peptide containing SEQ ID NO: 6), a splicing XBP1 peptide from group A (e.g., a splicing XBP1 peptide containing SEQ ID NO: 10), a CD138 peptide from group A (e.g., a CD138 peptide containing SEQ ID NO: 12), and a CS-1 peptide from group A (e.g., a CS-1 peptide containing SEQ ID NO: 16). In one embodiment, the subject is administered at least two, for example, two, three, or four peptides from group B. For example, the subject may be administered a non-splicing XBP1 peptide from group B, a splicing XBP1 peptide from group B, a CD138 peptide from group B, a CS-1 peptide from group B, and combinations thereof. In one embodiment, the subject is administered a non-splicing XBP1 peptide from Group B (e.g., a non-splicing XBP1 peptide containing SEQ ID NO: 29), a splicing XBP1 peptide from Group B (e.g., a splicing XBP1 peptide containing SEQ ID NO: 30), a CD138 peptide from Group B (e.g., a CD138 peptide containing SEQ ID NO: 31), and a CS-1 peptide from Group B (e.g., a CS-1 peptide containing SEQ ID NO: 32).In one embodiment, the subject is administered at least two peptides from group C (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more). In another embodiment, the subject is administered two or more peptides from group A and group C or group B and group C.

[0069] The above method may also include a step of measuring whether an immune response occurred in a subject after delivering one or more peptides or compositions to the subject. The one or more peptides may be delivered to the subject as a pharmaceutical composition, for example, a pharmaceutical composition described herein. The subject may be, for example, a mammal (e.g., human) or any other subject described herein. The subject may have, be suspected of having, be at risk of developing, or be in remission from cancer, for example, cancers described herein, such as breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma. In one embodiment, the subject has a precancerous condition, for example, smoldering multiple myeloma. In one embodiment, the cancer is a cancer described herein. For example, these cancers include bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, and lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0070] In some embodiments, the above method may include a step of determining whether cancer cells (or multiple cancer cells) express one or more of XBP1, CD138, or CS-1.

[0071] In some embodiments, the above method may further include the step of administering one or more further treatments to the subject, such as a chemotherapeutic agent, ionizing radiation, surgery, or one or more further immunotherapeutic agents. One or more forms of ionizing radiation may be, for example, gamma irradiation, X-ray irradiation, or beta irradiation. One or more chemotherapeutic agents may be chemotherapeutic agents described herein, for example, chemotherapeutic agents selected from the group consisting of platinum-based agents, taxanes, topoisomerase inhibitors, antimetabolites, alkylating agents, protease inhibitors, and vinca alkaloids. Examples of chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide, verampil, podophyllotoxin, taxol, transplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, and any of the analogs described herein. The above method may also include the step of administering one or more immunostimulants, for example, one or more immunostimulants described herein, to a subject.

[0072] In one embodiment, the method further comprises administering a further immunogenic peptide, for example, an immunogenic peptide derived from WT1 or a derivative thereof, such as the immunogenic WT1 peptide described herein, together with one or more peptides described herein. Other immunogenic peptides include, but are not limited to, immunogenic peptides derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0073] In some embodiments, the delivery step includes administering one or more peptides from Group A, Group B, and / or Group C or compositions described herein to a subject. In some embodiments, the delivery step includes administering one or more nucleic acids to a subject, each of which comprises a nucleotide sequence encoding one or more peptides, the nucleotide sequence being operably linked to an expression regulatory sequence. The nucleic acid may be present in recombinant cells that have been transfected with the nucleic acid and are expressing one or more peptides. The recombinant cells may be transfected cells or offspring of transfected cells, produced by transfecting cells obtained from a subject. The recombinant cells may be antigen-presenting cells (e.g., dendritic cells, macrophages, monocytes, or B cells, but not limited to these).

[0074] In some embodiments of any of the methods described above, the delivery step includes contacting one or more peptides with cells; and, after contacting the cells with the one or more peptides, delivering the cells to a subject. The cells may be, for example, antigen-presenting cells (e.g., any of those described herein). The cells may be, for example, cells or offspring of cells obtained from a subject. In some embodiments, the cells may be cells or offspring of cells obtained from another subject of the same species as the subject. The other subject may express at least one MHC molecule common to the subject. The at least one MHC molecule may be, for example, an MHC class I molecule (e.g., an HLA-A2 molecule and / or an HLA-A24 molecule).

[0075] In another embodiment, the Disclosure features methods for treating subjects having cancer, for example, cancers described herein, such as breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, for example, AML or CML or multiple myeloma, or a precancerous condition, for example, a subject having smoldering multiple myeloma. In one embodiment, the method comprises administering to a subject one or more peptides or compositions described herein from Group A (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18), wherein the subject has or is at risk of developing cancer, e.g., cancers described herein, e.g., breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., colon adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma, or the subject has a precancerous condition, e.g., smoldering multiple myeloma. In one embodiment, the method comprises administering to a subject one or more (e.g., 1, 2, 3, or 4) arbitrary peptides or compositions described herein from Group B, the subject having or being at risk of developing cancer, cancers described herein, e.g., lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., colon adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma.In one embodiment, the method comprises administering to a subject one or more peptides or compositions described herein from group C (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of any of the peptides or compositions described herein, wherein the subject has or is at risk of developing cancer, e.g., cancers described herein, e.g., breast cancer (e.g., invasive lobular carcinoma, invasive ductal carcinoma, mixed lobular-ductal carcinoma, intraductal cribriform carcinoma, invasive lobular-ductal carcinoma, invasive carcinoma), colon cancer (e.g., colon adenocarcinoma, e.g., mucinous adenocarcinoma), pancreatic cancer, prostate cancer, leukemia, e.g., AML or CML or multiple myeloma, or the subject has a precancerous condition, e.g., smoldering multiple myeloma.

[0076] In one embodiment, cancer is a cancer described herein. For example, such cancer is bladder cancer (including advanced and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). This may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), digestive system cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), and head and neck cancers.

[0077] In one embodiment, the subject is administered at least two, for example, two, three, or four peptides from Group A. For example, the subject may be administered two or more of the following: unsplicing XBP1 peptide from Group A, splicing XBP1 peptide from Group A, CD138 peptide from Group A, CS-1 peptide from Group A, and combinations thereof. In one embodiment, the subject is administered unsplicing XBP1 peptide from Group A (e.g., unsplicing XBP1 peptide containing SEQ ID NO: 6), splicing XBP1 peptide from Group A (e.g., splicing XBP1 peptide containing SEQ ID NO: 10), CD138 peptide from Group A (e.g., CD138 peptide containing SEQ ID NO: 12), and CS-1 peptide (e.g., CS-1 peptide containing SEQ ID NO: 16). One or more of these peptides may be delivered to the subject as a pharmaceutical composition, for example, a pharmaceutical composition from Group A.

[0078] In one embodiment, the subject is administered at least two, for example, two, three, or four peptides from Group B. For example, the subject may be administered a non-splicing XBP1 peptide from Group B, a splicing XBP1 peptide from Group B, a CD138 peptide from Group B, a CS-1 peptide from Group B, or a combination thereof. In one embodiment, the subject is administered a non-splicing XBP1 peptide from Group B (e.g., a non-splicing XBP1 peptide containing SEQ ID NO: 29), a splicing XBP1 peptide from Group B (e.g., a splicing XBP1 peptide containing SEQ ID NO: 30), a CD138 peptide from Group B (e.g., a CD138 peptide containing SEQ ID NO: 31), and a CS-1 peptide from Group B (e.g., a CS-1 peptide containing SEQ ID NO: 32).

[0079] In one embodiment, the subject is administered at least two peptides from group C (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more). In another embodiment, the subject is administered two or more peptides from group A and group C or from group B and group C.

[0080] In one embodiment, the method further comprises the step of administering a further agent to a subject, for example, a chemotherapeutic agent and / or an immunostimulant and / or immunomodulator. In one embodiment, the further agent is an immunostimulant, for example, an immunostimulant described herein. In one embodiment, the immunostimulant is an adjuvant comprising carboxymethylcellulose, polyinosinate-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., Hiltonol); an adjuvant comprising a water-oil emulsion (e.g., Montanaide); or an adjuvant comprising a protein (e.g., cytokines, complement, GCSF, GM-CSF). In one embodiment, the further agent is an immunomodulator, for example, an immunomodulator described herein. In one embodiment, the immunomodulator is a protein, for example, an antibody that activates the immune system (e.g., anti-CTLA4 antibody, e.g., ipilimumab or tremelimumab, anti-PD-1 antibody, anti-PDL-1 antibody); or a small molecule adjuvant (e.g., thalidomide or a thalidomide derivative, e.g., lenalidomide). In one embodiment, the method includes administering a further immunogenic peptide, for example, an immunogenic peptide derived from WT1 or a derivative thereof, e.g., a WT1 peptide described herein, together with one or more of the above peptides. Other immunogenic peptides include, but are not limited to, immunogenic peptides derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0081] In one embodiment, the method further comprises an administered step of administering one or more additional doses of peptides from Group A, Group B and / or Group C or compositions described herein. In one embodiment, the subject receives one or more further additional doses approximately 14 days after the previous dose, for example, the subject receives 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of peptides from Group A, Group B and / or Group C or compositions described herein at one-week intervals.

[0082] In another embodiment, the Disclosure features a method for selecting a treatment for a mammal in need of treatment. The method includes the steps of: determining whether one or more cancer cells of a cancer in a mammal, e.g., cancers described herein, e.g., breast cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, blood cells, e.g., plasma cells, express XBP1; and, if one or more of those cancer cells express XBP1, selecting one or more peptides from Group A, Group B and / or Group C, fusion proteins containing such peptides, or compositions described herein as a therapeutic agent for that mammal. The method may also include the step of delivering one or more peptides from Group A, Group B and / or Group C, fusion proteins containing such peptides, or compositions described herein to a subject after determining that one or more of the cancer cells express XBP1.

[0083] In another embodiment, the Disclosure features a method for selecting a treatment for a mammal having cancer. The method includes the steps of: determining whether one or more cancer cells of a cancer in a mammal, e.g., cancers described herein, e.g., breast cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, blood cells, e.g., plasma cells, express CD138; and, if one or more of those cancer cells express CD138, selecting one or more peptides from group A, group B and / or group C, fusion proteins containing such peptides, or compositions described herein as a therapeutic agent for that mammal. The method may also include the step of delivering one or more peptides from group A, group B and / or group C, fusion proteins containing such peptides, or compositions described herein to a subject after determining that one or more of the cancer cells express CD138.

[0084] In another embodiment, the Disclosure features a method for selecting a treatment for a mammal in need of treatment. The method includes the steps of: determining whether one or more cancer cells of a cancer in a mammal, e.g., cancers described herein, e.g., breast cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, blood cells, e.g., plasma cells, express CS-1; and, if one or more of those cancer cells express CS-1, selecting one or more peptides from group A, group B and / or group C, fusion proteins containing such peptides, or compositions described herein as a therapeutic agent for that mammal. The method may also include the steps of delivering one or more peptides selected from group A, group B and / or group C, fusion proteins containing such peptides, or compositions described herein to a subject after determining that one or more of the cancer cells express CS-1.

[0085] In another embodiment, the Disclosure features a method for selecting a therapeutic agent for a mammal having cancer, for example, cancers described herein, such as breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, or a precancerous disorder, such as smoldering multiple myeloma. The method includes the step of selecting one or more peptides from group A, group B and / or group C, a fusion protein containing such peptides, or a composition described herein as a therapeutic agent for a mammal if one or more cancer cells of the mammal express XBP1, CD138 and / or CS-1. The method may also include the step of delivering one or more peptides selected from group A, group B and / or group C, a fusion protein containing such peptides, or a composition described herein to a subject after determining that one or more of the cancer cells express XBP1, CD138 and / or CS-1.

[0086] In some embodiments of any of the above methods, the subject or mammal may be a subject or mammal that has been treated for cancer, for example, cancers described herein, such as breast cancer, colon cancer, pancreatic cancer, or prostate cancer, and has been unresponsive to such treatment, and for example, peptides from group A, group B and / or group C, fusion proteins containing such peptides, or compositions described herein may be a second-choice, third-choice, or fourth-choice treatment.

[0087] In yet another embodiment, the Disclosure comprises (ii) compositions described herein and (ii) major histocompatibility complex (MHC) molecular polymers, wherein the polymer comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) peptide-binding domains of an MHC molecule. In some embodiments, each peptide-binding domain has a peptide from group A, group B, and / or group C bound thereto. In some embodiments, each peptide-binding domain has a peptide from group A, group B, and / or group C bound thereto non-covalently or covalently. The MHC molecular polymer may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) whole MHC molecules. The MHC molecular polymer may comprise human MHC molecules. The MHC molecular polymer may comprise MHC class I molecules, e.g., HLA-A molecules, e.g., HLA-A2 molecules or HLA-A24 molecules.

[0088] In some embodiments, the two or more peptide-binding regions may originate from the same MHC molecule. In some embodiments, the two or more peptide-binding regions may originate from different MHC molecules. In some embodiments, the two or more peptide-binding regions may be a mixture of at least two regions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) originating from the same MHC molecule and at least one region (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) originating from different MHC molecules.

[0089] In some embodiments, the MHC molecular polymer is one or more of the above composition. It can bind to at least one of the peptides (of more).

[0090] In some embodiments, the above composition may be detectably labeled. For example, one or more of the peptides and / or one or more of the peptide-binding regions may be detectably labeled. In some embodiments, at least one of the one or more MHC molecular polymers or at least one of the one or more peptides may be detectably labeled.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. In case of any conflict, this document, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in the practice or testing of this invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative and not intended to limit the scope of this invention. The present invention also provides, for example, the following items: (Item 1) A pharmaceutical composition comprising the non-splicing type XBP1 peptide, the splicing type XBP1 peptide, the CD138 peptide, and the CS-1 peptide as described herein, and optionally a pharmaceutically acceptable carrier. (Item 2) The pharmaceutical composition according to item 1, wherein the non-splicing type XBP1 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 6. (Item 3) The pharmaceutical composition according to item 1, wherein the splicing-type XBP1 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 10. (Item 4) The pharmaceutical composition according to item 1, wherein the CD138 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 12. (Item 5) The pharmaceutical composition according to item 1, wherein the non-splicing type CS-1 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 16. (Item 6) The pharmaceutical composition according to item 1, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6. (Item 7) The pharmaceutical composition according to item 1, wherein the splicing-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10. (Item 8) The pharmaceutical composition according to item 1, wherein the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12. (Item 9) The pharmaceutical composition according to item 1, wherein the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16. (Item 10) The pharmaceutical composition according to item 1, wherein the non-splicing type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 6; the splicing type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 10; the CD138 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 12; and the CS-1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 16. (Item 11) The pharmaceutical composition according to item 1, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6; the splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10; the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12; and the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16. (Item 12) A pharmaceutical composition according to any of the preceding items, further comprising one or more immunostimulants or immunomodulators. (Item 13) The pharmaceutical composition according to item 12, wherein the immunostimulant is selected from an adjuvant comprising carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., hiltonol); an adjuvant comprising a water-oil emulsion (e.g., montanaide); and an adjuvant comprising a protein (e.g., cytokine, GCSF, GM-CSF). (Item 14) The pharmaceutical composition according to item 12, wherein the immunomodulator is selected from an antibody that activates the immune system (e.g., anti-CTLA4 antibody, e.g., ipilimumab or tremelimumab); an anti-PD-1 antibody, an anti-PDL-1 antibody, or a small molecule adjuvant (e.g., thalidomide or a thalidomide derivative, e.g., lenalidomide). (Item 15) A kit comprising a composition described in any of items 1 to 14 and instructions for administering the composition to a subject. (Item 16) The kit described in item 15 further includes one or more pharmaceutically acceptable carriers. (Item 17) A kit as described in item 15 or 16, further comprising one or more immunostimulants and / or immunomodulators. (Item 18) The kit according to any one of items 15-17, wherein the subject has cancer, for example, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia (for example, AML or CML), multiple myeloma, or Waldenstrom's macrogloulinemia. (Item 19) The kit described in any of items 15 to 17, wherein the subject has smoldering multiple myeloma. (Item 20) A manufactured product comprising a container and a composition described in any of items 1 to 14 contained within the container. (Item 21) The product according to item 20, wherein the container has an indication that the composition is intended for use in inducing an immune response in mammals. (Item 22) The product according to item 20, wherein the container has an indication that the composition is for use in treating cancer, for example, breast cancer, colon cancer, pancreatic cancer, leukemia (for example, AML or CML), multiple myeloma or Waldenström macroglobulinemia. (Item 23) The product according to item 20, wherein the container has an indication that the composition is for use in treating smoldering multiple myeloma. (Item 24) A manufactured product according to any one of items 20 to 23, further comprising instructions for administering the composition to the mammal. (Item 25) A method for inducing an immune response in a subject, the method comprising the step of delivering a composition described in any of items 1 to 14 to the subject. (Item 26) The method according to item 25, further comprising the step of determining whether an immune response occurred in the subject after delivering the composition to the subject. (Item 27) The method according to item 25 or 26, wherein the subject is human. (Item 28) The method according to any one of items 25-27, wherein the subject has or is suspected to have breast cancer, colon cancer, pancreatic cancer, leukemia (e.g., AML or CML), multiple myeloma, or Waldenström macroglobulinemia. (Item 29) The method according to any one of items 25 to 27, wherein the subject has smoldering multiple myeloma. (Item 30) The method according to item 28, further comprising the step of determining whether one or more cancer cells express XBP1, CD138, or CS1. (Item 31) The method according to item 28 or 30, wherein the subject is in remission from cancer, e.g., breast cancer, colon cancer, leukemia (e.g., AML or CML), multiple myeloma, or Waldenström macroglobulinemia. (Item 32) The method according to any one of items 25 to 31, further comprising administering the composition in combination with a further treatment, for example, one or more chemotherapeutic agents, one or more forms of ionizing radiation, or one or more immunotherapeutic agents. (Item 33) The method according to any one of items 25 to 31, further comprising the step of administering one or more immunostimulants and / or immunomodulators to the subject. (Item 34) The method according to item 33, wherein the immunostimulant is selected from adjuvants comprising carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., hiltonol); adjuvants comprising water-oil emulsions (e.g., montanaide); and adjuvants comprising proteins (e.g., cytokines, GCSF, GM-CSF). (Item 35) The method according to item 33, wherein the immunomodulator is selected from antibodies that activate the immune system (e.g., anti-CTLA4 antibodies, e.g., ipilimumab or tremelimumab, PD-1 antibodies, anti-PDL-1 antibodies); and small molecule adjuvants (e.g., thalidomide or thalidomide derivatives, e.g., lenalidomide). (Item 36) A method for treating cancer, e.g., breast cancer, colon cancer, pancreatic cancer, leukemia (e.g., AML or CML), multiple myeloma or Waldenström macroglobulinemia, the method comprising the step of administering one or more of the compositions described in any of items 1 to 14 to a subject, wherein the subject has or is at risk of developing cancer, e.g., breast cancer, colon cancer, pancreatic cancer, leukemia (e.g., AML or CML), multiple myeloma or Waldenström macroglobulinemia. (Item 37) The method according to item 36, further comprising the step of administering one or more immunostimulants and / or immunomodulators to the subject. (Item 38) The method according to item 37, wherein the immunostimulant is selected from adjuvants comprising carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., hiltonol); adjuvants comprising water-oil emulsions (e.g., montanaide); and adjuvants comprising proteins (e.g., cytokines, GCSF, GM-CSF). (Item 39) The method according to item 37, wherein the immunomodulator is selected from antibodies that activate the immune system (e.g., anti-CTLA4 antibodies, e.g., ipilimumab or tremelimumab, PD-1 antibody, PDL-1 antibody); and small molecule adjuvants (e.g., thalidomide or thalidomide derivatives, e.g., lenalidomide). (Item 40) A method for treating smoldering multiple myeloma, the method comprising the step of administering one or more of the compositions described in any of items 1 to 14 to a subject, wherein the subject has smoldering multiple myeloma. (Item 41) The method according to item 40, further comprising the step of administering one or more immunostimulants and / or immunomodulators to the subject. (Item 42) The method according to item 41, wherein the immunostimulant is selected from adjuvants comprising carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., hiltonol); adjuvants comprising water-oil emulsions (e.g., montanaide); and adjuvants comprising proteins (e.g., cytokines, GCSF, GM-CSF). (Item 43) The method according to item 41, wherein the immunomodulator is selected from antibodies that activate the immune system (e.g., anti-CTLA4 antibodies, e.g., ipilimumab or tremelimumab, PD-1 antibodies, anti-PDL-1 antibodies); and small molecule adjuvants (e.g., thalidomide or thalidomide derivatives, e.g., lenalidomide). (Item 44) A method for treating breast cancer in a subject, the method comprising the step of administering one or more of the non-splicing XBP1 peptide, the splicing XBP1 peptide, the CD138 peptide, and the CS-1 peptide described herein to the subject having breast cancer. (Item 45) The method according to item 44, wherein the non-splicing type XBP1 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 6. (Item 46) The method according to item 44, wherein the splice-type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 10. (Item 47) The method according to item 44, wherein the CD138 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 12. (Item 48) The method according to item 44, wherein the CS-1 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 16. (Item 49) The method according to item 44, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6. (Item 50) The method according to item 44, wherein the splice-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10. (Item 51) The method according to item 44, wherein the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12. (Item 52) The method according to item 44, wherein the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16. (Item 53) A method for treating colon cancer in a subject, the method comprising the step of administering one or more of the non-splicing XBP1 peptide, the splicing XBP1 peptide, the CD138 peptide, and the CS-1 peptide described herein to the subject having colon cancer. (Item 54) The method according to item 53, wherein the non-splicing type XBP1 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 6. (Item 55) The method according to item 53, wherein the splicing-type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 10. (Item 56) The method according to item 53, wherein the CD138 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 12. (Item 57) The method according to item 53, wherein the CS-1 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 16. (Item 58) The method according to item 53, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6. (Item 59) The method according to item 53, wherein the splice-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10. (Item 60) The method according to item 53, wherein the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12. (Item 61) The method according to item 53, wherein the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16. (Item 62) A method for treating pancreatic cancer in a subject, the method comprising the step of administering one or more of the non-splicing XBP1 peptide, splicing XBP1 peptide, CD138 peptide, and CS-1 peptide described herein to the subject having pancreatic cancer. (Item 63) The method according to item 62, wherein the non-splicing type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 6. (Item 64) The method according to item 62, wherein the splice-type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 10. (Item 65) The method according to item 62, wherein the CD138 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 12. (Item 66) The method according to item 62, wherein the CS-1 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 16. (Item 67) The method according to item 62, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6. (Item 68) The method according to item 58, wherein the splice-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10. (Item 69) The method according to item 62, wherein the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12. (Item 70) The method according to item 62, wherein the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16. (Item 71) A method for treating leukemia (e.g., AML) in a subject, the method comprising the step of administering one or more of the non-splicing XBP1 peptide, the splicing XBP1 peptide, the CD138 peptide, and the CS-1 peptide described herein to the subject having leukemia. (Item 72) The method according to item 71, wherein the non-splicing type XBP1 peptide is 35 amino acids or less in length and contains the amino acid sequence of SEQ ID NO: 6. (Item 73) The method according to item 71, wherein the splicing-type XBP1 peptide has a length of 35 amino acids or less and includes the amino acid sequence of SEQ ID NO: 10. (Item 74) The method according to item 71, wherein the CD138 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 12. (Item 75) The method according to item 71, wherein the CS-1 peptide has a length of 35 amino acids or less and contains the amino acid sequence of SEQ ID NO: 16. (Item 76) The method according to item 71, wherein the non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 6. (Item 77) The method according to item 71, wherein the splice-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 10. (Item 78) The method according to item 71, wherein the CD138 peptide consists of the amino acid sequence of SEQ ID NO: 12. (Item 79) The method according to item 71, wherein the CS-1 peptide consists of the amino acid sequence of SEQ ID NO: 16.

[0092] Other features and advantages of the present invention, for example, a method for inducing an immune response in a subject, are evident from the following description, drawings, and claims. [Brief explanation of the drawing]

[0093] [Figure 1a] Figure 1a is a bar graph showing the HLA-A2 binding activity of a multipeptide cocktail. The Y-axis represents the average fluorescence intensity, and the X-axis represents the peptide concentration of the cocktail. Influenza virus matrix protein 58-66 (IVMP58-66; GILGFVFTL) (SEQ ID NO: 25) was used as an HLA-A2 specific positive control peptide.

[0094] [Figure 1b] Figure 1b is a bar graph showing the HLA-A2 stability of a multipeptide cocktail using T2 cells. The Y-axis represents the average fluorescence intensity, and the X-axis represents the time after treatment with Brefeldin A (BFA). Influenza virus matrix protein 58-66 (IVMP58-66; GILGFVFTL) (SEQ ID NO: 25) was used as an HLA-A2 specific positive control peptide.

[0095] [Figure 2] Figure 2 is a series of bar graphs showing different phenotypes of multipeptide-specific CTLs (MP-CTLs). The Y-axis represents the percentage of cells in a given population.

[0096] [Figure 3] Figure 3 is a series of bar graphs showing IFN-γ production by MP-CTLs in response to HLA-A2+ MM cell lines. The Y-axis represents the percentage of IFN-γ+ cells in a given population.

[0097] [Figure 4]Figure 4 shows the induction of MP-CTL proliferation by stimulation using HLA-A2+ MM cells, including primary myeloma cells and multiple myeloma cell lines. The upper panel is a representative dot plot from flow cytometry analysis. The Y-axis shows CD8 expression, and the X-axis shows the decrease in CFSE staining, a direct measure of cell proliferation. The lower panel is a bar graph showing the proliferation response of MP-CTLs to primary multiple myeloma cells (lower left panel) and multiple myeloma cell lines (lower right panel). In the bar graph, the Y-axis shows the percentage of proliferating MP-CTLs, and the X-axis shows the origin of the stimulated MM cells tested.

[0098] [Figure 5] Figure 5 is a series of graphs showing the cytotoxic activity of MP-CTLs against HLA-A2+ MM cells, including primary MM cells and cell lines. The Y-axis represents the percentage of cytotoxicity, and the X-axis represents the ratio of effector cells (MP-CTLs) to target cells.

[0099] [Figure 6a] Figure 6a is a series of dot plots showing the peptide-specific response of multipeptide-specific CTLs generated from a single donor (Donor A). The Y-axis represents the expression level of CD107α, and the X-axis represents the expression level of IFN-γ.

[0100] [Figure 6b] Figure 6b is a series of bar graphs showing the peptide-specific responses of multipeptide-specific CTLs generated from three donors (Donor B, Donor C, and Donor D). The Y-axis shows the percentage of CD107α+ cells (upper panel) or IFN-γ+ cells (lower panel), and the X-axis shows the peptides presented by K562-A2 cells.

[0101] [Figure 7]Figure 7 is a table showing the relative expression of non-splicing and splicing XBP1 in various cancer cell lines. Relative expression levels are indicated by plus or minus signs, and the number of plus signs is also shown.

[0102] [Figure 8a] Figure 8a shows a series of histograms illustrating the proliferation response of XBP1-CTLs to an HLA-A2+ breast cancer cell line at day 6. The X-axis indicates the decrease in CFSE staining, a direct measure of cell proliferation. [Figure 8b] Figure 8b is a series of histograms showing the proliferation response of XBP1-CTLs to HLA-A2+ breast cancer cells on day 7. The X-axis indicates the decrease in CFSE staining, a direct measure of cell proliferation.

[0103] [Figure 9] Figure 9 is a series of dot plots showing IFN-γ production and cell activation (CD69 expression) of XBP1-CTLs against HLA-A2+ breast cancer cell lines. The Y-axis represents CD69 expression, and the X-axis represents IFN-γ expression.

[0104] [Figure 10] Figure 10 is a series of dot plots showing degranulation (CD107α) of XBP1-CTLs against HLA-A2+ breast cancer cell lines. The Y-axis represents CD107α expression, and the X-axis represents CD8 expression.

[0105] [Figure 11] Figure 11 shows a series of histograms illustrating the proliferation response of XBP1-CTLs to HLA-A2+ pancreatic cancer cell lines and colon cancer cell lines. The X-axis indicates the decrease in CFSE staining, a direct measure of cell proliferation.

[0106] [Figure 12]Figure 12 is a series of dot plots showing the IFN-γ production and degranulation response of XBP1-CTLs to HLA-A2+ pancreatic cancer cell lines and colon cancer cell lines. The Y-axis represents CD107α expression, and the X-axis represents IFN-γ expression.

[0107] [Figure 13] Figure 13 is a table showing the relative expression of CD138 in various cancer cell lines. Relative expression is indicated by a plus or minus sign, and the number of plus signs is also shown.

[0108] [Figure 14] Figure 14 is a table showing the relative expression of CS1 in various cancer cell lines. Relative expression is indicated by a plus or minus sign, and the number of plus signs is also shown.

[0109] [Figure 15a] Figures 15a and 15b are a series of bar graphs showing the increase in CD8+ CTLs induced by a cocktail of immunogenic XBP1 unsplicing, XBP1 splicing, CD138, and CS-1 HLA-A2 specific peptides from T cells of different smoldering multiple myeloma patients. The Y-axis in the left panel shows the percentage of CD3+CD8+ CTLs, the Y-axis in the right panel shows the percentage of CD4+ Th cells, and the X-axis shows the number of peptide stimulations prior to phenotypic analysis.

[0110] [Figure 15b]Figures 15a and 15b are a series of bar graphs showing the increase in CD8+ CTLs induced by a cocktail of immunogenic XBP1 unsplicing, XBP1 splicing, CD138, and CS-1 HLA-A2 specific peptides from T cells of different smoldering multiple myeloma patients. The Y-axis in the left panel shows the percentage of CD3+CD8+ CTLs, the Y-axis in the right panel shows the percentage of CD4+ Th cells, and the X-axis shows the number of peptide stimulations prior to phenotypic analysis.

[0111] [Figure 16a] Figure 16a is a series of histograms showing the proliferation response of MP-CTLs generated from smoldering multiple myeloma patients to myeloma cells in an HLA-A2-restricted manner. The X-axis represents the decrease in CFSE staining, a direct measure of cell proliferation. The response at 5 days after stimulation is shown in the top panel, the response at 6 days in the middle panel, and the response at 7 days in the bottom panel.

[0112] [Figure 16b] Figure 16b is a series of histograms showing the proliferation response of MP-CTLs generated from a second smoldering multiple myeloma patient in response to myeloma cells, in an HLA-A2-restricted manner. The Y-axis represents cell count, and the X-axis represents CFSE staining. The response 5 days after stimulation is shown in the top panel, the response 6 days after stimulation is shown in the middle panel, and the response 7 days after stimulation is shown in the bottom panel.

[0113] [Figure 17a] Figure 17a is a series of dot plots showing IFN-γ production of MP-CTLs generated from smoldering multiple myeloma patients in response to myeloma cell lines, in an HLA-A2-restricted manner. The Y-axis represents IFN-γ expression, and the X-axis represents CD8 expression.

[0114] [Figure 17b]Figure 17b is a series of bar graphs showing IFN-γ production of MP-CTLs generated from four smoldering multiple myeloma patients in response to myeloma cell lines in an HLA-A2-restricted manner. The Y-axis shows the percentage of IFN-γ+ cells, and the X-axis shows the type of cell used to stimulate the MP-CTLs.

[0115] [Figure 18a] Figure 18a is a series of dot plots showing degranulation of MP-CTLs generated from smoldering multiple myeloma patients in response to myeloma cell lines, in an HLA-A2-restricted manner. The Y-axis represents CD107α expression, and the X-axis represents CD8 expression.

[0116] [Figure 18b] Figure 18b is a series of bar graphs showing the degranulation of MP-CTLs generated from four smoldering multiple myeloma patients in response to myeloma cell lines in an HLA-A2-restricted manner. The Y-axis shows the percentage of CD107α+ cells, and the X-axis shows the type of cell used to stimulate the MP-CTLs.

[0117] [Figure 19a] Figure 19a is a series of dot plots showing multifunctional IFN-γ production and degranulation (CD107α) among CD3+CD8+CD137+MP-CTLs generated from smoldering multiple myeloma patients in response to K562-A2 cells presenting individual peptides. The Y-axis shows CD107α expression, the X-axis shows + cells, and the X-axis shows IFN-γ expression.

[0118] [Figure 19b]Figure 19b is a series of bar graphs showing multifunctional IFN-γ production and degranulation (CD107α) among CD3+CD8+CD137+MP-CTLs generated from three smoldering multiple myeloma patients in response to K562-A2 cells presenting individual peptides. The Y-axis shows the percentage of CD3+CD8+CD137+ cells expressing both IFN-γ and CD107α, and the X-axis shows the peptides presented by K562 A2+ cells.

[0119] [Figure 19c] Figure 19c is a summary bar graph showing both IFN-γ production and degranulation (CD107α) among CD3+CD8+CD137+MP-CTLs generated from three smoldering multiple myeloma patients in response to K562-A2 cells presenting individual peptides. The Y-axis shows the percentage of CD3+CD8+CD137+ cells expressing both IFN-γ and CD107α, and the X-axis shows the peptides presented by the K562-A2 cells.

[0120] [Figure 19d] Figure 19d is a bar graph summarizing from two separate experiments, showing both IFN-γ production and degranulation (CD107α) among CD3+CD8+CD137+MP-CTLs generated from three smoldering multiple myeloma patients in response to K562-A2 cells presenting individual peptides. The Y-axis shows the percentage of CD3+CD8+CD137+ cells expressing both IFN-γ and CD107α, and the X-axis shows the peptides presented by the K562-A2 cells.

[0121] [Figure 20a]Figure 20a shows a series of dot plots (top panel) and bar graphs (bottom panel) illustrating the increase in memory CD8+ T cells in MP-CTLs generated from four patients with smoldering multiple myeloma. In the dot plots, the Y-axis represents CCR7 expression, and the X-axis represents CD45RP expression. In the bar graphs, the Y-axis represents the percentage of positive cells for the T cell subset defined on the X-axis.

[0122] [Figure 20b] Figure 20b is a series of bar graphs showing the increase in effector memory (EM) cells among MP-CTLs generated from two patients with smoldering multiple myeloma. Increasing the number of peptide stimulations from 4 to 7 resulted in an increase in the percentage of EM-type cells. The Y-axis in the bar graphs represents the percentage of positive cells for the T cell subset defined on the X-axis.

[0123] [Figure 20c] Figure 20c is a series of histograms showing the increase in IFN-γ+CD107α+ among effector memory cells (EMs) and terminal effector cells (TEs) derived from three patients with smoldering multiple myeloma. The Y-axis shows the percentage of IFN-γ+CD107α+ double-positive cells, and the X-axis shows the type of cells used to stimulate MP-CTLs.

[0124] [Figure 20d] Figure 20d is a series of bar graphs showing CD69 activation against naive and memory CD8+ T cell subsets in MP-CTLs generated from four smoldering multiple myeloma patients in response to MM cell lines (HLA-A2+U266 cells or HLA-A2-RPMI cells). The Y-axis represents the percentage of CD69+ cells, and the X-axis represents the subpopulation of CTLs.

[0125] [Figure 21]Figure 21 is a graph showing the affinity of peptides derived from unsplicing XBP1, splicing XBP1, CD138, and CS1 for HLA-A24. T2 cells were exposed to the peptides shown at concentrations of 1 mg / ml. HIV envelope proteins 583-591 (RYLKDQQLL; SEQ ID NO: 537) were used as an HLA-A24-specific positive control peptide.

[0126] [Figure 22] Figure 22 is a graph showing the affinity of unsplicing XBP1 peptide 4 (SEQ ID NO: 35), unsplicing XBP1 peptide 7 (SEQ ID NO: 29), and splicing peptide 1 (SEQ ID NO: 30) for HLA-A24. T2 cells were exposed to the peptides at the concentrations shown.

[0127] [Figure 23] Figure 23 is a graph showing the affinity of CD138 peptides 1 (SEQ ID NO: 31), 3 (SEQ ID NO: 41), and 4 (SEQ ID NO: 42) for HLA-A24. T2 cells were exposed to the peptides at the concentrations shown.

[0128] [Figure 24] Figure 24 is a graph showing the affinity of CS1 peptides 3 (SEQ ID NO: 48) and 5 (SEQ ID NO: 32) for HLA-A24. T2 cells were exposed to the peptides at the concentrations shown.

[0129] [Figure 25] Figure 25 is a schematic diagram of the method used to generate peptide-specific CTLs. APCs presenting the shown peptides were used to stimulate donor-derived CD3+ T lymphocytes to generate peptide-specific CTLs.

[0130] [Figure 26a]Figure 26a is a bar graph showing the increase in CD8+ T cells induced by unsplicing XBP1 peptide 7 presented on T lymphocytes derived from two donors (Donor A and Donor B). The Y-axis represents the percentage of CD8+ T lymphocytes, and the X-axis represents the number of peptide stimulations prior to phenotypic analysis.

[0131] [Figure 26b] Figure 26b is a bar graph showing the increase in CD8+ T cells induced by splicing-type XBP1 peptide 1 presented on T lymphocytes derived from two donors (Donor A and Donor B). The Y-axis represents the percentage of CD8+ T lymphocytes, and the X-axis represents the number of peptide stimulations prior to phenotypic analysis.

[0132] [Figure 26c] Figure 26c is a bar graph showing the increase in CD8+ T cells induced by CD138 peptide 1 presented on T lymphocytes derived from two donors (Donor A and Donor B). The Y-axis represents the percentage of CD8+ T lymphocytes, and the X-axis represents the number of peptide stimulations prior to phenotypic analysis.

[0133] [Figure 26d] Figure 26d is a bar graph showing the increase in CD8+ T cells induced by CS1 peptide 1 presented on T lymphocytes derived from two donors (Donor A and Donor B). The Y-axis represents the percentage of CD8+ T lymphocytes, and the X-axis represents the number of peptide stimulations prior to phenotypic analysis.

[0134] [Figure 27] Figure 27 is a schematic diagram of the methods used to evaluate the response of peptide-specific CTLs to various multiple myeloma tumor cells. Peptide-specific CTLs were incubated with KMS, OPM1, or U266 multiple myeloma cells for 5 hours and assayed for IFN-γ production, CD107α upregulation, CD8 T cell proliferation, or IL-2 production.

[0135] [Figure 28] Figure 28 is a series of dot plots showing IFN-γ production and CD8 expression of peptide-specific CTLs in response to multiple myeloma cells. The Y-axis represents the IFN-γ expression level, and the X-axis represents CD8 expression. The peptide specificity of the analyzed CTL population is shown on the left. The CD8+, IFN-γ+ populations are represented by the areas enclosed by squares.

[0136] [Figure 29a] Figure 29a shows a series of dot plots illustrating IFN-γ expression in CTL populations and subpopulations. CTLs specific to unsplicing XBP1 peptide 7 (SEQ ID NO: 29) were stimulated in KMS11 cells. Populations represented by the squared areas are shown on the graph.

[0137] [Figure 29b] Figure 29b is a series of dot plots showing IFN-γ expression in CTL populations and subpopulations. CTLs specific to splicing-type XBP1 peptide 1 (SEQ ID NO: 30) were stimulated in KMS11 cells. The populations represented by the areas enclosed in squares are shown on the graph.

[0138] [Figure 29c] Figure 29c is a series of dot plots showing IFN-γ expression in populations and subpopulations of CTLs. CTLs specific to CD138 peptide 1 (SEQ ID NO: 31) were stimulated in KMS11 cells. The populations represented by the areas enclosed in squares are shown on the graph.

[0139] [Figure 29d] Figure 29d is a series of dot plots showing IFN-γ expression in CTL populations and subpopulations. CTLs specific to CS1 peptide 5 (SEQ ID NO: 32) were stimulated in KMS11 cells. The populations represented by the squares are shown on the graph.

[0140] [Figure 30] Figure 30 is a schematic diagram of the CD107α detection assay used to measure degranulation. Release of lytic granules due to degranulation of tumor cells causes upregulation of CD107α in CTLs, which can be detected by anti-CD107α antibodies.

[0141] [Figure 31] Figure 31 is a series of dot plots showing IFN-γ expression and degranulation in peptide-specific CTLs. CTLs were either not stimulated or stimulated with KMS11 cells or OPM1 cells as shown at the top. The peptide specificity of the CTLs is shown on the left. The Y-axis represents the expression of CD107α and the X-axis represents the expression of IFN-γ.

[0142] [Figure 32] Figure 32 is a schematic diagram of the assay used to measure the proliferation of peptide-specific CTLs in response to multiple myeloma cells. Peptide-specific CTLs and irradiated multiple myeloma cells were incubated together for 6 or 8 days, and the proliferation of CTLs was measured by CFSE incorporation.

[0143] [Figure 33a] Figure 33a is a series of histograms showing the 6-day proliferation response of peptide-specific CTLs to myeloma cells. The peptide specificity of the CTLs is shown on the left. The X-axis shows the decrease in CFSE staining, which is a direct measure of cell proliferation.

[0144] [Figure 33b] Figure 33b is a series of histograms showing the 8-day proliferation response of peptide-specific CTLs to myeloma cells. The peptide specificity of the CTLs is shown on the left. The X-axis shows the decrease in CFSE staining, which is a direct measure of cell proliferation.

[0145] [Figure 34] Figure 34 is a series of dot plots showing IL-2 production by peptide-specific CTLs in response to myeloma cells. The peptide specificity of the CTLs is shown on the left. The Y-axis represents IL-2 expression, and the X-axis represents CD8 expression. The areas enclosed by squares represent the IL-2+CD8+ population.

[0146] [Figure 35] Figure 35 is a series of dot plots showing IFN-γ expression of peptide-specific CTLs derived from donor A in response to various colon cancer cell lines. CTLs were either unstimulated or stimulated with SW80, WiDr, or LS180 cells, as shown at the top. The peptide specificity of the CTLs is shown on the left. The Y-axis represents IFN-γ expression, and the X-axis represents CD8 expression. Areas enclosed by squares represent the IFN-γ+CD8+ population.

[0147] [Figure 36] Figure 36 is a series of dot plots showing IFN-γ expression and degranulation in peptide-specific CTLs derived from donor A in response to various colon cancer cell lines. CTLs were either unstimulated or stimulated with SW80, WiDr, or LS180 cells, as shown at the top. Peptide specificity of the CTLs is shown on the left. The Y-axis represents CD107α expression, and the X-axis represents IFN-γ expression.

[0148] [Figure 37] Figure 37 is a series of dot plots showing IFN-γ expression of peptide-specific CTLs derived from donor B in response to various colon cancer cell lines. CTLs were either unstimulated or stimulated with SW80, WiDr, or LS180 cells, as shown at the top. The peptide specificity of the CTLs is shown on the left. The Y-axis represents IFN-γ expression, and the X-axis represents CD8 expression. Areas enclosed by squares represent the IFN-γ+CD8+ population.

[0149] [Figure 38] Figure 38 is a series of dot plots showing IFN-γ expression and degranulation in peptide-specific CTLs derived from donor B. CTLs were either unstimulated or stimulated with SW80, WiDr, or LS180 cells, as shown at the top. The peptide specificity of the CTLs is shown on the left. The Y-axis represents CD107α expression, and the X-axis represents IFN-γ expression.

[0150] [Figure 39a] Figure 39a shows a series of histograms illustrating the degranulation response of CD138 peptide-specific CTLs to SW480 tumor cells. CTLs and SW480 tumor cells were co-incubated in various cell-to-cell ratios, as shown on the left, and the expression of CD107α, IFN-γ, and IL-2 was analyzed, as shown at the top.

[0151] [Figure 39b] Figure 39b shows a series of histograms illustrating various responses of CD138 peptide-specific CTLs to LS180 tumor cells. CTLs and LS180 tumor cells were co-incubated in various cell-to-cell ratios, as shown on the left, and the expression of CD107α, IFN-γ, and IL-2 was analyzed, as shown at the top.

[0152] [Figure 40a] Figure 40a is a series of dot plots showing IFN-γ expression in peptide-specific CTLs derived from donor A in response to different cancer cell types. IFN-γ expression was analyzed after incubation of CTLs either alone or co-incubating them with SW480 colon cancer cells or KMS11 multiple myeloma cells. The peptide specificity of the CTLs is shown on the left. The Y-axis represents IFN-γ expression, and the X-axis represents CD8 expression. Areas enclosed by squares represent IFN-γ+CD8+ populations.

[0153] [Figure 40b]Figure 40b is a series of dot plots showing degranulation and IFN-γ expression of peptide-specific CTLs from donor A in response to different cancer cell types. CTLs were incubated alone or co-incubated with SW480 colon cancer cells or KMS11 multiple myeloma cells, and analyzed for IFN-γ expression. The peptide specificities of the CTLs are shown on the left. The Y-axis indicates CD107α expression, and the X-axis indicates IFN-γ expression.

[0154] [Figure 41a] Figure 41a is a series of dot plots showing IFN-γ expression of peptide-specific CTLs from donor B in response to different cancer cell types. CTLs were incubated alone or co-incubated with SW480 colon cancer cells or KMS11 multiple myeloma cells, and analyzed for IFN-γ expression. The peptide specificities of the CTLs are shown on the left. The Y-axis indicates IFN-γ expression, and the X-axis indicates CD8 expression. The area surrounded by the square represents the IFN-γ+CD8+ population.

[0155] [Figure 41b] Figure 41b is a series of dot plots showing degranulation and IFN-γ expression of peptide-specific CTLs from donor B in response to different cancer cell types. CTLs were incubated alone or co-incubated with SW480 colon cancer cells or KMS11 multiple myeloma cells, and analyzed for IFN-γ expression. The peptide specificities of the CTLs are shown on the left. The Y-axis indicates CD107α expression, and the X-axis indicates IFN-γ expression.

[0156] [Figure 42]Figure 42 shows a series of dot plots illustrating IFN-γ expression in peptide-specific CTLs derived from donor B in response to various pancreatic cancer cell lines. IFN-γ expression was analyzed after incubation of CTLs either alone or co-incubating them with 8902, PL45, or MiaPaca cells. The peptide specificity of the CTLs is shown on the left. The Y-axis represents IFN-γ expression, and the X-axis represents CD8 expression. Areas enclosed by squares represent the IFN-γ+CD8+ population.

[0157] [Figure 43] Figure 43 shows a series of histograms and dot plots illustrating the diverse responses of CD138 peptide-specific CTLs to Panc1 pancreatic tumor cells. CTLs and Panc1 cells were co-incubated in various cell-to-cell ratios, such as 1:1 or 1:5, as shown on the left, and the expression of CD107α, IFN-γ, and IL-2 was analyzed, as shown at the top. The Y-axis of the dot plots represents CD107α expression, and the X-axis of the histograms and dot plots represents IFN-γ or IL-2 expression, as shown below each graph.

[0158] [Figure 44] Figure 44 is a series of dot plots showing phenotypic changes in T cells (naive T cells, central memory (CM), effector cells, and effector memory (EM) CD8+ T cells) in CTLs induced with a cocktail of non-splicing heterocritic XBP1 peptide and splicing heterocritic XBP1 peptide. In the dot plots, the Y axis represents CCR7 expression and the X axis represents CD45RP expression.

[0159] [Figure 45]Figure 45 is a series of bar graphs showing the generation of central memory CD3+CD8+ T cells in three donors in response to a cocktail of non-splicing heterocritic XBP1 peptides and splicing heterocritic XBP1 peptides.

[0160] [Figure 46] Figure 46 is a series of bar graphs showing the generation of effector memory CD3+CD8+ T cells in three donors in response to a cocktail of non-splicing heterocritic XBP1 peptide and splicing heterocritic XBP1 peptide.

[0161] [Figure 47] Figure 47 shows a series of histograms illustrating the proliferation response of XBP1 peptide cocktail-specific CTLs to MB231 breast cancer cells. The response results in the proliferation of CD45RO- non-memory cells and CD45RO+ memory cells, with their proportions within memory cells being those of CD45RO+, CCR7+ central memory T cells and CD45RO+, CCR7-effector memory T cells.

[0162] [Figure 48] Figure 48 shows a series of histograms illustrating the proliferation response of XBP1 peptide cocktail-specific CTLs to LS180 colon cancer cells. The response results in the proliferation of CD45RO- non-memory cells and CD45RO+ memory cells, with their proportions within memory cells being those of CD45RO+, CCR7+ central memory T cells and CD45RO+, CCR7-effector memory T cells.

[0163] [Figure 49]Figure 49 shows a series of histograms illustrating the proliferation response of XBP1 peptide cocktail-specific CTLs to Panc1 pancreatic cancer cells. The response results in the proliferation of CD45RO- non-memory cells and CD45RO+ memory cells, with their proportions within memory cells being those of CD45RO+, CCR7+ central memory T cells and CD45RO+, CCR7-effector memory T cells.

[0164] [Figure 50] Figure 50 shows a series of histograms illustrating the diverse responses of XBP1 peptide cocktail-specific CTLs to various tumor cells (MB231, MCF7, LS180, SW480, Panc1, and PL45). Central memory T cells and effector memory T cells were analyzed for IFN-γ expression.

[0165] [Figure 51] Figure 51 is a bar graph showing the expression of IFN-γ by effector memory T cells and central memory T cells of XBP1 cocktail-specific CTLs against various tumor cells (MB231, MC7, LS180, SW480, Panc1, and PL45).

[0166] [Figure 52] Figure 52 shows a series of histograms illustrating the diverse responses of XBP1 peptide cocktail-specific CTLs to various tumor cells (MB231, MCF7, LS180, SW480, Panc1, and PL45). Central memory T cells and effector memory T cells were analyzed for IL-2 expression.

[0167] [Figure 53] Figure 53 is a bar graph showing IL-2 expression by effector memory T cells and central memory T cells of XBP1 cocktail-specific CTLs against various tumor cells (MB231, MC7, LS180, SW480, Panc1, and PL45).

[0168] [Figure 54] Figure 54 shows a series of histograms illustrating the diverse responses of XBP1 peptide cocktail-specific CTLs to various tumor cells (MB231, MCF7, LS180, SW480, Panc1, and PL45). Central memory T cells and effector memory T cells were analyzed for cytotoxicity.

[0169] [Figure 55] Figure 55 is a bar graph showing the cytotoxicity of effector memory T cells and central memory T cells by XBP1 cocktail-specific CTLs against various tumor cells (MB231, MC7, LS180, SW480, Panc1, and PL45).

[0170] [Figure 56] Figure 56 is a bar graph showing Tbet expression by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs.

[0171] [Figure 57] Figure 57 is a bar graph showing the expression of Tbet by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs.

[0172] [Figure 58] Figure 58 is a bar graph showing the expression of Tbet and IFN-γ by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs against various tumor cells (MB231, SW480, and Panc1).

[0173] [Figure 59]Figure 59 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against MB231 breast cancer cells.

[0174] [Figure 60] Figure 60 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against Panc1 pancreatic cancer cells.

[0175] [Figure 61] Figure 61 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against SW480 colon cancer cells.

[0176] [Figure 62] Figure 62 is a bar graph showing Eomes expression by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs.

[0177] [Figure 63] Figure 63 is a bar graph showing the expression of Eomes by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs.

[0178] [Figure 64] Figure 64 is a bar graph showing the expression of Eomes and IFN-γ by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs against various tumor cells (MB231, SW480, and Panc1).

[0179] [Figure 65]Figure 65 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against MB231 breast cancer cells.

[0180] [Figure 66] Figure 66 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against Panc1 pancreatic cancer cells.

[0181] [Figure 67] Figure 67 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against SW480 colon cancer cells.

[0182] [Figure 68] Figure 68 is a bar graph showing the expression of granzyme B by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs against various tumor cells (MB231, SW480, and Panc1).

[0183] [Figure 69] Figure 69 is a bar graph showing the expression of granzyme B and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against MB231 breast cancer cells.

[0184] [Figure 70] Figure 70 is a bar graph showing the expression of granzyme B and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against Panc1 pancreatic cancer cells.

[0185] [Figure 71] Figure 71 is a bar graph showing the expression of granzyme B and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs against SW480 colon cancer cells.

[0186] [Figure 72] Figure 72 is a bar graph showing the number of non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs in the presence and absence of the adjuvant lenalidomide.

[0187] [Figure 73] Figure 73 is a bar graph showing the number of central memory T cells and effector memory T cells in XBP1 cocktail-specific CTLs in the presence and absence of the adjuvant lenalidomide.

[0188] [Figure 74] Figure 74 is a bar graph showing the expression of CD40L, CD69, and CD38 in XBP1 cocktail-specific CTLs in the presence and absence of the adjuvant lenalidomide.

[0189] [Figure 75] Figure 75 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in MB231 breast cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0190] [Figure 76]Figure 76 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in MB231 breast cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0191] [Figure 77] Figure 77 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in Panc1 pancreatic cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0192] [Figure 78] Figure 78 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in Panc1 pancreatic cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0193] [Figure 79] Figure 79 is a bar graph showing the expression of Tbet and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in SW480 colon cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0194] [Figure 80] Figure 80 is a bar graph showing the expression of Eomes and IFN-γ by naive T cells, central memory cells, effector memory cells, and effector T cells of XBP1 cocktail-specific CTLs in SW480 colon cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0195] [Figure 81] Figure 81 is a bar graph showing the expression of granzymes and IFN-γ by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs in MB231 breast cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0196] [Figure 82] Figure 82 is a bar graph showing the expression of granzymes and IFN-γ by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs in Panc1 pancreatic cancer cells, in the presence or absence of the adjuvant lenalidomide.

[0197] [Figure 83] Figure 83 is a bar graph showing the expression of granzymes and IFN-γ by non-memory T cells and memory T cells of XBP1 cocktail-specific CTLs in SW480 colon cancer cells, in the presence or absence of the adjuvant lenalidomide. [Modes for carrying out the invention]

[0198] Detailed explanation This disclosure features immunogenic peptides (and their pharmaceutically acceptable compositions) derived from XBP1, CD138, and CS-1, which can be used, for example, to induce an immune response (e.g., to stimulate a CTL response) or to stimulate antibody production in a subject. These peptides can be used in a variety of applications (e.g., methods for inducing an immune response, methods for producing antibodies, and methods for treating cancer (e.g., lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia (e.g., AML or CML) and plasma cell damage (e.g., multiple myeloma or Waldenström macroglobulinemia)) or precancerous disorders (e.g., smoldering multiple myeloma)). These peptides can also be incorporated into MHC molecular multimer compositions and used, for example, in methods for detecting T cells in a cell population.

[0199] A detailed description of the above-mentioned peptides, as well as exemplary methods for producing and using them, is provided below.

[0200] peptide Group A peptides. This disclosure features isolated peptides ("Group A peptides") having an amino acid sequence that is sufficiently identical to or identical to any one of SEQ ID NOs: 1 to 18 as shown in Table 1. [Table 1]

[0201] Preferably, the peptides isolated from group A are at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and contain amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NOs. 1-18. Other preferred peptides may be at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and may include the amino acid sequences of SEQ ID NOs. 1-18, or amino acid sequences having 1, 2, 3, or 4 substitutions of the amino acid sequences of SEQ ID NOs. 1-18. These substitutions may be conserved or non-conserved.

[0202] The "unsplicing XBP1" peptides from Group A include the peptides shown in Table 1, which consist of 261 amino acids and the following sequence: MVVVAAAPNPADGTPKVLLLSGQPASAAGAPAGQALPLMVPAQRGASPEAASGGLPQARKRQRLTHLSPEEKALRRKLKNRVAAQTARDRKKARMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQ ELRQRLGMDALVAEEEAEAKGNEVRPVAGSAESAALRLRAPLQQVQAQLSPLQNISPWILAVLTLQIQSLISCWAFWTTWTQSCSSNALPQSLPAWRSSQRSTQKDPVPYQPPFLCQWGRHQPSWKPLMN( This refers to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids from the unspliced ​​form of human XBP1 protein having SEQ ID NO: 19, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conserved substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 19. The amino acid positions mentioned in Table 1 are based on SEQ ID NO: 19.

[0203] The "splicing-type XBP1" peptides from Group A include the peptides shown in Table 1, which consist of 376 amino acids and the following sequence: MVVVAAAPNPADGTPKVLLLSGQPASAAGAPAGQALPLMVPAQRGASPEAASGGLPQARKRQRLTHLSPEEKALRRKLKNRVAAQTARDRKKARMSELEQQVVDLEEENQKLLENQLLREKTHGLVVENQELRQRLGMDALVAEEEAEAKGNEVRPVAGSA ESAAGAGPVVTPPEHLPMDSGGIDSSDSESDILLGILDNLDPVMFFKCPSPEPASLEELPEVYPEGPSSLPASLSLSVGTSSAKLEAINELIRFDHIYTKPLVLEIPSETESQANVVVKIEEAPLSPSENDHPEFIVSVKEEPVEDDLVPELGISNLLSSSHC This refers to peptides having an amino acid sequence of at least five (e.g., 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, 31, 32, 33, 34, or 35) consecutive amino acids from the spliced ​​form of human XBP1 (XBP1 splicing type) protein having PKPSSCLLDAYSDCGYGGSLSPFSDMSSLLGVNHSWEDTFANELFPQLISV (Sequence ID 20; Genbank accession number NP_001073007), as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of Sequence ID 20. The amino acid positions mentioned in Table 1 are based on Sequence ID 20.

[0204] The "CD138" peptide from Group A includes the peptides shown in Table 1, which consist of 310 amino acids and the following sequence: MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQAST TTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMK This refers to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids derived from the amino acid sequence of SEQ ID NO: 21, as well as peptides having one, two, three, four, or five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 21. The amino acid positions mentioned in Table 1 are based on SEQ ID NO: 21.

[0205] The "CS-1" peptides from Group A include the peptides shown in Table 1, which consist of 335 amino acids and the following sequence: MAGSPTCLTLIYILWQLTGSAASGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTC VTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSMVLLCLLLVPLLLSLFVLGLFLWFLKRERQEEYIEEKKRVDICRETPNICPHSGENTEYDTIPHTN This refers to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids derived from the amino acid sequence of SEQ ID NO: 22, as well as peptides having one, two, three, four, or five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 22. The amino acid positions mentioned in Table 1 are based on SEQ ID NO: 22.

[0206] Group B peptides. The disclosure also features isolated peptides ("Group B peptides") having an amino acid sequence that is sufficiently identical to or identical to any one of SEQ ID NOs. 29-50 as shown in Table 2. [Table 2-1] [Table 2-2]

[0207] Preferably, the peptides isolated from group B are at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and contain amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NOs. 29-50. Other preferred peptides may be at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and may include the amino acid sequences of SEQ ID NOs. 29-50, or amino acid sequences having one, two, three, or four substitutions of the amino acid sequences of SEQ ID NOs. 29-50. These substitutions may be conserved or non-conserved.

[0208] The “unsplicing XBP1” peptides from Group B include the peptides shown in Table 2, which refer to peptides having an amino acid sequence of at least five (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) consecutive amino acids from the unspliced ​​form of human XBP1 protein having 261 amino acids and the amino acid sequence of SEQ ID NO: 19, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conserved substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 19. The unsplicing XBP1 peptides from Group B include peptides having an amino acid sequence from SEQ ID NO: 19 that includes part or all of any one of SEQ ID NOs. 29 and 33-37, for example, a sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminal and / or C-terminal end of any one of SEQ ID NOs. 29 and 33-37. The amino acid positions mentioned in Table 2 are based on Sequence ID No. 19.

[0209] The "splicing XBP1" peptides from Group B include the peptides shown in Table 2, which refer to peptides having an amino acid sequence of at least five (e.g., 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, 31, 32, 33, 34, or 35) consecutive amino acids from the spliced ​​form of human XBP1 (XBP1 splicing type) protein having 376 amino acids and the amino acid sequence of SEQ ID NO: 20, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 20. Splicing-type XBP1 peptides from Group B include peptides having an amino acid sequence from SEQ ID NO: 20 that includes part or all of any one of SEQ ID NOs: 30, 38, and 39, for example, a sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminal and / or C-terminal end of any one of SEQ ID NOs: 30, 38, and 39. The amino acid positions mentioned in Table 2 are based on SEQ ID NO: 20.

[0210] The "CD138" peptides from Group B include the peptides shown in Table 2, which refer to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids derived from the amino acid sequence of SEQ ID NO: 21, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 21. CD138 peptides from Group B include peptides having amino acid sequences from SEQ ID NO: 21 that include some or all of any one of SEQ ID NOs: 31 and 40-45, for example, sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminal and / or C-terminal end of any one of SEQ ID NOs: 31 and 46-50. The amino acid positions mentioned in Table 2 are based on SEQ ID NO: 21.

[0211] The "CS-1" peptides from Group B include the peptides shown in Table 2, which refer to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids derived from the amino acid sequence of SEQ ID NO: 22, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 22. CS1 peptides from Group B include peptides having amino acid sequences from Sequence ID No. 22 that include some or all of one of Sequence ID No. 32 and 46-50, for example, sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminal and / or C-terminal end of one of Sequence ID No. 32 and 46-50. The amino acid positions mentioned in Table 2 are based on Sequence ID No. 22.

[0212] Group C peptides. This disclosure features isolated peptides ("Group C peptides") having an amino acid sequence that is sufficiently identical to or identical to any one of SEQ ID NOs. 51-536 as shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21

[0213] Preferably, the peptides isolated from group C are at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and contain amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NOs. 51-536. Other preferred peptides may be at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acid lengths (e.g., 9-35 amino acid lengths, e.g., 9-30, 9-25, 9-20, 9-15 amino acid lengths) and may include the amino acid sequences of SEQ ID NOs. 51-536, or amino acid sequences having one, two, three, or four substitutions of the amino acid sequences of SEQ ID NOs. 51-536. These substitutions may be conserved or non-conserved.

[0214] The “unsplicing XBP1” peptides from Group C include those peptides shown in Table 3, which refer to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids from the unspliced ​​form of human XBP1 protein having 261 amino acids and the amino acid sequence of SEQ ID NO: 19, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conserved substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 19. The unsplicing XBP1 peptides from Group C include peptides having an amino acid sequence from SEQ ID NO: 19, including some or all of SEQ ID NOs: 51-206. The amino acid positions mentioned in Table 3 are based on SEQ ID NO: 19.

[0215] The "CD138" peptides from Group C include those peptides shown in Table 3, which refer to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids from the 310-amino acid sequence of the human CD138 protein and the amino acid sequence of SEQ ID NO: 21, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 21. The CD138 peptides from Group C include peptides having an amino acid sequence from SEQ ID NO: 21, including some or all of SEQ ID NOs: 207-371. The amino acid positions mentioned in Table 3 are based on SEQ ID NO: 21.

[0216] The "CS-1" peptides from Group C include those peptides shown in Table 3, which refer to peptides having an amino acid sequence of at least five (e.g., 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, or 35) consecutive amino acids from the human CS-1 protein having 335 amino acids and the amino acid sequence of SEQ ID NO: 22, as well as peptides having one, two, three, four, five or fewer substitutions (e.g., conservative substitutions) of amino acids derived from the amino acid sequence of SEQ ID NO: 22. The CS-1 peptides from Group C include peptides having an amino acid sequence from SEQ ID NO: 22, including some or all of SEQ ID NOs: 372-536. The amino acid positions mentioned in Table 3 are based on SEQ ID NO: 22.

[0217] Peptides in general. Peptides described herein are often referred to by the residue numbers of the N and C-terminal amino acids of those peptides, if the relevant sequences exist as full-length, wild-type mature human proteins having sequence numbers 19-22 (e.g., XBP1). 118-126These peptides may frequently have sequences identical to the corresponding segments of the wild-type, full-length mature protein having sequence numbers 19-22. However, the terms "unsplicing XBP1 peptide" (e.g., unsplicing XBP1 peptides having amino acid positions: 118-136, 185-193, 186-194, 190-198, 193-200 or 111-119), "splicing XBP1 peptide" (e.g., splicing XBP1 peptides having amino acid positions: 197-205, 194-202, 224-232, 368-376), "CD138 peptide" (e.g., amino acid positions It is understood that the CD138 peptide (having amino acid positions 256-264, 265-273, 260-268, 5-13 or 7-15) and the CS1 peptide (e.g., the CS-1 peptide having amino acid positions 236-245, 240-248, 239-247, 232-240 or 9-17) of non-human species may be peptide fragments of the XBP1 unsplicing peptide, XBP1 splicing peptide, CD138 or CS-1 polypeptide, respectively. As recognized by those skilled in the art, the numbering of the N and C-terminal amino acids of such non-human polypeptide peptide fragments is not necessarily the same as the numbering in the corresponding peptide fragment of the human polypeptide. Furthermore, the length and / or amino acids of the peptide fragments of non-human polypeptides will not necessarily be the same as those in the corresponding peptide fragment of the human polypeptide. Those skilled in the art will be familiar with methods for establishing the N-terminal and C-terminal amino acids, lengths, and amino acid sequences of peptides derived from non-human unsplicing XBP1, splicing XBP1, CD138, and CS-1 polypeptides. One useful method for doing this is sequence alignment, in particular, maximum homology sequence alignment.

[0218] The percentage of identity between two peptide sequences (e.g., peptides SEQ ID NOs. 1-18 and 29-536, and other amino acid sequences that may be at least 66% identical to those peptides) can be determined using a variety of algorithms and computer programs, including but not limited to Clustal W (The European Bioinformatics Institute (EMBL-EBI)), BLAST-Protein (National Center for Biotechnology Information (NCBI), United States National Institutes of Health) and PSAlign (University of Texas A&M; Sze et al. (2006) Journal of Computational Biology 13:309-319).

[0219] Variants of the human and non-human peptides described above are also disclosed herein. Variants of the human and non-human peptides described herein may include peptide forms having (i) at most four (e.g., three, two, or one) amino acid substitutions (e.g., conserved or non-conserved substitutions); (ii) terminal or internal deletions; or (iii) terminal or internal additions (all of which are described in detail below).

[0220] The disclosure also features peptides that contain, consist of, or are essentially derived from any of the amino acid sequences of Sequence IDs 1-18 and 29-536 (as shown in Tables 1-3), but have at most four substitutions (e.g., at most three, at most two, or at most one). These substitutions may be, for example, conserved or non-conserved (as described above).

[0221] Conservative substitutions include substitutions within the following groups: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. Nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the polar, basic, or acidic groups by another member of the same group may be considered a conservative substitution. In contrast, a non-conservative substitution is the substitution of one amino acid for another amino acid that has different characteristics.

[0222] In some embodiments, one or more of the 3, 4, 5, 6, 7, and 8 positions of any of the above peptides (e.g., 1, 2, 3, 4, or all 5) are not substituted. In some embodiments, one or more of the 3, 4, 5, 6, 7, and 8 positions of any of the above peptides are identical to the amino acids of the peptides in Tables 1-3.

[0223] The invention also features a fusion protein comprising a first amino acid sequence of a peptide described herein (e.g., the unsplicing XBP1 peptide described herein, the splicing XBP1 peptide described herein, the CD138 peptide described herein, and / or the CS-1 peptide described herein); and a second amino acid sequence heterogeneous to the first amino acid sequence.

[0224] The second heterologous amino acid sequence of the above peptide typically does not (and is selected not to) adversely affect the production of any of the immunogenic peptides of SEQ ID NOs: 1-18 and 29-536 in cells. The cellular mechanism is expected to yield any of the immunogenic peptides of SEQ ID NOs: 1-18 and 29-536 by removing any further sequences within the peptide, and that peptide, upon presentation by a class I or class II MHC molecule, stimulates an immune response against cancer cells expressing XBP1, CD138, or CS1.

[0225] An amino acid sequence that is “heterogeneous” with the first amino acid sequence, or the term “heterogeneous amino acid sequence,” is any amino acid sequence other than those adjacent to the first amino acid sequence if it exists in nature. For example, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more) and / or fewer than 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) carboxy-terminal and / or amino-terminal amino acids directly adjacent to LLREKTHGL (SEQ ID NO: 1) in human XBP1 are not considered heterogeneous with SEQ ID NO: 1. It is understood that fusion proteins containing a first amino acid sequence that is less than 100% identical to any of the amino acid sequences of sequence numbers 1-18 and 29-536, or that contain 1-4 conserved substitutions in those sequences, may never exist in nature.

[0226] In some embodiments, the second amino acid sequence may be a single amino acid. An amino acid that is “heterogeneous” with respect to the first amino acid sequence, or the term “heterogeneous amino acid,” is understood to be any amino acid other than those adjacent to the first amino acid sequence if it were naturally occurring. For example, the two amino acids directly adjacent to LLREKTHGL (SEQ ID NO: 1) in human XBP1 are not considered heterogeneous with SEQ ID NO: 1.

[0227] The heterologous sequence may be, for example, a sequence used for the purification of recombinant proteins (e.g., FLAG, polyhistidine (e.g., hexahistidine) (SEQ ID NO: 544), hemagluttanin (HA), glutathione-S-transferase (GST), or maltose-binding protein (MBP)). The heterologous sequence may also be a protein useful as a diagnostic marker or detectable marker, such as luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). In some embodiments, the fusion protein may include a signal sequence derived from another protein, such as the KDEL (SEQ ID NO: 23) sequence, or any other such sequence described herein. In some embodiments, the fusion protein may include all or part of an immunoglobulin molecule (e.g., all or part of the constant region of an immunoglobulin heavy chain; see below). In some embodiments, the fusion protein may include a therapeutic polypeptide or immunostimulatory polypeptide (e.g., a T helper epitope (e.g., a PADRE epitope or a tetanus toxoid universal T helper cell epitope) or all or part of a cytokine or chemokine) and / or a carrier useful in inducing an immune response (e.g., antibody production) (e.g., KLH). In some embodiments, the fusion protein may include one or more linkers, e.g., linkers containing peptide sequences (see below). The fusion protein may also include a targeted polypeptide. The heterologous sequence may be of varying lengths and may, in some cases, be longer than the first amino acid sequence to which the heterologous amino acid sequence is linked. It should be understood that the fusion protein, comprising the first amino acid sequence and the second amino acid sequence heterologous to the first, does not correspond in order to naturally occurring proteins.

[0228] Targeted polypeptides, as used herein, are polypeptides that target a specific tissue (e.g., lymph node) or cell (e.g., antigen-presenting cells or other immune cells), or, in vitro, a specific isolated molecule or molecular complex, by the portion to which they are attached (e.g., a first amino acid sequence). Targeted polypeptides may be, for example, antibodies (immunoglobulins) or their antigen-binding fragments, or ligands for cell surface receptors. Antibodies (or their antigen-binding fragments) may be, for example, monoclonal antibodies, polyclonal antibodies, humanized antibodies, fully human antibodies, single-chain antibodies, chimeric antibodies, or Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, or scFv fragments of antibodies. Antibody fragments containing or being Fc regions (with or without antigen-binding regions) may also be used to target reagents to cells expressing Fc receptors (e.g., antigen-presenting cells, e.g., finger-entangled dendritic cells, macrophages, monocytes, or B cells). Ligands for cell surface receptors may be, for example, chemokines, cytokines (e.g., interleukin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) or death receptor ligands (e.g., FasL or TNFα).

[0229] In some embodiments, heterologous sequences may be “transport sequences” that facilitate the delivery of peptides to cells or specific compartments within cells (e.g., the endoplasmic reticulum or Golgi apparatus). Examples of transport sequences include membrane transposition sequences, transportan sequences, Antennapedia sequences, cyclic integrin-binding peptides and Tat-mediated peptides or modified versions thereof.

[0230] A linker, for example, a linker peptide, can directly or indirectly connect a first amino acid sequence to one or more heterologous amino acid sequences. For example, a linker can connect a first amino acid sequence to a second amino acid sequence. A linker peptide may be, for example, a sequence of amino acids in which at least 4 to 6 amino acids are glycine (see, for example, Mancebo et al. (1990) Mol. Cell. Biol. 10:2492-2502). A linker peptide may also be, or contain, six or more (e.g., 7, 8, 9, 10, 11 or 12 or more) histidine residues. The linker peptide may be, or contain, at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 or more) protease cleavage sites. The protease sites may be, for example, trypsin, chymotrypsin, or factor Xa cleavage sites. Such protease sites may be useful, for example, for cleaving a first amino acid sequence from a heterologous sequence. For instance, after the expression and purification of a fusion protein containing a first amino acid sequence linked to a polyhistidine sequence (used in this case for purification) by a trypsin protease cleavage site, the polyhistidine sequence can be removed from the first amino acid sequence by contacting the fusion protein with trypsin.

[0231] The first amino acid sequence and the second amino acid sequence can associate with each other in various ways. As used herein, “associated with” in the context of interactions between two or more atoms or molecular units includes any covalent, non-covalent, or physical mixture of two or more atoms or molecular units (e.g., the first amino acid sequence and the second amino acid sequence). The chemical properties of covalent bonds (where two atoms share one or more valence pairs) are known in the art and include, for example, disulfide bonds or peptide bonds. Non-covalent bonds are chemical bonds between atoms or molecules that do not involve the sharing of valence pairs. Examples of non-covalent interactions include, for example, hydrophobic interactions, hydrogen bonding interactions, ionic bonds, van der Waals bonds, or dipole interactions. Examples of such non-covalent interactions include antibody-antigen complexation or binding pair interactions (interactions between the first and second members of a binding pair, e.g., the interaction between streptavidin and biotin). Therefore, it is understood that the term "associated with" (for example, in the context of the first amino acid sequence and the second amino acid sequence) has the same scope as the term "comprising".

[0232] In some embodiments, the first amino acid sequence and the second amino acid sequence may be encoded by a single nucleic acid sequence (and may be expressed as a fusion protein from a single nucleic acid sequence). In some cases, the first amino acid sequence and the second amino acid sequence may be encoded by two or more (e.g., three, four, five, or six or more) different nucleic acid sequences. For example, the first amino acid sequence may be encoded by a first nucleic acid sequence, and the second amino acid sequence may be encoded by a second nucleic acid sequence (see "Nucleic Acids and Methods for Peptide Production" below).

[0233] When expressed or produced separately, the first and second amino acid sequences can be crosslinked together using one of several known chemical crosslinkers. An example of such a chemical crosslinker is one that links two amino acid residues via a bond containing a "hindered" disulfide bond. In these bonds, the disulfide bond within the crosslinking unit is protected from reduction, for example, by the action of reduced glutathione or the enzyme disulfide reductase (by hindering the group on either side of the disulfide bond). One suitable chemical crosslinker, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes the terminal lysine in one of the two amino acid sequences and the terminal cysteine ​​in the other to form such a bond between those sequences. Heterobifunctional reagents crosslink by different binding sites in each amino acid sequence. In this way, the resulting "dimer" is neither a homodimer (e.g., two first amino acid sequences or two second amino acid sequences) nor a mixture of homodimers and heterodimers, but a heterodimer (a peptide containing the first and second amino acid sequences). Therefore, the binding site in the first amino acid sequence can be a cysteine ​​residue, or a lysine residue. Other useful crosslinking agents include, but are not limited to, chemicals that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), chemicals that link two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), chemicals that link an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), chemicals that link an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamide]butylamine), and chemicals that link an amino group and a guanadium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0234] The above-mentioned binding sites are preferably located at the terminal (C or N) of each amino acid sequence. These binding sites may be cysteine ​​residues in each amino acid sequence, or cysteine ​​in one and lysine in the other, as shown above. If the binding sites are two cysteine ​​residues, crosslinking can be achieved, for example, by exposing the amino acid sequence to oxidative conditions.

[0235] The fusion protein may contain a first amino acid sequence and a second amino acid sequence, or it may contain more than one (e.g., two, three, four, five, six, seven, or eight or more) additional heterologous amino acid sequences. These additional heterologous amino acid sequences may be adjacent to or ligated to the amino and / or carboxyl terminals of the first amino acid sequence.

[0236] When more than two amino acid sequences are linked together, at least one of those amino acid sequences may have more than one crosslinking portion. For example, the first amino acid sequence may have crosslinking portions at its amino and carboxyl termini. Such polymers can be constructed "continuously." Thus, each amino acid sequence is linked to the next amino acid sequence such that only the terminal amino acid sequence in its chain has one residue involved in inter-domain (or inter-agent) binding, while each of the "internal" amino acid sequences has two portions involved in inter-domain binding. Alternatively, one amino acid sequence (e.g., the first amino acid sequence) may be linked to multiple (e.g., two, three, four, or five) other amino acid sequences.

[0237] Peptide compositions comprising a first component and a second component are also characterized, where the first component is a peptide described herein. The second component may be, for example, a heterologous amino acid sequence (as described above), any other antigenic peptide (e.g., peptides other than those described herein), a detectable label (see below), a therapeutic agent, a diagnostic agent or prophylactic agent (see below). For example, a peptide composition may comprise an amino acid sequence consisting of or essentially derived from any of SEQ ID NOs: 1-18 and 29-536, as well as a detectable label such as a radionuclide.

[0238] In some embodiments, the peptides described herein are heterogeneous and number up to 200 (for example, 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, 3 It is understood that a molecule may have 8, 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 at its amino and / or carboxyl termini.

[0239] The peptides described herein may bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I molecules or MHC class II molecules). “Major histocompatibility complex” or “MHC” refers to a group of genes that play a role in regulating cell interactions involved in physiological immune responses. In humans, MHC is known as the HLA complex (e.g., Paul et al., FUNDAMENTAL IMMUNOLOGY, 3). rd Edition, Raven Press, New York, (1993) and Stites et al., IMMUNOLOGY, 8 th See Edition, Lange Publishing, Los Altos, Calif. (1994).

[0240] Where used herein, “HLA supertype or family” refers to a set of HLA molecules grouped based on shared peptide bond specificity. HLA class I molecules that share somewhat similar binding affinity to peptides having a particular amino acid motif are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecule (where xx represents a specific HLA type) are synonymous. Examples of HLA class I molecule types include HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-B7, HLA-B27, HLA-B44, HLA-B58, or HLA-B62. Such HLA molecules are described in detail in U.S. Patent No. 7,026,443 (the entire disclosure thereof is incorporated by reference).

[0241] Peptides can bind to MHC molecules with high affinity or intermediate affinity. As used herein, “high affinity” binding of a peptide to an HLA class I molecule is defined as having a dissociation constant (K) less than 50 nM (e.g., less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1 nM, or 0.05 nM). D ) is defined as a bond at approximately 50 nM to approximately 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) K DThis refers to the binding of peptides to HLA class I molecules. "High affinity" binding of peptides to HLA class II molecules is less than 100 nM (e.g., 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1 nM or less than 0.05 nM) D It is defined as binding at [a specific point]. The "intermediate affinity" of a peptide to an HLA class II molecule is approximately 100 to approximately 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, K (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) D The binding is at [location]. Methods for determining the binding affinity of peptides and MHC molecules are known in the art and are shown in the attached examples. Preferred methods are also described, for example, in U.S. Patent No. 7,026,443.

[0242] The peptides described herein can be recognized by antigen-specific T cell receptors on T cells when in association with MHC molecules. By using various suitable methods, it can be determined whether a peptide is recognized by a T cell receptor on a T cell when in association with an MHC molecule. For example, peripheral blood lymphocytes (PBL) from a normal subject can be cultured in vitro for several weeks with a test peptide in the presence of antigen-presenting cells. T cells specific for the peptide become activated during this time and can be detected using, for example, a proliferation assay (carboxyfluoroscein succinimidyl ester (CFSE) assay or 3 H-thymidine assay), limiting dilution assay, cytotoxicity assay (e.g., calcein release assay) or cytokine release assay (e.g., IFNγ), lymphokine release assay or 51 Cr release assay (see, for example, Wentworth, P.A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998, each of which disclosures is incorporated herein by reference in its entirety). Suitable in vivo methods include immunizing HLA transgenic mice, where the peptide in adjuvant is administered subcutaneously to the HLA transgenic mice, and several weeks after immunization, splenocytes are removed and cultured in vitro for approximately one week in the presence of the test peptide, and peptide-specific T cells are, for example, 51Detection is performed using a Cr-releasing assay (see, for example, 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 (each of these disclosures is incorporated by reference in whole)). Preferred methods are also shown in the accompanying examples. For example, T cell activation by peptides (in association with MHC molecules) can be determined by IFN-γ cytokine production, CD107α degranulation, or calcein-releasing cytotoxicity assays (see, for example, Examples 13 and 14).

[0243] Furthermore, direct quantification of antigen-specific T cells can be performed by staining T cells with either a detectably labeled MHC complex (e.g., an MHC molecular multimer composition described herein (see below) or an HLA-I tetramer (e.g., as described by Altman, JD et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993 and Altman, JD et al., Science 274:94, 1996 (each of these disclosures is incorporated by reference in whole))).

[0244] In some embodiments, the peptides described herein may be modified to modulate one or more properties of those peptides (e.g., to increase or decrease them) (e.g., amino acids of those peptides may be substituted). For example, one or more (e.g., two, three, or four) amino acids of one of the peptides shown in Table 1 may be substituted to increase the affinity of that peptide to an MHC molecule. In some embodiments, one amino acid of one of the peptides described herein (e.g., a T cell receptor that contacts the amino acid residue of that peptide) may be modified to enhance the binding interaction between the T cell receptor and the peptide (in relation to an MHC molecule). Such modified peptides are often referred to as “modified peptide ligands” (see, for example, Kalergis et al. (2000) J Immunol. 165(1):280; Conlon et al. (2002) Science 1801; and International Publication No. WO02070003 (each of these disclosures is incorporated by reference)).

[0245] Preferred methods for modifying peptides and determining the effects of such modifications are shown in the accompanying examples and, for example, described in Collins et al. (Immunological Reviews (1998) 163:151-160 (this disclosure is incorporated by reference in its entirety)).

[0246] Nucleic acids and methods for producing peptides This disclosure also features nucleic acid sequences (and nucleic acid vectors comprising nucleic acid sequences) encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) any peptides (or fusion proteins) described herein, and methods for producing them. Such methods may optionally include providing cells (or a group of cells) comprising a nucleic acid vector comprising nucleic acid sequences encoding one or more of the peptides (or fusion proteins) described herein (the nucleic acid sequences being operably linked to an expression regulatory sequence), and culturing the cells under conditions that enable the expression of the peptides (or fusion proteins). The methods may also include isolating one or more peptides (or proteins) from the cells or the culture medium in which the cells were cultured.

[0247] Preferred methods for constructing nucleic acid sequences and vectors (e.g., expression vectors) for the recombinant expression of one or more peptides (or fusion proteins) described herein are well known to those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring Harbor Laboratory. The information is presented in Press: Cold Spring Harbor, New York, USA, Nov. 1989 (this disclosure is incorporated by reference in its entirety). These nucleic acids and vectors can be used, for example, to express their peptides (or fusion proteins) in a wide variety of host cells (including, for example, bacterial, yeast, or mammalian cells). These nucleic acids and vectors can also be used in in vivo and exo vivo methods, for example, as described below.

[0248] A peptide-encoding sequence (or a fusion protein-encoding sequence) can be operably linked to a promoter and / or enhancer element that directs the expression of the nucleic acid-encoded peptide (or fusion protein). The enhancer provides specificity of expression with respect to time, location, and level. Unlike promoters, enhancers can function even when located at an indeterminate distance from the transcription start site, under the conditions of a promoter's presence. Enhancers can also be located downstream of the transcription start site or within the exons of the associated gene. For the coding sequence to be controlled by a promoter, the translation start site of the peptide's translation reading frame must be located 1 to approximately 50 nucleotides downstream (3') of the promoter. Target promoters include, but are not limited to, the cytomegalovirus hCMV pre-early gene, the early or late promoter of SV40 adenovirus, the lac, trp, TAC, TRC systems, major operator and promoter regions of phage A, the regulatory region of fd coat proteins, promoters for 3-phosphoglycerate kinase, promoters for acid phosphatases, and promoters for yeast α-conjugation factors, the adenovirus E1b minimal promoter, or the thymidine kinase minimal promoter.

[0249] A sequence encoding a peptide, a sequence encoding a fusion protein, or a vector containing such a sequence may include a leader sequence encoding a signal peptide. The leader sequence may be located at the 5' end of the sequence encoding one or more of the peptides or fusion proteins described herein. The signal peptide may be immediately N-terminal to a given peptide (or fusion protein), or it may be cleaved from the peptide by one or more amino acids (e.g., 2, 3, 4, 6, 8, 10, 15, or 20) provided that the leader sequence is in-frame with the nucleic acid sequence encoding the peptide or fusion protein. The signal peptide is typically cleaved from the peptide (or fusion protein) before secretion (unless, of course, the signal peptide is directed towards transmembrane protein insertion), directing the bound peptide (or fusion protein) into the lumen of the host cell's endoplasmic reticulum (ER) during translation, and then secreted into the host cell's environment via secretory vesicles. Useful signal peptides include, for example, natural leader sequences of cytokines or growth factors, KDEL (SEQ ID NO: 23), or any signal sequence described, for example, U.S. Patent No. 5,827,516 (the disclosure thereof is incorporated herein by reference in its entirety).

[0250] In some embodiments, the 5' end of the peptide-coding sequence (or fusion protein-coding sequence) may include a non-natural ATG "start sequence." That is, for example, an ATG sequence may be added to the nucleic acid encoding the peptide (or fusion protein) to ensure that the peptide (or fusion protein) is correctly transcribed and translated. The leader sequence typically includes an ATG start sequence, but in embodiments where the leader sequence does not include an ATG start sequence, an ATG sequence may be added to the 5' end of the nucleic acid encoding the leader sequence.

[0251] Preferred methods for constructing peptide-encoding sequences and expression vectors are well known to those skilled in the art, for example, Sambrook et al., Molecular Cloning: A The disclosure is contained herein by reference in the Laboratory Manual Second Edition vol. 1, 2, and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989.

[0252] Recombinant vectors can be introduced into cells using a variety of methods, and these methods may depend at least in part on the type of cell into which the nucleic acid is introduced. For example, bacterial cells can be transformed using methods such as electroporation or heat shock. Methods for transfecting yeast cells include, for example, the spheroplast method or the whole-cell lithium chloride yeast transformation method (see, e.g., U.S. Patent No. 4,929,555; Hinnen et al. (1978) Proc. Nat. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163; U.S. Patent No. 4,879,231; and Sreekrishna et al. (1987) Gene 59:115 (each of these disclosures is incorporated herein by reference in whole)). Transfection of animal cells may be characterized by introducing a vector into the cells, for example, by using calcium phosphate, electroporation, heat shock, liposomes, or transfection reagents (e.g., FUGENE® or LIPOFECTAMINE®), or by contacting a naked nucleic acid vector with cells in solution (see, for example, Sambrook et al., previously cited).

[0253] Expression systems that can be used for small-scale or large-scale production of the peptides (or fusion proteins) described herein include: microorganisms transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors, e.g., bacteria (e.g., Escherichia coli and B. subtilis); fungi transformed with recombinant yeast expression vectors (e.g., yeast (e.g., Saccharomyces and Pichia)); insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus); plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); or mammalian cell lines (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38 and NIH) having recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, CMV promoter, SV40 promoter, or vaccinia virus 7.5K promoter); Examples include, but are not limited to, 3T3 cells. Primary or secondary cells obtained directly from mammals, transfected with plasmid vectors, or infected with viral vectors (e.g., viral vectors, such as herpesviruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary poxviruses, adenoviruses, and adeno-associated viruses) are also useful as host cells.

[0254] As described above, after expression of any peptide (or fusion protein) described herein, those peptides (or fusion proteins) can be isolated from cultured cells or from the culture medium in which those cells were cultured using standard methods (see Sambrook et al., previously cited). 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.

[0255] Smaller peptides (e.g., peptides having fewer than 200 amino acids (e.g., fewer than 175, 150, 125, 100, 90, 80, 70, or 60)) can be chemically synthesized by standard chemical means such as FMOC solid-phase synthesis (see Example 1).

[0256] The peptides (and fusion proteins) described herein may be isolated, but are not necessarily required. The term “isolated,” when applied to any peptide (or fusion protein) described herein, means a peptide, its fragment (or, in the case of a composition, a macromolecular complex) that has been separated or purified from its naturally associated components (e.g., proteins or other naturally occurring biological or organic molecules). Recombinant molecules (e.g., recombinant peptides) are always understood to be “isolated.” Typically, a peptide (or fragment or macromolecular complex) is isolated if it constitutes at least 60% by weight of all molecules of the same type in a preparation, for example, 60% of all molecules of the same type in a sample. For example, a peptide described herein is considered isolated if it constitutes at least 60% by weight of all proteins in a preparation or sample. In some embodiments, molecules in a preparation constitute at least 75% by weight, at least 90% by weight, or at least 99% by weight of all molecules of the same type in the preparation.

[0257] Similarly, peptide-coding sequences, fusion protein-coding sequences, or vectors containing such sequences, as described herein, can also be isolated. The term “isolated,” when applied to any of the peptide-coding sequences, fusion protein-coding sequences, or vectors described herein, refers to peptide-coding sequences, fusion protein-coding sequences, or vectors, or their fragments, that have been separated or purified from their naturally occurring components (e.g., nucleic acids, proteins, or other naturally occurring biological or organic molecules). Recombinant molecules (e.g., recombinant vectors, or peptide-coding sequences or fusion protein-coding sequences) are always understood to be “isolated.” Typically, a peptide-coding sequence, fusion protein-coding sequence, or vector (or their fragments) is isolated if it constitutes at least 60% by weight of all molecules of the same type in a preparation, for example, 60% of all molecules of the same type in a sample. For example, a peptide-coding sequence or vector described herein is considered isolated if it constitutes at least 60% by weight of all nucleic acids in a preparation or sample. In some embodiments, molecules in the preparation constitute at least 75% by weight, at least 90% by weight, or at least 99% by weight of all molecules of the same type in the preparation.

[0258] In some embodiments, isolated peptides, fusion proteins, peptide-encoding sequences, fusion protein-encoding sequences, or vectors may be frozen, lyophilized, or immobilized and stored under suitable conditions that allow the molecules to retain their activity (e.g., the ability of the peptide to bind to MHC molecules such as MHC class I molecules, or the ability of the vector to support peptide expression in cells).

[0259] Further processing of peptides Following the expression or synthesis of any peptide (or fusion protein) described herein, such peptide (or fusion protein) may be further processed. Further processing may include chemical or enzymatic modification of the peptide (or fusion protein), or, if the peptide (or fusion protein) is modified, enzymatic or chemical modification of existing modifications, or both. Further processing of the peptide may include the addition (covalent or noncovalent linking) of heterologous amino acid sequences (e.g., any heterologous amino acid sequences described above, but not limited to these). Enzymatic processing may include contacting the peptide with, for example, one or more proteases, phosphatases, or kinases under conditions that allow the peptide to be modified. Enzymatic processing may include contacting the peptide with one or more enzymes (e.g., oligosaccharide transferases or mannosidases) that can glycosylate the peptide or modify its glycosylation.

[0260] The above processing may include the addition of, for example, a detectable label to the peptide. For example, the peptide may be an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase), a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride, allophycocyanin (APC) or phycoerythrin), a luminescent material (e.g., lanthanides or their chelates), a bioluminescent material (e.g., luciferase, luciferin or aequorin) or a radionuclide (e.g., 3 H, 32 P, 33 P, 125 I or 35 It can be labeled in a detectable manner as S).

[0261] The above process may also include conjugating the peptide (or fusion protein) to a polymer (e.g., a polyalkylene glycol moiety, e.g., a polyethylene glycol moiety). In some embodiments, the polymer is conjugated to the peptide at its N-terminal site. In some embodiments, the peptide may include one or more internal amino acid insertions that provide an internal polymer conjugation site to which the polymer can be conjugated.

[0262] Pharmaceutical composition Any of the peptides, fusion proteins, and nucleic acids encoding those peptides or fusion proteins described herein may be incorporated into a pharmaceutical composition. Such a composition typically comprises one or more of those peptides (and / or nucleic acids encoding those peptides) and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for administration of a pharmacopoeia. One or more peptides may be formulated as a pharmaceutical composition in the form of a syrup, elixir, suspension, powder, granules, tablet, capsule, lozenge, troche, solution, cream, ointment, lotion, gel, emulsion, etc. Auxiliary active compounds (e.g., one or more chemotherapeutic agents) may also be incorporated into the composition. Preferably, the composition comprises two or more (e.g., two, three, four, five, or six) peptides described herein. The composition may also include immunogenic peptides other than those disclosed herein, such as peptides derived from WT1 or its derivatives, such as immunogenic peptides described herein. Other immunogenic peptides include, but are not limited to, those derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0263] Pharmaceutical compositions are typically formulated to suit the intended route of administration. Examples of routes of administration include oral, rectal, and parenteral administration, such as intravenous, intramuscular, intradermal, subcutaneous, inhalation, percutaneous, or permucosal administration. Solutions or suspensions used for parenteral administration may contain the following components: sterile diluents (e.g., water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); antimicrobial agents (e.g., benzyl alcohol or methylparaben); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid); buffers (e.g., acetates, citrates, or phosphates, and active ingredients to adjust osmotic pressure, such as sodium chloride or dextrose). The pH may be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). Compositions may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0264] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate 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 enough to allow for easy syringability. The pharmaceutical composition must be stable under manufacturing and storage conditions and protected from any contamination by microorganisms (e.g., bacteria and fungi). Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial contamination can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is desirable to include isotonic agents, such as sugars and polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of injectable compositions can be promoted by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0265] Sterile injectable solutions may be prepared by incorporating one or more peptides (or one or more nucleic acids encoding those peptides) in the required amounts into a suitable solvent, along with one or a combination of the components listed above, if required, and then by filtration sterilization. Generally, dispersions are prepared by incorporating the peptide (or fusion protein, or nucleic acid encoding the peptide) into a sterile vehicle containing a basic dispersion medium and other required components from the components listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method may involve vacuum drying or lyophilization, yielding a powder of the active ingredient + any further desired components from its pre-filtered solution.

[0266] Oral compositions typically contain an inert diluent or edible carrier. For therapeutic oral administration, one or more peptides (or fusion proteins) may be incorporated with excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any or similar compounds of the following components: binders (e.g., microcrystalline cellulose, tragacanth gum, or gelatin); excipients (e.g., starch or lactose); disintegrants (e.g., alginic acid, primogel, or corn starch); lubricants (e.g., magnesium stearate or sterotes); glidants (e.g., colloidal silicon dioxide); sweeteners (e.g., sucrose or saccharin); flavorings (e.g., peppermint, methyl salicylate, or orange flavor).

[0267] Powders and tablets may contain 1% to 95% (w / w) of individual peptides or mixtures of two or more peptides. In certain embodiments, the peptides may be in the range of about 5% to 70% (w / w). Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point wax, and cocoa butter. The term “preparation” is intended to include formulations of peptides (or nucleic acids) accompanied by encapsulating material as a carrier, which provides a capsule in which the peptide is surrounded by a carrier, with or without other carriers, and thereby associated with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges may be used as solid dosage forms suitable for oral administration.

[0268] Aqueous solutions suitable for oral use can be prepared by dissolving the active components in water and adding suitable colorants, fragrances, stabilizers, and thickeners as desired. Aqueous suspensions suitable for oral use can be produced by dispersing the pulverized active components in water with a viscous material, such as natural or synthetic rubber, resin, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0269] When administered by inhalation, the peptide (or fusion protein or nucleic acid) may be delivered in the form of a suitable spray, such as an aerosol spray from a pressurized container or dispenser or sprayer containing a gas such as carbon dioxide.

[0270] Systemic administration may also be carried out by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, an appropriate penetrating agent for the barrier to be penetrated is used in the formulation. Such penetrating agents are widely known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives in the case of transmucosal administration. Transmucosal administration can be achieved by using nasal drops or suppositories. In the case of transdermal administration, the peptide (or fusion protein or nucleic acid) may be formulated into an ointment, plaster, gel, or cream, as are widely known in the art.

[0271] The above peptides (or fusion proteins or nucleic acids) may also be prepared in the form of suppositories for rectal delivery (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0272] In one embodiment, the peptides (or fusion proteins or nucleic acids) may be prepared using a carrier that protects the peptides (fusion proteins or nucleic acids) from rapid elimination from the body (e.g., controlled-release formulations including implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) may be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (e.g., containing liposomes targeted to APC by monoclonal antibodies against APC-specific antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0273] For ease of administration and for dose uniformity, it may be beneficial to formulate oral or parenteral compositions in dosage unit form. Where used herein, a dosage unit form refers to a physically distinct unit suitable as a unit dose to the subject being treated; each unit contains a predetermined amount of peptide (or fusion protein or nucleic acid) calculated to produce the desired therapeutic effect, along with the required pharmaceutical carrier. Dosage units may also be accompanied by instructions for use.

[0274] The nucleic acid molecule encoding the above peptide (or fusion protein) may be inserted into a vector and used as a gene therapy vector (as described above). The gene therapy vector may be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutically appropriate preparation of the gene therapy vector may contain the gene therapy vector in an acceptable diluent or contain a sustained-release matrix in which the gene delivery vehicle is embedded. Alternatively, if the complete gene delivery vector can be produced intact from recombinant cells (e.g., a retroviral vector), the pharmaceutically appropriate preparation may contain one or more cells that produce the gene delivery system (see "Exovivo Method" below).

[0275] Further examples of gene delivery vehicles include, but are not limited to, other recombinant vehicles commonly used in the field, described for expression in various eukaryotic and prokaryotic hosts and potentially used for gene therapy and simple protein expression. These include liposomes, biocompatible polymers including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses (e.g., baculoviruses, adenoviruses, and retroviruses); bacteriophages; cosmids; plasmids; fungal vectors; and other recombinant vehicles commonly used in the field and described for expression in various eukaryotic and prokaryotic hosts.

[0276] Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphavirus vectors. Liposomes containing a targeting moiety, such as an antibody or a fragment thereof, can also be used to prepare a pharmaceutical composition of nucleic acids for delivery to a subject.

[0277] Any pharmaceutical composition described herein may be included in a container, pack, or dispenser, along with instructions for administration as described below.

[0278] MHC molecular polymer composition and method for using the composition The disclosure also features compositions comprising (i) one or more of the peptides described above, and (ii) a major histocompatibility complex (MHC) molecular polymer. The polymer comprises two or more (e.g., three, four, five, six, seven, eight, nine, or ten or more) MHC molecules or peptide-binding domains of MHC molecules. The one or more peptides may associate with the MHC molecular polymer (e.g., covalently or noncovalently).

[0279] The MHC molecule of the above-mentioned polymer may be an MHC class I molecule (e.g., an HLA-A molecule, e.g., an HLA-A2 or HLA-A24 molecule) or an MHC class II molecule. The MHC molecule may be an MHC molecule of a mammal (e.g., a rodent, a non-human primate, a human, or any other mammal described herein).

[0280] In the above-described polymer, two or more MHC molecules (or the peptide-binding regions of those MHC molecules) may originate from the same MHC molecule or from different MHC molecules. For example, an MHC molecule polymer 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 in the polymer is different. At least one of those MHC molecules may bind to at least one of the above-described peptides.

[0281] In some embodiments, the composition may comprise at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 15 or more) of the peptides described herein. The composition may also comprise immunogenic peptides other than those disclosed herein, such as peptides derived from WT1 or its derivatives. Other immunogenic peptides include, but are not limited to, those derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0282] The above compositions may also be associated with detectable labels. For example, one or more MHC molecules of the above polymer may be covalently or acovalently bound to the detectable label. Suitable detectable labels (e.g., enzymes, fluorescent materials, luminescent materials, bioluminescent materials, or radionuclides) and methods for linking the detectable labels to peptides or MHC molecules are described above.

[0283] MHC multimer compositions can be produced using the peptides described above, as follows: A tripolecular complex is formed by refolding a peptide that binds to an HLA molecule in the presence of the corresponding HLA heavy chain and β2-microglobulin. The complex is then biotinylated at the carboxyl terminus of the heavy chain at the site that has been previously manipulated into a heavy chain. Multimerization is then induced by adding streptavidin.

[0284] If a T cell receptor can recognize a specific peptide-MHC complex on a target cell among a wide variety of other peptide-MHC complexes, the MHC multimer compositions described herein may be used, for example, to detect antigen-specific T cells within an unrelated population of T cells (see below). For such assays, the multimers are generally labeled for detection (see above).

[0285] For example, multimeric MHC molecule / peptide complexes can be used in assays to evaluate peripheral blood mononuclear cells for the presence of antigen-specific CTLs after exposure to an immunogen. These MHC multimeric complexes can be used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al., Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996) and to determine the frequency of antigen-specific CTL populations in a sample of peripheral blood mononuclear cells. In one example, detectably labeled streptavidin, used to multimerize MHC multimers, can be used to label T cells that bind to the multimeric MHC molecule / peptide complex. To do this, cells treated with the multimer are exposed to a label (e.g., a biotin-conjugated fluorophore). These cells can then be readily isolated or detected, for example, using flow cytometry.

[0286] Purpose The peptides, fusion proteins (and their pharmaceutical compositions), compositions, kits, and products comprising MHC polymers described herein may be used in a variety of ways. For example, the peptides described herein may be used (i) to induce an immune response in a subject (e.g., a subject with cancer); (ii) to activate T cells in culture (e.g., central memory T cells and / or effector memory T cells); and / or (iii) to treat or prevent cancer. Examples of cancers include lung cancer, liver cancer, bile duct cancer, stomach cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, hematological cancers, such as plasma cell carcinoma, such as multiple myeloma and leukemia, such as AML or CML. The peptides described herein may also be used to treat precancerous conditions such as smoldering multiple myeloma. As described above, compositions containing MHC multimers can be used, for example, to detect antigen-specific T cells within a population of unrelated T cells.

[0287] The usefulness of compositions, kits, or products comprising the above-mentioned peptides (or their pharmaceutical compositions), MHC polymers is by no means limited to any specific embodiment described herein, but exemplary methods in which these reagents may be used are provided below.

[0288] Methods for inducing an immune response This disclosure also features various methods for inducing an immune response in a subject. Methods for inducing an immune response in a subject may include administering to the subject one or more peptides or any pharmaceutical composition described herein. The immune response is CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes (CTLs), T H 1 response, T H It can be two responses or a combination of both types of responses.

[0289] Any of the above methods may also be methods for treating or preventing cancer in a subject (e.g., plasma cell damage, e.g., multiple myeloma or Waldenström macroglobulinemia, or any other cancer expressing XBP1, CD138 or CS1) (prevention against cancer). When the terms “prevent,” “preventing,” or “prevention” are used herein in connection with a given treatment for a given condition, they mean that the treated subject does not develop the condition at all at a clinically observable level (e.g., the subject does not exhibit one or more symptoms of the condition, or, in the case of cancer, the subject does not develop cancer at a detectable level).

[0290] Where used herein, the terms “treat,” “treatment,” or “treating” a subject having a disorder, e.g., cancer, are used in relation to a given treatment for a given disorder, wherein at least one symptom of the disorder is treated, cured, alleviated, reduced, altered, cured, restored, or improved. Treatment includes the administration of an amount of composition effective in alleviating, reducing, altering, curing, restoring, improving, or influencing the disorder or its symptoms. Treatment may inhibit the exacerbation or worsening of the symptoms of the disorder, or cause the condition (e.g., fewer symptoms or fewer cancer cells in the subject) to develop more slowly and / or to a lower degree in the subject than would be possible without treatment. For example, treatment would be said to have “treated” a condition if, during the course of a condition, for example, during an early diagnosis of cancer that was expected to give rise to a given sign of that condition (advanced cancer) (e.g., detection of several cancer cells in a sample derived from the subject), the subject experiences fewer and / or milder symptoms of the condition than would have been expected otherwise. Treatment could also be said to have “treated” a cancer if the subject exhibits only mild, obvious symptoms of the cancer (e.g., plasma cell damage, e.g., multiple myeloma or Waldenström macroglobulinemia).

[0291] In one embodiment, cancer is a cancer described herein. For example, such cancer is bladder cancer (including advanced bladder cancer and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma)). These may include cancers of the genitourinary system, such as ovarian cancer (including fallopian tube cancer, endometrial cancer, and peritoneal cancer), cervical cancer, prostate cancer, and testicular cancer, cancers of the lymphatic system, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic carcinoma), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer, or lower gastric cancer), gastrointestinal cancer (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal cancer and glial cell cancer (e.g., glioblastoma multiforme), and head and neck cancer (e.g., nasopharyngeal cancer).

[0292] Generally, the peptide(s) delivered to the subject are suspended in a pharmaceutically acceptable carrier (e.g., saline) and administered orally, rectally, or parenterally, for example, by injection intravenously, subcutaneously, intramuscularly, intrathecally, intraperitoneally, rectally, vaginally, intranasally, intragastricly, intratracheally, or intrapulmonaryly (see below).

[0293] Administration may be by periodic bolus injection of the pharmaceutical composition, or by uninterrupted or continuous intravenous or intraperitoneal administration from an external reservoir (e.g., an IV bag) or an internal reservoir (e.g., a bioerodable implant, bioprosthetic organ, or implanted colony of reagent-producing cells). See, for example, U.S. Patents 4,407,957, 5,798,113, and 5,800,828 (each incorporated herein by reference in whole).

[0294] Generally, the required dose of peptide or nucleic acid depends on the choice of route of administration; the nature of the formulation; the nature or severity of the subject's disease; the subject's immune status; the subject's size, weight, surface area, age, and sex; other medications being administered; and the judgment of the healthcare professional in charge.

[0295] The appropriate dose of peptides for inducing an immune response is within the range of 0.000001 to 10 mg of the reagent or antigenic / immunogenic composition per kg of subject. Wide variations in the required dose should be anticipated, considering the diversity of reagents and the different efficiencies of various administration routes. For example, nasal or rectal administration may require higher doses than intravenous administration. These variations in dose levels can be adjusted using standard, empirical routines for optimization, as well as those well understood in the art. Dosage may be single or multiple (e.g., 2, 3, 4, 6, 8, 10, 20, 50, 100, 150 times or more). For example, the peptide(s) may be administered as a primary immunization, and then one or more subsequent booster immunizations.

[0296] To optimize the therapeutic effect (e.g., the effectiveness of one or more peptides that induce an immune response in a subject, or the nucleic acids encoding those peptides), compositions containing those peptides or nucleic acids may first be administered in various drug regimens. The unit dose and regimen depend on factors including, for example, the mammalian species, its immune status, and the mammal's body weight.

[0297] The frequency of administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is within the scope of the skills and clinical judgment of the healthcare professional (e.g., a physician or nurse). Typically, the administration regimen is established by clinical trials that can determine the optimal administration parameters. However, the professional may modify such an administration regimen according to the age, health status, weight, sex, and medical condition of the subject.

[0298] In some embodiments, the pharmaceutical composition may be administered to a subject at least twice (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 or more times). For example, the pharmaceutical composition may be administered to a subject once a month over a three-month period; once a week over a one-month period; once a year over a three-year period, once a year over a five-year period; once every five years; once every ten years; or once every three years over a lifetime.

[0299] In some embodiments, the reagent may be administered together with an immunomodulator (e.g., a Toll receptor ligand or adjuvant (see below)).

[0300] As defined herein, a “therapeutic effective dose” of a peptide or nucleic acid encoding a peptide is the amount of the peptide or nucleic acid that can elicit an immune response in the subject being treated. A therapeutic effective dose of a peptide (i.e., an effective dose) includes an amount of the reagent in milligrams, micrograms, nanograms, or picograms per kilogram of body weight of the subject or sample (e.g., about 1 nanogram to about 500 micrograms per kilogram, about 1 microgram to about 500 milligrams per kilogram, about 100 micrograms to about 5 milligrams per kilogram, or about 1 microgram to about 50 micrograms per kilogram). The therapeutically effective dose of nucleic acid also includes an amount of reagent in micrograms, nanograms, or picograms per kilogram of subject body weight or sample weight (e.g., approximately 1 nanogram to 500 micrograms per kilogram, approximately 1 microgram to 500 micrograms per kilogram, approximately 100 micrograms to 500 micrograms per kilogram, or approximately 1 microgram to 50 micrograms per kilogram).

[0301] As defined herein, the “preventive effective dose” of a peptide or nucleic acid encoding a peptide is the amount of the peptide or nucleic acid that can elicit an immune response in a treated subject against cancer cells (e.g., breast cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, multiple myeloma cells, or Waldenström macroglobulinemia cells) that can prevent the development of cancer in the subject or substantially reduce the probability that the subject will develop or continue to develop cancer (see above). The preventive effective dose (i.e., effective dose) of a peptide contains an amount of reagent in milligrams, micrograms, nanograms, or picograms per kilogram of body weight of the subject or sample (e.g., about 1 nanogram to about 500 micrograms per kilogram, about 1 microgram to about 500 milligrams per kilogram, about 100 micrograms to about 5 milligrams per kilogram, or about 1 microgram to about 50 micrograms per kilogram). The prophylactic effective dose of nucleic acid also includes an amount of reagent in micrograms, nanograms, or picograms per kilogram of subject body weight or sample weight (e.g., approximately 1 nanogram to 500 micrograms per kilogram, approximately 1 microgram to 500 micrograms per kilogram, approximately 100 micrograms to 500 micrograms per kilogram, or approximately 1 microgram to 50 micrograms per kilogram).

[0302] The subjects may be any animal capable of an immune response to the antigen (e.g., mammals, e.g., humans (e.g., human patients) or non-human primates (e.g., chimpanzees, baboons, or monkeys), mice, rats, rabbits, guinea pigs, gerbils, hamsters, horses, a species of domestic animal (e.g., cattle, pigs, sheep, or goats), dogs, cats, or whales). The subjects may have, be suspected of having, or be at risk of developing cancer (e.g., multiple myeloma, Waldenström macroglobulinemia, or any other type of cancer expressing XBP1, CD138, or CS-1 (e.g., lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, leukemia, e.g., AML or CML)). The subjects may be those in remission from cancer, such as breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, such as AML or CML, multiple myeloma, or Waldenström macroglobulinemia.

[0303] The above method may also include a step of determining whether one or more cancer cells of the subject's cancer (e.g., lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, or plasma cell disorder, e.g., multiple myeloma or Waldenström macroglobulinemia) express XBP1, CD138, or CS-1 before administering one or more peptides (or nucleic acids) to the subject. The expression of these proteins includes the expression of both mRNA and protein. Methods for detecting protein and mRNA expression in cells are known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), Western blotting and dot blotting or immunohistochemistry for detecting proteins, and reverse transcription polymerase chain reaction (RT-PCR) or Northern blotting for detecting mRNA (see Sambrook et al., cited above).

[0304] The peptides or compositions described above may be used in combination with other known treatments. “Administered in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s suffering due to the disorder, for example, that the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder is treated or eliminated or before the treatment is interrupted for any other reason. In some embodiments, the delivery of one treatment is still occurring when the delivery of a second treatment begins, resulting in an overlap in administration. This is sometimes referred to herein as “simultaneous” or “combined delivery.” In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective due to combined administration. For example, the second treatment may be more effective, for instance, with fewer doses of the second treatment achieving an equivalent effect, or the second treatment may reduce symptoms more significantly than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation to that achieved with the first treatment may be observed. In some embodiments, the delivery is such that the reduction in symptoms, or other parameters relating to the impairment thereof, is greater than what would be observed if one treatment were delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered.

[0305] Further treatments may include, for example, surgery, one or more chemotherapeutic agents, one or more forms of ionizing radiation, and / or one or more immunomodulatory agents.

[0306] One or more forms of ionizing radiation may be gamma-ray irradiation, X-ray irradiation, or beta-ray irradiation.

[0307] Examples of chemotherapeutic agents include, for example: Alkylating agents (including, but not limited to, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, and triazenes): Uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen) Mustard (registered trademark), Uracillost (registered trademark), Uracilmostaza (registered trademark), Uramustin (registered trademark), Uramustine (registered trademark), Chlormetine (Mustargen (registered trademark)), Cyclophosphamide (Cytoxan (registered trademark), Neosar (registered trademark), Clafen (registered trademark), Endoxan (registered trademark), Procytox (registered trademark), Revimmune (trademark)), Ifosfamide (Mitoxana (registered trademark)), Melphalan (Alkeran (registered trademark)), Chlorambucil (Leukeran (registered trademark)), Pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).

[0308] Anti-EGFR antibodies (e.g., cetuximab (Erbitux®) and panitumumab (Vectibix®)).

[0309] Anti-HER-2 antibodies (e.g., trastuzumab (Herceptin®)).

[0310] Antimetabolites (including, but not limited to, folate antagonists (also referred to herein as antifolate agents), pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), cytarabine (Cytosar-U®, Tarabine PFS), 6-mercaptopurine (Puri-Nethol®), 6-thioguanine Tabloid®, fludarabine phosphate (Fludara®), pentostatin (Nipent®), pemetrexed (Alimta®), raltitrexed (Tomudex®), cladribine (Leustatin®), clofarabine (Clofarex®, Clolar®), mercaptopurine (Puri-Nethol®), capecitabine (Xeloda®), nelarabine (Arranon®), azacitidine (Vidaza®), and gemcitabine (Gemzar®). Preferred antimetabolites include, for example, 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), phloxuridine (FUDF®), capecitabine (Xeloda®), pemetrexed (Alimta®), larcitrexed (Tomudex®), and gemcitabine (Gemzar®).

[0311] Vinca alkaloids: Vinblastine (Velban®, Velsar®), Vincristine (Vincasar®, Oncovin®), Vindesine (Eldisine®), Vinorelbine (Navelbine®).

[0312] Platinum-based drugs: Carboplatin (Paraplat®, Paraplatin®), cisplatin (Platinol®), oxaliplatin (Eloxatin®).

[0313] Anthracyclines: Daunorubicin (Cerubidine®, Rubidomycin®), Doxorubicin (Adriamycin®), Epirubicin (Ellence®), Idarubicin (Idamycin®), Mitoxantrone (Novantrone®), Barurubicin (Valstar®). Preferred anthracyclines include Daunorubicin (Cerubidine®, Rubidomycin®) and Doxorubicin (Adriamycin®).

[0314] Topoisomerase inhibitors: Topotecan (Hycamtin®), Irinotecan (Camptosar®), Etoposide (Toposar®, VePesid®), Teniposide (Vumon®), Lamelarin D, SN-38, Camptothecin.

[0315] Taxanes: Paclitaxel (Taxol®), Docetaxel (Taxotere®), Larotaxel, Cabazitaxel.

[0316] Epothiron: ixabepilone, Epothiron B, Epothiron D, BMS310705, dehydelone, ZK-EPO.

[0317] Poly-ADP-ribose polymerase (PARP) inhibitors: (e.g., BSI201, Olaparib (AZD-2281), ABT-888, AG014699, CEP9722, MK4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide).

[0318] Antibiotics: Actinomycin (Cosmegen®), Bleomycin (Blenoxane®), Hydroxyurea (Droxia®, Hydrea®), Mitomycin (Mitozytrex®, Mutamycin®).

[0319] Immunomodulatory substances: Lenalidomide (Revlimid®), Thalidomide (Thalomid®).

[0320] Immune cell antibodies: alemtuzumab (Campath®), gemtuzumab (Myelotarg®), rituximab (Rituxan®), tositumomab (Bexxar®).

[0321] Interferon (e.g., IFN-alpha (Alferon®, Roferon-A®, Intron®-A) or IFN-gamma (Actmme®)).

[0322] Interleukins: IL-1, IL-2 (Proleukin®), IL-24, IL-6 (Sigosix®), IL-12.

[0323] HSP90 inhibitors (e.g., geldanamycin or any derivative thereof). In certain embodiments, the HSP90 inhibitor is selected from geldanamycin, 17-alkylamino-17-desmethoxygeldanamycin ("17-AAG"), or 17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin ("17-DMAG").

[0324] Pharmaceutical drugs (A6 (Angstrom Pharmacueticals), ABT-510 (Abbott Laboratories), ABT-627 (Atrasentan) (Abbott Laboratories / Xinlay), ABT-869 (Abbott Laboratories, Actimide (CC4047), Pomalidomide (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex). Technologies, AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP). O2) (Alnylam Pharmaceuticals), AMG386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Ridaforolimus / MK8669) (Ariad Pharmaceuticals, AQ4N (Novavea), ARQ197 (ArQule), ASA404 (Novartis / Antisoma), Atiprimod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals, Avastin (Bevacizumab) (Genentech), AZD2171 (Cediranib / Recentin) (AstraZeneca), BAY 57-9352 (Telatinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim). Pharmaceuticals) 、BIBW2992(Boehringer Ingelheim Pharmaceuticals) 、BMS-275291(Bristol-Myers Squibb) 、BMS-582664(Brivanib)(Bristol-Myers).Squibb)、BMS-690514(Bristol-Myers Squibb)、Calcitriol、CCI-779(Torisel)(Wyeth)、CDP-791(ImClone Systems)、Ceflatonin(Homoharringtonine / HHT)(ChemGenex Therapeutics)、Celebrex(Celecoxib)(Pfizer)、CEP-7055(Cephalon / Sanofi)、CHIR-265(Chiron Corporation)、NGR-TNF、COL-3(Metastat)(Collagenex Pharaceuticals)、Combretastatin(Oxigene)、CP-751,871(Figitumumab)(Pfizer)、CP-547,632(Pfizer)、CS-7017(Daiichi Sankyo Pharma)、CT-322(Angiocept)(Adnexus)、Curcumin、Dalteparin(Fragmin)(Pfizer)、Disulfiram(Antabuse)、E7820(Eisai Limited)、E7080(Eisai Limited)、EMD121974(Cilengitide)(EMD Pharmaceuticals)、ENMD-1198(EntreMed)、ENMD-2076(EntreMed)、Endostar(Simcere)、Erbitux(ImClone / Bristol-Myers Squibb)、EZN-2208(Enzon Pharmaceuticals)、EZN-2968(Enzon Pharmaceuticals)、GC1008(Genzyme)、Genistein、GSK1363089(Foretinib)(GlaxoSmithKline)、GW786034(Pazopanib)(GlaxoSmithKline)、GT-111(Vascular Biogenics Ltd.)、IMC--1121B(Ramucirumab)(ImClone Systems)、IMC-18F1(ImClone Systems)、IMC-3G3(ImClone LLC)、INCB007839(Incyte Corporation)、INGN241(Introgen Therapeutics)、Iressa(ZD1839 / Gefitinib)、LBH589(Faridak / Panobinostst)(Novartis)、Lucentis(Ranibizumab)(Genentech / Novartis)、LY317615(Enzastaurin)(Eli Lilly and Company)、Macugen(Pegaptanib)(Pfizer)、MEDI522(Abegrin)(MedImmune)、MLN518(Tandutinib)(Millennium)、Neovastat(AE941 / Benefin)(Aeterna Zentaris)、Nexavar(Bayer / Onyx)、NM-3(Genzyme Corporation)、Noscapine(Cougar Biotechnology)、NPI-2358(Nereus Pharmaceuticals)、OSI-930(OSI)、Palomid529(Paloma Pharmaceuticals,Inc.)、Panzem Capsules(2ME2)(EntreMed)、Panzem NCD(2ME2)(EntreMed)、PF-02341066(Pfizer)、PF-04554878(Pfizer)、PI-88(Progen Industries / Medigen Biotechnology)、PKC412(Novartis)、Polyphenon E(Green Tea Extract)(Polypheno E International,Inc)、PPI-2458(Praecis Pharmaceuticals)、PTC299(PTC Therapeutics)、PTK787(Vatalanib)(Novartis)、PXD101(Belinostat)(CuraGen Corporation)、RAD001(Everolimus)(Novartis)、RAF265(Novartis)、Regorafenib(BAY73-4506)(Bayer)、Revlimid(Celgene)、Retaane(Alcon Research)、SN38(Liposomal)(Neopharm)、SNS-032(BMS-387032)(Sunesis)、SOM230(Pasireotide)(Novartis)、Squalamine(Genaera)、Suramin、Sutent(Pfizer)、Tarceva(Genentech)、TB-403(Thrombogenics)、Tempostatin(Collard Biopharmaceuticals)、Tetrathiomolybdate(Sigma-Aldrich)、TG100801(TargeGen)、Thalidomide(Celgene Corporation)、Tinzaparin Sodium、TKI258(Novartis)、TRC093(Tracon Pharmaceuticals Inc.This includes, but is not limited to, VEGF Trap (Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK474.

[0325] Anti-androgens (including, but not limited to, nilutamide (Nilandron®) and bicalutamide (Caxodex®)).

[0326] Anti-estrogens (including, but not limited to, tamoxifen (Nolvadex®), toremifene (Fareston®), letrozole (Femara®), testolactone (Teslac®), anastrozole (Arimidex®), bicalutamide (Casodex®), exemestane (Aromasin®), flutamide (Eulexin®), fulvestrant (Faslodex®), raloxifene (Evista®, Keoxifene®), and raloxifene hydrochloride).

[0327] Anti-hypercalcemia agents (including, but not limited to, gallium(III) nitrate hydrate (Ganite®) and disodium pamidronate (Aredia®)).

[0328] Apoptosis-inducing factors (including, but not limited to, ethanol, 2-[[3-(2,3-dichlorophenoxy)propyl]amino]-(9Cl), gambogic acid, envelope, and arsenic trioxide (Trisenox®)).

[0329] Aurora kinase inhibitors (including, but not limited to, binucleine 2).

[0330] Bruton's tyrosine kinase inhibitors (including, but not limited to, terreic acid).

[0331] Calcineurin inhibitors (including, but not limited to, cypermethrin, deltamethrin, fenvalerate, and tyrophostine 8).

[0332] CaM kinase II inhibitors (including, but not limited to, 5-isoquinoline sulfonic acid, 4-[{2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-{4-phenyl-1-piperazinyl)propyl]phenyl ester, and benzenesulfonamides).

[0333] CD45 tyrosine phosphatase inhibitors (including, but not limited to, phosphonic acids).

[0334] CDC25 phosphatase inhibitors (including, but not limited to, 1,4-naphthalenedione, 2,3-bis[(2-hydroxyethyl)thio]-(9Cl)).

[0335] CHK kinase inhibitors (including, but not limited to, debromohimenialdisine).

[0336] Cyclooxygenase inhibitors (1H-indole-3-acetamide, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-N-(2-phenylethyl)-(9Cl), 5-alkyl-substituted 2-arylaminophenylacetic acid and its derivatives (e.g., celecoxib (Celebrex®), rofecoxib (Vioxx®), etoricoxib (Arcoxia®), lumiracoxib (Prexige®), valdecoxib (Bextra®), or 5-alkyl-2-arylaminophenylacetic acid, but not limited to these)).

[0337] cRAF kinase inhibitors (including, but not limited to, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindole-2-one and benzamide, 3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)amino]-4-methylphenyl]-(9Cl)).

[0338] Cyclin-dependent kinase inhibitors (including, but not limited to, olomoucine and its derivatives, pluvaranol B, roascovitine (Seliciclib®), indirubin, kenpaullone, pluvaranol A, and indirubin-3'-monoxime).

[0339] Cysteine ​​protease inhibitors (including, but not limited to, 4-morpholine carboxamide, N-[(1S)-3-fluoro-2-oxo-1-(2-phenylethyl)propyl]amino]-2-oxo-1-(phenylmethyl)ethyl]-(9Cl)).

[0340] DNA intercalators (including, but not limited to, Plicamycin (Mithracin®) and Daptomycin (Cubicin®)).

[0341] DNA strand cleavage agents (including, but not limited to, bleomycin (Blenoxane®)).

[0342] E3 ligase inhibitors (including, but not limited to, N-((3,3,3-trifluoro-2-trifluoromethyl)propionyl)sulfanilamides).

[0343] EGF pathway inhibitors (including, but not limited to, thyrophostin 46, EKB-569, erlotinib (Tarceva®), gefitinib (Iressa®), lapatinib (Tykerb®), and compounds generally and in detail disclosed in WO97 / 02266, EP0564409, WO99 / 03854, EP0520722, EP0566226, EP0787722, EP0837063, U.S. Patent No. 5,747,498, WO98 / 10767, WO97 / 30034, WO97 / 49688, WO97 / 38983, and WO96 / 33980).

[0344] Farnesyltransferase inhibitors (including, but not limited to, A-hydroxyfarnesylphosphonic acid, butanoic acid, 2-[(2S)-2-[[(2S,3S)-2-[[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-1-methylethyl ester (2S)-(9Cl), and manumycin A).

[0345] Flk-1 kinase inhibitors (including, but not limited to, 2-propenamide, 2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl)-(2E)-(9Cl)).

[0346] Glycogen synthase kinase-3 (GSK3) inhibitors (including, but not limited to, indirubin-3'-monoxime).

[0347] Heat shock protein 90 (Hsp90) chaperone regulators (including, but not limited to, AUY922, STA-9090, ATI13387, MCP-3100, IPI-504, IPI-493, SNX-5422, Debio0932, HSP990, DS-2248, PU-H71, 17-DMAG (Alvespimycin), and XL888).

[0348] Histone deacetylase (HDAC) inhibitors (including, but not limited to, suberoylanilide hydroxamic acid (SAHA), [4-(2-amino-phenylcarbamoyl)-benzyl]-carbamate pyridine-3-yl methyl ester and its derivatives, butyric acid, pyroxamide, trichostatin A, oxamflatin, apicidine, depsipeptide, depudecin, trapoxin, and compounds disclosed in WO02 / 22577).

[0349] I-copper B-alpha kinase inhibitors (IKK) (including, but not limited to, 2-propennitrile, 3-[(4-methylphenyl)sulfonyl]-(2E)-(9Cl)).

[0350] Imidazone (including, but not limited to, temozolomide (Methazolastone®, Temodar® and its derivatives (e.g., as generally and in detail disclosed in U.S. Patent No. 5,260,291)) and mitozolomide).

[0351] Insulin-like growth factor pathway inhibitors (e.g., IGF inhibitors or IGF receptor (IGFR1 or IGFR2) inhibitors include small molecule inhibitors such as OSI-906; and anti-IGF antibodies or anti-IGFR antibodies such as AVE-1642, MK-0646, IMC-A12 (cixutumab), R1507, CP-751, 871 (figitumumab)).

[0352] Insulin tyrosine kinase inhibitors (including, but not limited to, hydroxyl-2-naphthalenylmethylphosphonic acid).

[0353] c-Jun-N-terminal kinase (JNK) inhibitors (including, but not limited to, pyrazoleanthrone and epigallocatechin gallate).

[0354] Mitogen-activated protein kinase (MAP) inhibitors (including, but not limited to, benzenesulfonamides, N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methyl]aminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxy-(9Cl)).

[0355] MDM2 inhibitors (including, but not limited to, trans-4-iodine and 4'-voranyl chalcone).

[0356] MEK inhibitors (including, but not limited to, but but also butanedinitrile and bis[amino[2-aminophenyl]thio]methylene]-(9Cl)).

[0357] MMP inhibitors (including, but not limited to, Actinonin, epigallocatechin gallate, collagen peptide mimetic and non-peptide mimetic inhibitors, tetracycline derivatives marimastat (Marimastat®), prinomastat, incyclinide (Metastat®), shark cartilage extract AE-941 (Neovastat®), Tanomastat, TAA211, MMI270B, or AAJ996).

[0358] mTor inhibitors (including, but not limited to, rapamycin (Rapamune®) and its analogs and derivatives, AP23573 (also known as ridaforolimus, deforolimus, or MK-8669), CCI-779 (also known as temsirolimus) (Torisel®), and SDZ-RAD).

[0359] NGFR tyrosine kinase inhibitors (including, but not limited to, tyrophostin AG879).

[0360] p38 MAP kinase inhibitors (including, but not limited to, phenol, 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole-2-yl]-(9Cl) and benzamide, 3-(dimethylamino)-N-[3-[(4-hydroxylbenzoyl)amino]-4-methylphenyl]-(9Cl)).

[0361] p56 tyrosine kinase inhibitors (including, but not limited to, damnacanthal and tyrophostin 46).

[0362] PDGF pathway inhibitors (including, but not limited to, thyrophostin AG1296, thyrophostin 9, 1,3-butadiene-1,1,3-tricarbonitric, 2-amino-4-(1H-indole-5-yl)-(9Cl), imatinib (Gleevec®), and gefitinib (Iressa®), as well as compounds generally and in detail disclosed in European Patent No. 0564409 and PCT Publication No. WO99 / 03854).

[0363] Phosphatidylinositol 3-kinase inhibitors (including, but not limited to, wartmannin and quercetin dihydrate).

[0364] Phosphatase inhibitors (including, but not limited to, cantharidinate, cantharidin, and L-leucinamide).

[0365] PKC inhibitors (including, but not limited to, 1-H-pyrrolo-2,5-dione, 3-[1-[3-(dimethylamino)propyl]-1H-indole-3-yl]-4-(1H-indole-3-yl)-(9Cl), bisindolylmaleimide IX, sphinogosine, staurosporine, and hypericin).

[0366] PKC delta kinase inhibitors (including, but not limited to, rottlerin). Polyamine synthesis inhibitors (including, but not limited to, DMFOs).

[0367] Proteasome inhibitors (including, but not limited to, acrasinomycin A, gliotoxin, and bortezomib (Velcade®)).

[0368] Protein phosphatase inhibitors (including, but not limited to, cantharidinate, cantharidin, LP-bromotetramisole oxalate, 2(5H)-furanone, 4-hydroxy-5-(hydroxymethyl)-3-(1-oxohexadecyl)-(5R)-(9Cl), and benzylphosphonic acid).

[0369] Protein tyrosine kinase inhibitors (including, but not limited to, thyrophostin Ag216, thyrophostin Ag1288, thyrophostin Ag1295, geldanamycin, genistein, and 7H-pyrrolo[2,3-d]pyrimidine derivatives);

[0370] PTP1B inhibitors (including, but not limited to, L-leucinamide).

[0371] SRC family tyrosine kinase inhibitors (including, but not limited to, PP1 and PP2).

[0372] Syk tyrosine kinase inhibitors (including, but not limited to, piceatanl).

[0373] Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors (including, but not limited to, thyrophostine AG490 and 2-naphthyl vinyl ketone).

[0374] Retinoids (including, but not limited to, isotretinoin (Accutane®, Amnesteem®, Cistane®, Claravis®, Sotret®) and tretinoin (Aberel®, Aknoten®, Avita®, Renova®, Retin-A®, Retin-A MICRO®, Vesanoid®)).

[0375] RNA polymerase II elongation inhibitors (including, but not limited to, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole).

[0376] Serine / threonine kinase inhibitors (including, but not limited to, 2-aminopurines).

[0377] Sterol biosynthesis inhibitors (including, but not limited to, squalene epoxidase and CYP2D6). VEGF pathway inhibitors (including, but not limited to, anti-VEGF antibodies such as bevacizumab and small molecules such as sunitinib (Sutent®), sorafenib (Nexavar®), ZD6474 (also known as vandetanib) (Zactima®), SU6668, CP-547632, AV-951 (tivozanib), and AZD2171 (also known as cediranib) (Recentin®)).

[0378] For example, one or more chemotherapeutic agents may be selected from the group consisting of cisplatin, carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, thalidomide, lenalidomide, proteosome inhibitors (e.g., bortezomib), HSP90 inhibitors (e.g., tenespinmycin), transplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, or any of the analogs mentioned above. Examples of immunomodulatory agents include various chemokines and cytokines (e.g., interleukin-2 (IL-2), granulocyte / macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL-12)).

[0379] In one embodiment, the further treatment is one or more additional immunogenic peptides, for example, one or more immunogenic peptides derived from WT1 or its derivatives. Examples of WT1 immunogenic peptides include, but are not limited to, peptides containing (or consisting of) the WT1 class 1 epitope; RMFPNAPYL (SEQ ID NO: 538) (WT1 126-134); YMFPNAPYL (SEQ ID NO: 539) (or consisting of) YMFPNAPYL (SEQ ID NO: 539); RSDELVRHHNMHQRNMTKL (SEQ ID NO: 540) (WT1 427-445) (or consisting of) PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 541) (WT1 331-352) (or consisting of) PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 541) (WT1 331-352) (or consisting of) SGQARMFPNAPYLPSCLES (SEQ ID NO: 542) (WT1 122-140) (or consisting of) SGQAYMFPNAPYLPSCLES (SEQ ID NO: 543) (or consisting of) SGQAYMFPNAPYLPSCLES (SEQ ID NO: 543). Other WT1 immunogenic peptides are described in U.S. Patent No. 7,598,221 (which is incorporated herein by reference). Other immunogenic peptides include, but are not limited to, those derived from MUC1, gp100, TRP-2, MAG1, NY-ESO1, HER-2, and AIM2.

[0380] A subject may have, be suspected of having, or be at risk of developing cancer (e.g., lung cancer, liver cancer, bile duct cancer, stomach cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, multiple myeloma, or Waldenström macroglobulinemia). A subject "suspected of having cancer" is a subject who has one or more symptoms of cancer. The symptoms of cancer are well known to those skilled in the art and typically include, but are not limited to, pain, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, coughing up blood, hematuria, bloody stools, nausea, vomiting, abdominal distension, bloating, ascites (fluid in peritoneal cavity), vaginal bleeding, constipation, abdominal distension, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, excessive sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, and dysphagia. The symptoms of multiple myeloma, in detail, include, for example, bone pain (e.g., 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 recurrent infections. Symptoms of Waldenström macroglobulinemia include, for example, weakness, swollen lymph nodes, severe fatigue, nosebleeds, weight loss, and neurological problems.

[0381] As used herein, a subject “at risk of developing cancer” is a subject who has a predisposition to cancer, i.e., a genetic predisposition to developing cancer (for example, a mutation in a tumor suppressor gene (e.g., a mutation in BRCA1, p53, RB, or APC), or who has been exposed to a situation that may cause cancer, or who is currently affected by a situation that may cause cancer). Therefore, if a subject has been exposed to certain compounds at mutagenic or carcinogenic levels (e.g., carcinogenic compounds in cigarette smoke, e.g., acrolein, 4-aminobiphenyl, aromatic amines, benzene, benzanthracene, benzopyrene, formaldehyde, hydrazine, polonium-210 (radon), urethane, or vinyl chloride), that subject may also be a subject “at risk of developing cancer.” If a subject has, for example, been exposed to high doses of ultraviolet or X-rays, or has been exposed to (e.g., infected with) a tumor-causing / tumor-associated virus (e.g., papillomavirus, Epstein-Barr virus, hepatitis B virus, or human T-cell leukemia / lymphoma virus), that subject may be at risk of developing cancer. Furthermore, if a subject has inflammation (e.g., chronic inflammation), that subject may be at risk of developing cancer. For example, if a subject has monoclonal immunoglobulinemia of unknown significance (MGUS), that subject may be at risk of developing multiple myeloma.The subjects may have any of the cancers described herein, e.g., bladder cancer (including advanced bladder cancer and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., renal cell carcinoma (e.g., papillary renal cell carcinoma, clear cell carcinoma, chromophobic carcinoma)), liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma and large cell There is a risk of developing cancers including cancer, genitourinary cancers such as ovarian cancer (including fallopian tube cancer, endometrial cancer and peritoneal cancer), cervical cancer, prostate cancer and testicular cancer, lymphatic system cancers, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), gastric cancer (e.g., gastroesophageal cancer, upper gastric cancer or lower gastric cancer), gastrointestinal cancers (e.g., anal cancer or bile duct cancer), gallbladder cancer, thyroid cancer, leukemia (e.g., acute myeloid leukemia), neuronal and glial cell cancers (e.g., glioblastoma multiforme), head and neck cancer or multiple myeloma.

[0382] Multiple myeloma (PMS) is a blood cancer that affects approximately 45,000 people annually in the United States. PMS is characterized by multifocal and clonal proliferation of plasma cells within the bone marrow, which can lead to skeletal dysfunction, serum monoclonal immunoglobulinemia, immunosuppression, and end-organ sequelae. Patients with smoldering PMS (SMM) are at high risk of progression to active PMS. Treatments, including bone marrow transplantation and chemotherapy, are used to treat patients who develop active PMS, but the prognosis for patients with the disease at these stages is often poor.

[0383] Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferation disorder characterized by monoclonal proliferation of plasma cells in the bone marrow, and monoclonal proteins in the blood and / or urine, without renal impairment, hypercalcemia, bone disease, or anemia. Diagnosis of SMM requires serum monoclonal (M) protein levels of ≥3 g / dL and / or >10% bone marrow clonal plasma cells (BMPCs), as well as the absence of terminal organ damage (i.e., hypercalcemia, renal failure, anemia, or bone lesions [CRAB]). Despite being asymptomatic, SMM is associated with a high risk of progression to symptomatic multiple myeloma (MM) or amyloidosis.

[0384] Currently, there is no aggressive treatment for SMM. Instead, a "cautious" wait-and-see approach is taken, with treatment initiated only after the disease has progressed to a symptomatic stage. In most patients, progression is indicated by increased anemia (hemoglobin below the lower limit of normal, or <10 g / dL) and / or skeletal complications (including bone lesions and / or diffuse osteoporosis).

[0385] Subjects with smoldering multiple myeloma (SMM) are also at risk of developing SMM. Smoldering multiple myeloma can be determined, for example, by high levels of monoclonal protein in the subject's urine or blood. Furthermore, subjects with SMM may show an increased number of myeloma cells in the bone marrow. Currently, SMM is estimated to account for approximately 15% of all newly diagnosed cases of MM. The median time from diagnosis to symptomatic MM is in the range of 2–3 years, and the one-year risk of progression from SMM to symptomatic MM requiring treatment is estimated to be 10%. The risk of progression depends on 1) monoclonal protein levels of ≥3 g / dL; 2) BMPC ≥10%; and 3) the presence of an abnormal serum-free light chain (FLC) ratio. As shown in Table 1, the median time to progression is significantly shorter (1.9 years) in patients meeting all three of these prognostic criteria compared to patients meeting only one of these criteria (10 years) or two of these criteria (5.1 years). Similarly, the proportion of patients progressing to symptomatic MM by 5 years is significantly higher (76%) in patients meeting all three prognostic criteria compared to patients meeting only one or two of these criteria (25% and 51%, respectively). [Table 4-1]

[0386] Recent data demonstrate the importance of other prognostic criteria for SMM, including the presence of an abnormal phenotype of BMPC, defined as a decrease in one or two of the uninvolved immunoglobulin (Ig) isotypes, as well as whether M protein remains stable or progressively deteriorates over time. In the latter case, referred to as evolving SMM, patients with progressively elevated serum M protein levels have a shorter median time to progression (TTP) compared to patients with stable M protein (1.3 years vs. 3.9 years, respectively).

[0387] In some embodiments, the above method may also include a step of determining whether an immune response has occurred in a subject after administering the peptide, nucleic acid, or composition described herein to the subject. A preferred method for determining whether an immune response has occurred in a subject includes the use of an immunoassay to detect, for example, the presence of antibodies specific to the peptide in a biological sample derived from the subject. For example, after administering the peptide or composition to a subject, a biological sample (e.g., a blood sample) may be obtained from the subject and tested for the presence of antibodies specific to the peptide. An immune response may 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 release assays or 51 Examples include Cr release assays (as described above).

[0388] In some embodiments, the above method may also include a step of determining whether the subject has cancer. Preferred methods for such determination are known in the art, depending on the type of cancer detected in the subject. Such methods may be qualitative or quantitative. For example, a healthcare professional may diagnose a subject having multiple myeloma if the subject exhibits two or more (e.g., three, four, five, or six or more) symptoms of multiple myeloma (e.g., any of the symptoms described herein). A subject may also be determined to have multiple myeloma by measuring blood calcium levels, white blood cell count or red blood cell count, or the amount of protein in the subject's urine.

[0389] Exovivo approach. An exovivo strategy for inducing an immune response in a subject may involve contacting a suitable APC (e.g., dendritic cells, monocytes, or macrophages) obtained from the subject with any peptide or composition described herein. Alternatively, these cells may be transfected with a nucleic acid encoding one or more of the peptides (e.g., an expression vector) and cultured, if necessary, for a period of time and under conditions that allow for the expression of those peptides. The transfection method depends on the cell type and the nucleic acid to be transfected into the cells. (See "Nucleic Acids and Methods for Peptide Production" above and Sambrook et al., previously cited). The cells are then returned to the subject after contact or transfection.

[0390] The cells described above can be any of the broad types of cells expressing MHC class I or MHC class II molecules. For example, these cells include myeloid cells, macrophages, monocytes, dendritic cells, and T cells (e.g., T helper cells, CD4). + cells, CD8 + Examples include cells (or cytotoxic T cells) or B cells.

[0391] An ex vivo method for stimulating an immune response may include, in vitro, contacting T cells (e.g., T cells from a population of lymphocytes obtained from a subject) with antigen-presenting cells expressing an MHC molecule that binds to one of the peptides described herein, for a period of time (and under conditions) sufficient to activate the T cells. After contact, the activated T cells are reintroduced into the subject from which the cells were obtained. A method for generating APCs expressing an MHC molecule that binds to one of the peptides described herein is described above.

[0392] In some embodiments of any exovivo method, cells may be obtained from a subject of the same species other than the subject (homogeneous), may be contacted with a reagent (or immunogenic / antigenic composition), and may be administered to the subject.

[0393] Method for producing antibodies in a test subject Methods for producing antibodies specific to immunogens (e.g., any one or more peptides described herein) are known in the art and are described in detail below. For example, antibodies or antibody fragments specific to the peptides described herein may be produced by immunization, for example, using animals, or by in vitro methods such as phage display. All or part of the peptides described herein may be used to produce antibodies or antibody fragments.

[0394] Antibodies can be prepared by using a peptide and immunizing a suitable subject (e.g., rabbits, goats, mice, or other mammals such as humans) with that peptide. A suitable immunogenic preparation may include, for example, any of the reagents described herein. The preparation may further include an adjuvant (e.g., Freund's complete or incomplete adjuvant, alum, RIBI, or similar immunostimulants). Adjuvants include, for example, cholera toxin (CT), Escherichia coli thermolabile toxin (LT), mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185:1203-1210), mutant Escherichia coli thermolabile toxin (MLT) (Di Tommaso et al. (1996) Infect. Immunity 64:974-979), carboxymethylcellulose, polyinosinate-polycytidylic acid and poly-L-lysine double-stranded RNA combinations (e.g., polyIC-LC, e.g., hiltonol), water-oil emulsions (e.g., montanaide), and proteins (e.g., cytokines, complement, GCSF, GM-CSF). MCT and MLT contain point mutations that substantially reduce toxicity compared to the parent molecule without substantially impairing adjuvant activity. A polyclonal antipeptide antibody response is induced by immunizing a suitable subject with an immunogenic peptide preparation (e.g., any of the reagents described herein).

[0395] As used herein, the term antibody refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule (i.e., a molecule containing an antigen-binding site that specifically binds to a peptide (e.g., a peptide described herein)). An antibody that specifically binds to a peptide described herein is an antibody that binds to that peptide but substantially does not bind to other molecules in the sample. Examples of immunologically active portions of immunoglobulin molecules include, for example, F(ab) fragments, F(ab')2 fragments, or any other antibody fragment described herein (see below).

[0396] Antipeptide antibodies can be monoclonal antibodies or preparations of polyclonal antibodies. The term monoclonal antibody, as used herein, refers to a group of antibody molecules that contain only one type of antigen-binding site capable of immunely reacting with a peptide. Therefore, monoclonal antibody compositions typically exhibit a single binding affinity to a specific peptide with which they immunely react.

[0397] Polyclonal antipeptide antibodies can be prepared as described above by immunizing a suitable subject with a peptide immunogen. The antipeptide antibody titer in the immunized subject can be monitored over time by standard methods (e.g., enzyme-linked immunosorbent assay (ELISA) using immobilized peptides). If desired, the antibody molecule against the peptide can be isolated from a mammal (e.g., blood) and further purified by methods such as protein A chromatography to obtain the IgG fraction. After a suitable time has elapsed since immunization, for example when the antipeptide antibody titer is highest, antibody-producing cells can be obtained from the subject and used by standard methods (e.g., Kohler and The hybridoma method, first described by Milstein (1975) Nature 256:495-497, the human B-cell hybridoma method (Kozbor et al. (1983) Immunol. Today 4:72), or the EBV-hybridoma method (Cole et al. (1985), Monoclonal Antibodies and Cancer Monoclonal antibodies can be prepared by Therapy, Alan R. Liss, Inc., pp. 77-96). Any of the many well-known protocols used to fuse lymphocytes with immortalized cell lines can be applied for the purpose of generating antipeptide monoclonal antibodies (see, for example, Current Protocols in Immunology, cited above; Galfre et al. (1977) Nature 266:55052; RH Kenneth, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol. Med., 54:387-402 (each of these disclosures is incorporated by reference in whole)).

[0398] As an alternative to preparing hybridomas that secrete monoclonal antibodies, monoclonal antipeptide antibodies can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) using the peptides described herein to isolate immunoglobulin library members that bind to the peptide.

[0399] Anti-peptide antibodies (e.g., monoclonal antibodies) may be used to isolate peptides by techniques such as affinity chromatography or immunoprecipitation. Furthermore, anti-peptide antibodies may be used to detect peptides in screening assays described herein. The antibody may, if necessary, be conjugated to a detectable label (e.g., any of the labels described herein) or to the first or second member of a conjugation pair (e.g., streptavidin / biotin or avidin / biotin) (the second member may be conjugated to the detectable label).

[0400] Non-human antibodies against target peptides (e.g., peptides described herein) can also be produced in non-human hosts (e.g., rodents) and then humanized as described, for example, in U.S. Patent No. 6,602,503, EP239400, U.S. Patent No. 5,693,761 and U.S. Patent No. 6,407,213 (each of which disclosures are incorporated by reference in whole).

[0401] Methods for choosing a treatment A method for selecting a treatment for a subject having cancer (e.g., plasma cell damage, e.g., multiple myeloma and / or Waldenström macroglobulinemia, or any cancer expressing XBP1, CD138, or CS1 (e.g., lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, leukemia, e.g., AML or CML)) or a precancerous condition (e.g., smoldering multiple myeloma) includes, if necessary, the step of determining whether one or more cells (e.g., plasma cells) of the subject's cancer express XBP1; and, if one or more cells express XBP1, selecting a peptide or composition described herein, e.g., an XBP1 peptide or a composition containing an XBP1 peptide, as a treatment for the subject.

[0402] A method for selecting a treatment for a subject with cancer may include, if necessary, the steps of determining whether one or more cells (e.g., plasma cells) of the subject's cancer express CD138; and, if one or more cells express CD138, selecting a peptide or composition described herein, for example, a CD138 peptide or a composition containing a CD138 peptide, as a treatment for the subject.

[0403] A method for selecting a treatment for a subject with cancer may include, if necessary, the steps of determining whether one or more cells (e.g., plasma cells) of the subject's cancer express CS-1; and, if one or more cells express CS-1, selecting a peptide or composition described herein, for example, a CS-1 peptide or a composition containing a CS-1 peptide, as a treatment for the subject.

[0404] If one or more cells (e.g., plasma cells) of a subject's cancer express two or more of XBP1, CD138, and CS-1, it is understood that a suitable combination of peptides can be delivered to the subject, for example, via a composition described herein. For example, if it is determined that one or more cells (e.g., plasma cells) of a subject's cancer express XBP1 and CD138, a method for selecting a treatment may include the step of selecting, as a treatment for the subject, at least one XBP1 peptide and at least one CD138 peptide, or a composition containing such peptides, as described herein.

[0405] Methods for determining whether one or more cells express XBP1, CD138, or CS-1 are known in the art and described above. For example, the presence or amount of XBP1, CD138, or CS-1 polypeptide expressed by cells (or cell lysates) can be detected by testing a biological sample obtained from a subject (e.g., a blood sample or lymph node tissue sample) with antibodies specific to XBP1, CD138, or CS-1, produced by the methods described herein. (See, e.g., Examples and Sambrook et al., previously cited). Methods for assaying a biological sample for the presence or amount of polypeptide include, for example, ELISA, immunohistochemistry, flow cytometry, Western blotting assay, or dot blotting assay.

[0406] In some embodiments, any method described herein may also include the steps of providing a biological sample from a subject and / or obtaining a biological sample from a subject. Suitable biological samples for the methods described herein include any biological fluid, cells, tissues, or fractions thereof containing the subject protein of interest (e.g., XBP1, CD138, or CS-1 protein). The biological sample may be, for example, a specimen obtained from or derived from a subject (e.g., a mammal such as a human). For example, the sample may be a tissue section obtained by biopsy, or cells arranged in or adapted to tissue culture. The biological sample may also be a biological fluid containing cells (e.g., urine, blood, plasma, serum, saliva, semen, sputum, cerebrospinal fluid, tears, mucus, or aspirates (e.g., lung or papillary aspirates)) or such a sample absorbed onto paper or a polymer substrate. The 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 fractions containing specific types of blood cells (e.g., red blood cells or white blood cells (leukocytes)). If desired, the sample may be a combination of sample types derived from the subject, such as a combination of tissue and biological fluid.

[0407] Biological samples can be obtained from subjects who have, are suspected of having, or are at risk of developing cancer (e.g., lung cancer, liver cancer, bile duct cancer, stomach cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia (e.g., AML or CML), multiple myeloma, and / or Waldenström macroglobulinemia). Any suitable method can be used to obtain biological samples, but exemplary methods include, for example, venotomy, swab (e.g., buccal swab), aspiration, or fine-needle aspiration biopsy. Non-limiting examples of tissues that are easily aspirated with a fine needle include lymph nodes, lungs, thyroid, breast, and liver. Samples can also be recovered by, for example, microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder lavage, smear (PAP smear), or ductal lavage.

[0408] For example, after detecting cancer (e.g., lung cancer, liver cancer, bile duct cancer, stomach cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, leukemia, multiple myeloma and / or Waldenström macroglobulinemia) or a precancerous condition, e.g., smoldering multiple myeloma, in a subject using the methods described above, a healthcare professional (e.g., a physician) may, for example, (i) write a prescription for the medicine; (ii) administer the medicine to the subject (but not necessarily administer it) (e.g., hand the patient a sample of the prescribed medicine while the patient is in the physician's office); (iii) communicate with the patient (oral, in writing (other than a prescription) or electronically (e.g., by email, electronically posting to a secure site) the proposed or recommended mode of treatment (e.g., treatment including one or more of the peptides described herein). (iv) A suitable mode of treatment for the subject can be identified and, for example, disseminated to other healthcare professionals via patient records, thereby enabling the selection of an appropriate mode of treatment for the subject (e.g., a treatment comprising one or more of the peptides described herein). (iv) at the end may be useful, for example, when two or more treatments or therapeutic agents are administered to a patient by different healthcare professionals.

[0409] After detecting the presence or amount of XBP1, CD138, or CS-1 in a subject (using any of the methods described above); and / or after selecting a treatment for the subject, a healthcare professional (e.g., a physician) may administer an appropriate mode of treatment to the subject. A method for administering a treatment comprising one or more peptides described herein, as detailed above.

[0410] Furthermore, healthcare professionals may also select, prescribe, and / or administer one or more additional therapeutic agents to treat cancer, or one or more medications to treat side effects of anticancer drugs. Suitable chemotherapeutic agents for treating multiple myeloma and / or Waldenström macroglobulinemia include, for example, melphalan, cyclophosphamide, vincristine, doxorubicin, prednisone, dexamethasone, proteosome inhibitors (e.g., bortezomib), thalidomide, or lenalidomide.

[0411] Side effects of anticancer drugs include, for example, anemia, gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea), leukopenia (a decrease in the number of white blood cells, which can lead to infection), temporary hair loss, or thrombocytopenia (a decrease in the number of platelets, which can lead to bleeding). Therefore, physicians may prescribe or administer chemotherapeutic agents such as vincristine to subjects together with anti-anemia drugs (e.g., epoetin alfa (e.g., Procrit® or Epogen®)).

[0412] Kits and manufactured products This disclosure also features various kits, which may include, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) any peptides or compositions described herein (or an expression vector comprising nucleic acid sequences encoding one or more peptides); and instructions for administering the peptides or compositions to a subject. The kit may include one or more pharmaceutically acceptable carriers and / or one or more immunostimulants and / or one or more immunomodulators. The immunostimulants may be, for example, T helper epitopes, modified peptide ligands, or adjuvants. In one embodiment, the immunostimulant may be a combination of carboxymethylcellulose, polyinosinate-polycytidylic acid, and poly-L-lysine double-stranded RNA (e.g., polyIC-LC, e.g., Hiltonol); a water-oil emulsion (e.g., Montanaide); or a protein (e.g., cytokines, complement, GCSF, GM-CSF). In one embodiment, the immunomodulator is a protein, for example, an antibody that activates the immune system (e.g., anti-CTLA4 antibody, e.g., ipilimumab or tremelimumab, anti-PD-1 antibody, anti-PDL-1 antibody); or a small molecule adjuvant (e.g., thalidomide or a thalidomide derivative, e.g., lenalidomide).

[0413] The above kit may also include one or more therapeutic, diagnostic, or prophylactic agents. These one or more therapeutic, diagnostic, or prophylactic agents may include: (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, thiazide, amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine, verapamil, digoxin, isorudyl, etc.) (iii) Drugs that affect gastrointestinal function (e.g., omeprazole or sucralfate); (iv) Antibiotics (e.g., tetracycline, clindamycin, amphotericin B, quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, streptomycin) (v) Anticancer agents (e.g., cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, or dacarbazine); (vi) Immunomodulators (e.g., For example, interleukins, interferons (e.g., interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), cyclosporine, FK506, azathioprine, steroids); (ix) drugs that act on the blood and / or hematopoietic 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 that affect calcification and bone metabolism (e.g., calcium, phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride), etc.) are included, but are not limited to these.

[0414] A container; and a product comprising a composition contained within the container, wherein the composition comprises an active ingredient for inducing an immune response in a mammal (e.g., human), the active ingredient comprising one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten or more) of any peptides described herein, and the container has a marking indicating that the composition is for use in inducing an immune response in a mammal (e.g., any of the mammals described herein). The marking may further indicate that the composition should be administered to a mammal having, suspected of having, or at risk of developing, cancer, such as lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, multiple myeloma, smoldering multiple myeloma, and / or Waldenström macroglobulinemia. The composition of the product may be dried or freeze-dried, and may include, for example, one or more solutions (and / or instructions) for solubilizing the dried or freeze-dried composition.

[0415] The above-mentioned manufactured products may also include instructions for administering the above-mentioned compositions to mammals (for example, those described above).

[0416] The following examples are intended to illustrate, rather than limit, the present invention. [Examples]

[0417] Example 1: Materials and Method Cell lines. Multiple myeloma cell lines: McCAR, MM1S, and U266 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The human acute myeloid leukemia (AML) cell line ML-2 was kindly provided by Dr. Y. Matsuo, Fujisaki Cell Center, Okayama, Japan. The T2 cell line, a human B-cell and T-cell hybrid expressing the HLA-A2.1 molecule (Zweerink et al. (1993) J Immunol. 150(5):1763-71), was provided by Dr. J. Moldrem (University of Texas MD Anderson Cancer Center, Houston, TX) and used as the source of antigen-presenting cells (APCs). K562-A * O201 cells were provided by Karen Anderson (Dana Farber Cancer Institute, Boston, MA) and used in immunomonitoring assays that presented individual peptides to CTLs. Various cancer cells, including LnCap, VCap, MB231, MCF7, BT474, LS180, SW480, WiDRr, OCI, U937, HEL, UT7, HL60, Nomo1, and THP1, were obtained from ATCC. All cell lines were treated with 10% fetal bovine serum (FCS; BioWhittaker, Walkersville, MD), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Gibco-Life). The cells were cultured in RPMI-1640 medium (Gibco-Life Technologies, Rockville, MD) supplemented with [technology name missing].

[0418] Reagents. Mouse anti-human CD80 or CD83 monoclonal antibodies (mAbs) conjugated to phycoerythrin (PE) were purchased from Immunotech (Hialeigha, FL). Mouse anti-human CD3, CD4, CD8, CCR7, CD45RO, CD69, CD107α, IFN-γ, and HLA-A2 mAbs conjugated to FITC, PE, PerCP, PerCP-Cy5.5, APC, Pacific Blue, APC-H7, or PE-Cy7 were purchased from Becton Dickinson (BD) / Pharmingen or BD / Biosciences (San Diego, CA). Recombinant human IL-2, IL-4, IFN-α, and TNF-α were purchased from R&D Systems (Minneapolis, MN), and GM-CSF was obtained from Immunex (Seattle, WA).

[0419] Synthetic peptide. Influenza virus protein matrix peptide. 58-66 (GILGFVFTL; SEQ ID NO: 25) and MAGE-3 peptide (FLWGPRALV; SEQ ID NO: 26) were used as control HLA-A2 binding peptides. Six naturally occurring unsplicing XBP1 peptides: XBP1 118-126 (LLREKTHGL; Sequence ID 1); XBP1 185-193 (NISPWILAV(sequence_2));XBP1 190-198 (ILAVLTLQI(sequence number 3));XBP1 193-201 (VLTLQIQSL(Sequence ID 4));XBP1 111-119 (KLLLENQLL(Sequence ID 5));XBP1 94-102 (RMSELEQQV(Sequence ID 27));SP XBP1 197-205 (GILDNLDPV (Sequence ID 7)), SP XBP1 194-202 (ILLGILDNL(Sequence ID 8)), SP XBP1 368-376Three naturally occurring splicing XBP1 peptides, including (ELFPQLISV (SEQ ID NO: 9)); heterocritic XBP1 (YISPWILAV (SEQ ID NO: 6)); and splicing heterocritic XBP1 (YILDNLDPV (SEQ ID NO: 24)); and YLFPQLISV (SEQ ID NO: 10)) peptides, were designed and investigated as potential HLA-A2 binding peptides. As used herein, “heterocritic” (e.g., heterocritic peptide) refers to a form of peptide in which one or more amino acids are modified from the wild-type or original sequence to produce a peptide that is more immunogenic than the corresponding wild-type peptide. For example, in the exemplary heterocritic peptides described above, the amino acids in bold indicate amino acids modified from the wild-type sequence of XBP1.

[0420] Four natural CD138 peptides: CD138 256-264 (VIAGGLVGL(Sequence ID 11));CD138 260-268 (GLVGLIFAV(Sequence ID 12));CD138 5-13 (ALWLWLCAL(Sequence ID 13)); and CD138 7-15 We designed (WLWLCALAL (SEQ ID NO: 14)) and investigated it as a potential HLA-A2 binding peptide.

[0421] Four natural CS1 peptides: CS1-P1: CS1 236-245 (LLLSLFVLGL(Sequence ID 15));CS1-P2:CS1 239-247 (SLFVLGLFL(Sequence ID 16));CS1-P3:CS1 232-240 (LLVPLLLSL(Sequence ID 17)); and CS1-P4:CS1 9-17 (TLIYILWQL (SEQ ID NO: 18)) was designed (using three different databases: RANKPEP, BIMAS, and NetMHC) and investigated as a potential HLA-A2 binding peptide. (See, e.g., Reche et al. (2002) Human Immunology 63:710-709).

[0422] XBP-1 and CD138 peptides were synthesized using standard FMOC (9-fluorenylmethyl-oxycarbonyl) chemistry (Biosynthesis, Lewisville, TX), purified to >85% purity using reverse-phase chromatography, and their molecular weight was verified by mass spectrometry. The CS1 peptide was synthesized by New England Peptides LLC with a purity of over 95%.

[0423] Heterocritical XBP1 US 185-193 (YISPWILAV)(Sequence ID 6), Heterocritical XBP1 SP 368-376 (YLFPQLISV)(Sequence ID 10), Natural CD138 260-268 (GLVGLIFAV)(SEQ ID NO: 12) and natural CS1 239-247 The (SLFVLGLFL)(SEQ ID NO: 16) peptide was derived from XBP1 unsplicing (US), XBP1 splicing (SP), CD138, and CS1 antigens, respectively. Influenza virus matrix proteins 58-66 (GILGFVFTL) (SEQ ID NO: 25) and CMV pp65 (NLVPMVATV) (SEQ ID NO: 28) were selected as HLA-A2 specific control peptides. All peptides were synthesized by standard fmoc (9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >90% using reverse-phase chromatography, and their molecular weight was verified by mass spectrometry (Biosynthesis, Lewisville, TX). Lyophilized peptides were dissolved in DMSO (Sigma, St. Louis, MO), diluted in AIM-V medium (Gibco-Life Technologies), and stored at -140°C.

[0424] Peptide binding assay. Four HLA-A2 peptides, heterocritical XBP1 US 185-193 Heterocritic XBP1 SP 368-376 CD138 260-268 and CS1 239-247The binding affinity of the cocktail was evaluated using a T2 cell line. In the assay, T2 cells were washed three times and 1 × 10⁶ 6 The cells were resuspended in serum-free AIM-V medium (Gibco-Life Technologies) to the final cell / ml concentration and transferred to 48-well tissue culture plates. These cells were pulsed with a cocktail of the four peptides listed above at a total peptide concentration ranging from 0 to 50 μg / ml + 3 μg / ml of human β2-microglobulin (Sigma, St. Louis, MO) and incubated in humidified air at 37°C and 5% CO2. After overnight incubation, the cells were washed, stained with mouse anti-human HLA-A2-FITC mAb for 15 minutes at 4°C, and analyzed using a FACSCanto® II flow cytometer (Becton Dickinson, San Jose, CA).

[0425] Peptide stability assay. The HLA-A2 stability of the above multipeptide cocktail was investigated over time. After overnight incubation of T2 cells pulsed with the multipeptide cocktail (25 μg / ml; 6.25 μg / ml / peptide), the cells were washed to remove unbound peptides, and the cell surface expression of newly synthesized HLA-A2 molecules was blocked by incubation with 10 μg / ml Brefeldin A (Sigma) at 37°C and 5% CO2 for 1 hour. The stability of the peptide / HLA-A2 complex was measured by staining cells with mouse anti-human HLA-A2-FITC mAb and analyzing them by flow cytometry 0, 2, 4, 6, and 14 hours after BFA treatment.

[0426] Generation of mature dendritic cells derived from monocytes. Peripheral blood mononuclear cells (PBMCs) are converted to HLA-A2 +Leukopaks obtained from normal organisms were isolated by standard density gradient centrifugation on Ficoll-Paque® Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden). To generate dendritic cells (DCs), monocytes isolated as the adherent cell fraction were cultured for 7 days in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS in the presence of 1,000 U / ml GM-CSF and 1,000 U / ml IL-4. Fresh medium + GM-CSF and IL-4 were added to the culture every other day. On day 7, 1,000 U / ml IFN-α + 10 ng / ml TNF-α were added to 10% FCS-RPMI along with fresh GM-CSF and IL-4, and the cultures were incubated for a further 3 days to obtain mature DCs (mDCs).

[0427] CD3 + T cell isolation. CD3 + T cells were obtained by negative selection from the non-adherent cell fraction using EasySep® magnets and Robosep® from StemCell Technologies (Vancouver, Canada). In short, T cell enrichment is achieved by depleting non-CD3 T cells, including B cells, monocytes, NK cells, erythrocytes, platelets, and basophils, by labeling with bispecific tetrameric antibody complexes against CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A. After removing magnetically labeled unwanted cells, the enriched CD3 + The T cells were washed and examined by flow cytometry.

[0428] Primary CD138 from bone marrow mononuclear cells of MM patients + Cell isolation. Bone marrow mononuclear cells (BMMCs) were isolated from bone marrow cells obtained from MM patients by standard density gradient centrifugation on a Ficoll-Paque® Plus. CD138 +MM cells were isolated from BMMCs using RoboSep® CD138 immunomagnetic positive selection (StemCell Technologies).

[0429] Induction of peptide-specific CTLs. CTLs specific to individual peptides (peptide-specific CTLs) or CTLs specific to multiple peptides (MP-CTLs) are induced in normal HLA-A2. + Alternatively, CD3 obtained from an HLA-A24+ donor. + T lymphocytes were generated ex vivo by repeated stimulation (see Figure 25). In short, APCs (mDC or T2 cells) were generated in heterocritical XBP1 US at 37°C and 5% CO2 in humidified air. 185-193 Heterocritic XBP1 SP 368-376 CD138 260-268 and CS1 239-247 The APCs were pulsed overnight with individual peptides or cocktails of peptides (25 μg / ml total peptide). The loaded APCs were collected, washed, irradiated at 20 Gy, and resuspended in AIM-V medium supplemented with 10% human AB serum (Gemni Bio Products, West Sacramento, CA). Using mp-pulsed and irradiated mDCs, APC / mp and CD3 were mixed in AIM-V medium supplemented with 10% human AB serum. + T cells in a 1:20 ratio with their own CD3 + T cells were initially stimulated. The culture was then re-stimulated every 7 days with irradiated APC / mp for a total of 4 cycles to generate mp-specific CTLs. IL-2 (50 U / ml) was added to the culture 2 days after the second stimulation and continued until the culture was complete.

[0430] Phenotypic analysis of XBP1-CTL, CD138-CTL, or target cells. One week after the fourth stimulation, MP-CTL and control T cells were stained with CD3-PacBlue, CD8-APC-H7, CCR7-PeCy7, CD45RO-PE and / or CD69-PerCP mAb for 30 minutes at 4°C, thereby enabling total CD3 + CD8+ T cells or naive T cells, effector memory T cells and activated CD3 + CD8 + T cells were evaluated. After staining, the cells were washed, fixed in 2% paraformaldehyde-PBS, and analyzed by flow cytometry.

[0431] Western blotting. Approximately 100 μg of protein solubilizes from each cell line (U266, McCAR, ML-2, and MM1S) were suspended in Laemmli's sample buffer (0.1 M Tris-HCl buffer, pH 6.8, containing 1% sodium dodecyl sulfate (SDS), 0.05% β-mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue), boiled for 2 minutes, and subjected to 8-16% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 2 hours (Xcell Surelock Mini Cell, Invitrogen, Carlsbad, CA). The molecular weight of the peptides was determined by using a protein ladder (a mixture of proteins with known molecular weights) as a size marker in the gel (Invitrogen, Carlsbad, CA). The gel was electroblotted in Tris-glycine buffer onto a nitrocellulose membrane (Trans-Blot, 0.2 micron transfer membrane, Bio-Rad Laboratories, CA) at 40 V for 2 hours. Protein transfer onto the nitrocellulose membrane was confirmed by Ponceau S staining. The membrane was incubated with mouse anti-human XBP1 antibody or anti-human CD138 antibody for 1 hour in phosphate-buffered saline containing 1% BSA and Tween 20 (PBST) with constant shaking. The membrane was washed three times with PBST and incubated with anti-mouse IgG-horseradish peroxidase conjugate in PBST containing 3% skim milk powder for 1 hour. After washing, specific proteins were detected using high-sensitivity chemiluminescence according to the instructions provided in the product manual (Amersham Life Sciences Inc., Arlington Heights, IL).

[0432] IFN-γ ELISA. IFN-γ release by XBP1-CTL, CD138-CTL, or CS1-CTL after co-culture with multiple myeloma (MM) cells (McCAR, MM1S), acute myeloid leukemia (AML) cells (ML-2), or T2 cells (see above) was measured using a human IFN-γ ELISA kit from BD Biosciences (San Diego, CA) (see Figure 27). Briefly, purified IFN-γ dilutions or CTL supernatants as standards were transferred to wells of a 96-well plate pre-coated with monoclonal anti-human IFN-γ capture antibody and incubated at room temperature for 2 hours. After several washes, buffer containing the detection antibody and avidin-horseradish peroxidase conjugate was added to each well and incubated at room temperature for 1 hour. The wells were washed, and then horseradish peroxidase substrate solution was added to each well and incubated at room temperature for 30 minutes. Stop solution was added to each well, and the absorbance at 450 nm was determined using a PerkinElmer Wallac Victor2 counter (PerkinElmer, Wellesley, MA). The amount of cytokines present in the CTL culture supernatant was calculated based on the IFN-γ calibration curve.

[0433] Cell proliferation was tracked by carboxyfluorescein succinimimidyl ester (CFSE). CTL proliferation was measured after co-culturing with multiple myeloma (MM) cells (McCAR, MM1S), acute myeloid leukemia (AML) cells (ML-2), or T2 cells (see above) (see Figures 27 and 32). Individual XBP1-CTLs, CD138-CTLs, CS1-CTLs, or CTLs generated by multipeptides were washed twice in PBS (Gibco-BRL) and measured at 1 × 10⁶. 6The cells were resuspended in RPMI-1640 culture medium at a concentration of cells / ml. CFSE (Molecular Probes, Eugene, OR) in the form of a 5 mM stock solution in DMSO was added to the CTLs to obtain a final concentration of 5 μM, and incubated at 37°C for 10 minutes in a light-protected CO2 incubator. After incubation, the reaction was quenched by adding ice-cold PBS (containing 2% FCS) equal to five times the volume of the CTL cells. The cells were incubated on ice for 5 minutes, centrifuged, washed a total of three times, and then resuspended in fresh PBS (containing 2% FCS). CFSE-labeled T cells were placed in RPMI culture medium at a rate of 2 × 10⁶ 6 Adjust the concentration to cells / ml, then 2 × 10 5 Primary multiple myeloma cells, MM cell lines, various cancer cell lines, or K562-A2 cells displaying individual peptides were stimulated at cell / ml concentrations. Stimulated, CFSE-labeled cells were examined by flow cytometry.

[0434] Cytotoxicity assay. The cytotoxic activity of CTLs generated by individual XBP1-CTLs, CD138-CTLs, CS1-CTLs, or multipeptides was measured by a calcein release assay, such as that described by Roden et al. (1999) J. Immunol Methods 226:29-41. Briefly, target cells (3 × 10⁶) including T2, U266 cells, McCAR cells, ML-2 cells, MM1S cell lines, or primary multiple myeloma cells were measured. 5 The cells were incubated at 37°C for 30 minutes in serum-free culture medium containing 10 mM calcein-AM (Molecular Probes), washed three times in cold PBS containing 5% FCS, and then placed in a 96-well U-bottom microtiter plate (set of 3 wells / sample) to extract effector cells (5 × 10⁶). 3Cells were incubated with various effector:target cell ratios (cells / well). The plates were incubated at 37°C and 5% CO2 for 3 hours. After incubation, the cells were pelleted by centrifugation at 1,000 rpm for 5 minutes, and 100 μl of the supernatant was transferred to the wells of a 96-well flat-bottom microtiter plate (Nunc). Calcein release was measured as the amount of fluorescence emitted from the cells (VICTOR). 2 - Using a 1420 multi-label counter (PerkinElmer, Boston, MA). Maximum calcein release was determined from the count of target cells released by the surfactant and the count of spontaneous release from target cells in the absence of effector cells. Cytotoxicity was calculated as follows: % specific lysis = [(experimental release - spontaneous release) ÷ (maximum release - spontaneous release)] × 100.

[0435] CD107α degranulation assay. CD107α degranulation was measured after co-culturing with multiple myeloma (MM) cells (McCAR, MM1S), acute myeloid leukemia (AML) cells (ML-2), or T2 cells (see above) (see Figures 27 and 30). The CD107α degranulation assay, which measures cytotoxic activity, was performed as described by Betts et al. (2003) and Mittendorf et al. (2005), with minor modifications as detailed below. Primary multiple myeloma cells, MM cell lines, various cancer cell lines, or K562-A2 cells pulsed with individual peptides were co-culturified with CTLs in various effector:target ratios. 10 μl aliquots of CD107α and CD107b (both conjugated to detectable labeled FITCs) were added to each well simultaneously with the addition of CD138-CTLs. The plates containing these cells were centrifuged at 1000 rpm for 5 minutes and incubated at 37°C for 1 hour. After incubation, Brefeldin A and Monensin were added to each well, and the cells were incubated at 37°C for a further 4 hours. The cells were harvested, washed, and stained with anti-human MAB conjugated to a fluorescent dye. The cells were washed and analyzed by flow cytometry.

[0436] CD107α upregulation and intracellular IFN-γ production. CD107α upregulation and IFN-γ production were measured after co-culturing with multiple myeloma (MM) cells (McCAR, MM1S), acute myeloid leukemia (AML) cells (ML-2), or T2 cells (see above) (see Figures 27 and 30). CD107α degranulation and IFN-γ production by CD8 + CTLs were identified by flow cytometry, using staining for cell surface markers and intracellular cytokines. In short, 1 × 10⁻⁶ 6 Each killer cell (responder cell) (MP-CTL, control T cell) is divided into 1 × 10⁻¹⁶ cells. 6 Individual stimulated cells (pulsed with each peptide, HLA-A2 + McCAR or U266MM cell line or K562-A* Cells were stimulated with 0201 cells. CD107α mAb was added to the culture, and the cells were placed in a 37°C, 5% CO2 incubator. After 1 hour of incubation, CD28 / CD49d mAb (BD) and the protein transport inhibitors Brefeldin A (BD) and Monensin (BD) were added to the cell culture, and the culture was incubated for a further 5 hours. As a baseline control, MP-CTLs were cultured in medium containing CD28 / CD49d mAb, Brefeldin A, and Monensin alone, without further stimulation. After incubation, these cells were harvested, washed, and their surfaces were stained with CD3-PacBlue and CD8-APC-H7, CCR7-PeCy7, CD45RO-PE, and / or CD69-PerCP anti-human mAbs for 30 minutes. Next, the cells were permeabilized, fixed using Cytofix / Cytoperm (BD), and intracellular cytokine production was detected by staining with anti-IFN-γ FITC mAb for 45 minutes. Finally, the cells were washed with Perm / Wash solution (BD), fixed in 2% paraformaldehyde, and acquired by flow cytometry.

[0437] IL-2 Production Assay. CD3+CD8+ CTLs producing CD107α degranulation and IL-2 were identified by flow cytometry using cell surface markers and intracellular cytokine staining. Briefly, peptide-specific CTLs or control T cells were stimulated with specific stimulators in the presence of CD107α mAb. After 1 hour incubation, CD28 / CD49d mAb (BD) and the protein transport inhibitors Brefeldin A and Monensin were added to the culture and incubated for a further 5 hours. As a baseline control, the CTLs were cultured in medium containing CD28 / CD49d mAb, Brefeldin A, and Monensin alone. After incubation, cells were stained with CD3-PacBlue and CD8-APC-H7 anti-human mAb, followed by fixation / permeabilization, and intracellular cytokine production was detected by staining with anti-IL-2 APC anti-human mAb. After staining, the cells were washed three times with Perm / Wash solution, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

[0438] Statistical analysis. Results are expressed as mean ± SE. Independent Student t-tests were used to compare groups. A difference was considered statistically significant if p < 0.05.

[0439] Example 2: A multipeptide (MP) cocktail of XBP1 unsplicing, XBP1 splicing, and CD138 and CS1-specific peptides exhibits high HLA-A2 binding affinity and stability.

[0440] Four immunogenic peptides, heterocritical XBP1 US 185-193 (YISPWILAV, Sequence ID 6), Heterocritical XBP1 SP 368-376 (YLFPQLISV, Sequence ID 10), Natural CD138 260-268 (GLVGLIFAV, Sequence ID 12) and natural CS1 239-247(SLFVLGLFL, SEQ ID NO: 16) (Table 1) has been individually proven to induce an immune response. Here, we evaluated them as an MP cocktail. The HLA-A2 specific binding and stability of the MP cocktail were evaluated by measuring the upregulation of HLA-A2 molecules on T2 cells by flow cytometry (27). The peptide binding assay showed an increase in mean fluorescence intensity (MFI) of HLA-A2 on T2 cells in a dose-dependent manner (0 to 50 μg / ml), reaching a plateau at a total peptide concentration of 25 μg / ml (6.25 μg / peptide / ml; MFI: 10,787.33 ± 2,371.71), which was similar to the highest total peptide concentration, 50 μg / ml (MFI: 10,889.33 ± 2,888.48) (Figure 1a). Therefore, an MP concentration of 25 μg / ml (6.25 μg / peptide / ml) was selected to evaluate HLA-A2 binding stability.

[0441] In the peptide binding stability assay, T2 cells were pulsed overnight with 25 μg / ml of the above MP cocktail, washed to remove unbound peptides, and then treated with Brefeldin A (BFA) to block the cell surface expression of newly synthesized HLA-A2 molecules. T2 cells were then evaluated for HLA-A2 MFI at 0, 2, 4, 6, or 14 hours after BFA treatment. Flow cytometry analysis demonstrated that the stability of the MP cocktail was highly maintained up to 6 hours after BFA treatment (MFI: 0 hours = 9,726.00 ± 1,373.24, 2 hours = 9,132.33 ± 1,435.51, 4 hours = 9,125.33 ± 1,130.62, 6 hours = 8,818.67 ± 413.50) (Figure 1b). 14 hours after BFA treatment, the HLA-A2 specific affinity of the MP cocktail was lower than that of the control influenza virus matrix protein (IVMP). 58-66It was higher (MFI: 6,793.67 ± 1,617.01) than the affinity of the peptide (MFI: 4,921.33 ± 1,428.16). Based on these results, the inventors confirmed the high level of HLA-A2-specific affinity and stability of the MP cocktail and proceeded to further evaluate the cocktail for its immunogenicity and ability to induce MM-specific CTLs.

Table 4-2

[0442] Example 3: Multi-peptide specific CTLs exhibit different phenotypes of specific T cell subtypes

[0443] MP-CTLs were generated by stimulating T cells from normal donors weekly with APCs pulsed with the above MP cocktail (total 25 μg / ml; 6.25 μg / ml / peptide) and HLA-A2 + One week after the fourth stimulation, the resulting MP-CTLs were evaluated for phenotype and functional activity. Flow cytometry analysis showed that the MP-CTLs contained a higher percentage of CD3 + CD8 + T cells (donor 1: 86%, donor 2: 74%) compared to control T cell cultures (donor 1: 25%, donor 2: 25%; Figure 2). The inventors also observed changes in different phenotypes in the CD3 + CD8 + T cell subset within the MP-CTLs. The frequency of effector memory T cells (EM: CD45RO + CCR7 - / CD3 + CD8 + ) was higher than that of naive T cells (CD45RO - ) CCR7 + / CD3 + CD8 +In association with the corresponding decrease (Donor 1: Control 74% vs. MP-CTL 8%, Donor 2: Control 60% vs. MP-CTL 6%), there was an increase (Donor 1: Control 5% vs. MP-CTL 44%, Donor 2: Control 4% vs. MP-CTL 35%). Furthermore, compared to the control T cell culture, the inventors found that activated CD69 in MP-CTLs was increased. + / CD3 + CD8 + An increase in T cell frequency was observed (Donor 1: Control 3% vs. MP-CTL 39%, Donor 2: Control 5% vs. MP-CTL 13%; Figure 2). Therefore, these results suggest that CD3 is affected by MP cocktails specific to XBP1, CD138, or CS1. + Repeated stimulation of T cells leads to different phenotypic changes and characteristic CD3 in antigen-specific CTLs. + / CD8 + This demonstrates that it leads to an expansion of the T cell subset.

[0444] Example 4: Multipeptide-specific CTLs are HLA-A2 + A high percentage of CD8 cells produce IFN-γ in response to MM cells. + Includes CTLs.

[0445] MP-CTL, HLA-A2 + The ability of MM cell lines to produce intracellular IFN-γ upon stimulation was analyzed by flow cytometry. EM(CD45RO) within MP-CTLs was also analyzed. + CCR7 - ) and activated (CD69 + )CD3 + CD8 + Both T cells are HLA-A2 + MM cell lines produced IFN-γ in response (Figure 3). The frequency of IFN-γ-producing cells was as follows: McCAR cells [Donor 1: Control vs. MP-CTL-0%] [Donor 1: Control vs. MP-CTL-0% vs. 8% EM cells, 0% vs. 11.2% activated cells; Donor 2: 0.4% vs. 2.9% EM cells, 1% vs. 3.9% activated cells] or U266 cells [Donor 1: Control vs. MP-CTL-0% vs. 8% EM cells, 0% vs. 11.2% activated cells; Donor 2: 0.4% vs. 2.9% EM cells, 1.3% vs. It was elevated upon stimulation in any of the 3.0% activated cells. Naive (CD45RO) in MP-CTLs - CCR7 + )CD3 + CD8 + T cells showed the lowest levels of IFN-γ production when stimulated with MM cell lines (data not shown).

[0446] Example 5: Multipeptide-specific CTLs are HLA-A2 + It exhibits cell proliferation in response to MM cells.

[0447] The function of MP-...

Claims

1. A non-splicing type XBP1 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 29; Splicing-type XBP1 peptides with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 30; and CD138 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 31 A composition containing the following:

2. The composition according to claim 1, further comprising a CS-1 peptide with a length of 35 amino acids or less, which contains the amino acid sequence of SEQ ID NO:

32.

3. The aforementioned non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 29, The splicing-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 30, and The CD138 peptide consists of the amino acid sequence of SEQ ID NO:

31. The composition according to claim 1 or 2.

4. The aforementioned non-splicing type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 29, The splicing-type XBP1 peptide consists of the amino acid sequence of SEQ ID NO: 30, The aforementioned CD138 peptide consists of the amino acid sequence of SEQ ID NO: 31, and The CS-1 peptide consists of the amino acid sequence of SEQ ID NO:

32. The composition according to claim 2.

5. A pharmaceutical composition comprising the composition according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. A non-splicing type XBP1 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 29; Splicing-type XBP1 peptides with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 30; and CD138 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 31 A composition for use in a method of inducing an immune response in a subject having cancer, including the following.

7. The composition for use according to claim 6, wherein the method includes a step of determining whether an immune response occurred in the subject after delivery of the composition to the subject.

8. A non-splicing type XBP1 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 29; Splicing-type XBP1 peptides with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 30; and CD138 peptide with a length of 35 amino acids or less, containing the amino acid sequence of SEQ ID NO: 31 A composition for use in a method of treating a subject having cancer, including the following.

9. The composition for use according to any one of claims 6 to 8, wherein the cancer is multiple myeloma, smoldering multiple myeloma, breast cancer, colon cancer, pancreatic cancer, prostate cancer, or leukemia.

10. The composition for use according to claim 9, wherein the breast cancer is estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, HER-2-positive breast cancer, HER-2-negative breast cancer, triple-negative breast cancer, or inflammatory breast cancer.

11. The composition for use according to any one of claims 6 to 10, wherein the subject is a human.

12. The composition or pharmaceutical composition according to any one of claims 1 to 5, or the composition for use according to any one of claims 6 to 11, wherein the composition further comprises a further treatment selected from one or more of chemotherapeutic agents, ionizing radiation, or immunotherapeutic agents.

13. The composition or pharmaceutical composition according to any one of claims 1 to 5, or the composition for use according to any one of claims 6 to 11, wherein the composition further comprises one or more immunostimulants and / or one or more immunomodulators.

14. The composition or pharmaceutical composition or composition for use according to claim 13, wherein the one or more immunostimulants are selected from carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA adjuvants; water-oil emulsion adjuvants; and protein adjuvants.

15. The composition or pharmaceutical composition or composition for use according to claim 14, wherein the adjuvant comprising carboxymethylcellulose, polyinosinic acid-polycytidylic acid, and poly-L-lysine double-stranded RNA is polyIC-LC.

16. The composition according to any one of claims 12 to 15, or a pharmaceutical composition or a composition for use, wherein the composition is combined with an anti-PD-1 antibody, an anti-PDL-1 antibody, or an HDAC inhibitor.

17. The composition or pharmaceutical composition according to any one of claims 12 to 16, or a composition for use, wherein the subject expresses HLA-A24.

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