Targeting a chimeric RNA for treating cancer

Abstract

Chimeric RNAs and their protein products, including BCR-ABL in chronic myelogenous leukemia and EML4-ALK4 in lung cancer, have been well-established as ideal biomarkers and drug targets for malignant cancers. Using RNA-Sequencing data from The Cancer Genome Atlas (TCGA) database, a chimeric RNA SLC2A11-MIF was identified that is present in nearly 50% of CRC samples but absent in non-cancer colon tissue. Disclosed herein is a method for diagnosing and treating cancers expressing this chimeric RNA and protein.

Description

TH Docket No.222117-2430 TARGETING A CHIMERIC RNA FOR TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 594,044, filed October 30, 2023, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “222117-2430 Sequence Listing” created on October 2, 2024, and having 38,447 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND OF THE INVENTION

[0003] Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths worldwide, affecting over 100,000 men and women in the United States every year. There is an urgent and unmet need for early biomarker discovery and the identification of novel therapeutic targets in CRC. SUMMARY OF THE INVENTION

[0004] Chimeric RNAs and their protein products, including BCR-ABL in chronic myelogenous leukemia and EML4-ALK4 in lung cancer, have been well-established as ideal biomarkers and drug targets for malignant cancers. Using RNA-Sequencing data from The Cancer Genome Atlas (TCGA) database, a chimeric RNA SLC2A11-MIF was identified that is present in nearly 50% of CRC samples but absent in non-cancer colon tissue. This chimeric RNA results from cis-splicing between adjacent genes, producing a novel chimeric protein. This protein includes a splice variant of the 5’ gene, which alters the reading frame of the 3’ gene, making a completely novel peptide sequence which may be immunogenic. The protein is predicted to be a transmembrane protein with the novel peptide sequence on the extracellular surface, making it an ideal target for antibody therapeutics.

[0005] Therefore, disclosed herein is a method for diagnosing a SLC2A11-MIF cancer in a subject that involves assaying a sample from the subject for a chimeric RNA comprising a fusion or splicing of SLC2A11 and MIF genes or transcripts or for a chimeric protein comprising a fusion of SLC2A11 and MIF proteins. In some embodiments, the method involves assaying a sample from the subject for a chimeric RNA encoding the amino acid sequence MEDELEPSLRPRTQIQGRILLLTICAAGIGGTFQFGYNLSIINAPTLHIQEFTNETWQARTGEPL PDHLVLLMWSLIVSLYPLGGLFGALLAGPLAITLGRKKSLLVNNIFVVSAAILFGFSRKAGSFE MIMLGRLLVGVNAGVSMNIQPMYLGESAPKELRGAVAMSSAIFTALGIVMGQVVGLRCTPTP PPCSGRQECRKRRSSTRSSGLGAASCSRRLLVTSRCTWSRTSSWPSAAPASRARSAACTASTH Docket No.222117-2430 ARSAARRTAPTASCCAACWPSACASARTGSTSTITT (SEQ ID NO:30) or for a peptide having the amino acid sequence SEQ ID NO:30.

[0006] In some embodiments, the method further involves treating the subject with an antibody drug conjugate that selectively binds chimeric SLC2A11-MIF protein. In some embodiments, the method further involves treating the subject with a vaccine comprising an antigenic fragment of CTPTPPPCSGRQECRKRRSSTRSSGLGAASCSRRLLVTSRCTWSRTSSWPSAAPASRARSA ACTASARSAARRTAPTASCCAACWPSACASARTGSTSTITT (SEQ ID NO:31), such as an antigenic peptide comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 contiguous amino acids of the amino acid sequence SEQ ID NO:31.

[0007] Also disclosed herein is an antigenic peptide comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 contiguous amino acids of the amino acid sequence SEQ ID NO:31. For example, in some embodiments the peptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 contiguous amino acids of any one of SEQ ID NOs:1-21.

[0008] In some embodiments the peptide is in the form of a pharmaceutically acceptable salt. For example, the pharmaceutically acceptable salt can be a chloride salt, acetate salt, or trifluoro-acetate salt. In some embodiments the peptide is acylated or pegylated. In some embodiments the peptide further comprises heterologous amino acids, or any combination thereof. For example, in some cases the heterologous amino acids is an adjuvant peptide.

[0009] Also disclosed is a vector comprising a polynucleotide encoding one or more of the peptides disclosed herein operably linked to a heterologous expression control sequence.

[0010] Also disclosed is a population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides disclosed herein or transfected with a vector comprising a polynucleotide encoding one or more of the peptides disclosed herein operably linked to a heterologous expression control sequence.

[0011] Also disclosed herein is a cancer (e.g. colorectal cancer) vaccine comprising one or more of the disclosed antigenic peptides in a physiologically acceptable buffer, carrier, or excipient. In some embodiments, the vaccine further contains an adjuvant or immunostimulant. For example, the adjuvant can be selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.TH Docket No.222117-2430

[0012] Also disclosed herein is an antibody that binds SLC2A11-MIF having a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences. In some embodiments the CDR1 sequence of the VHdomain comprises the amino acid sequence SYGVH (SEQ ID NO:22); the CDR2 sequence of the VHdomain comprises the amino acid sequence LIWRGSTNYNAAFMS (SEQ ID NO:23); the CDR3 sequence of the VHdomain comprises the amino acid sequence NFYGDYDAMDY (SEQ ID NO:24); the CDR1 sequence of the VLdomain comprises the amino acid sequence KSTKSLLNSDGFTYLD (SEQ ID NO:25); the CDR2 sequence of the VLdomain comprises the amino acid sequence LVSNRFS (SEQ ID NO:26); and the CDR3 sequence of the VLdomain comprises the amino acid sequence FQSNYLPYT (SEQ ID NO:27).

[0013] For example, in some embodiment the VHdomain comprises the amino acid sequence QVQLKQSGPSLVQPSQSLSITCTVSGFSLTSYGVHWVRSPGKGLEWLGLIWGRGSTNYNAAF MSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKNFYGDYDAMDYWGQGTSVTVSS SEQ ID NO:28 and the VLdomain comprises the amino acid sequence DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPHLLIYLVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPYTFGGGTKLEIK SEQ ID NO:29. In some embodiments the antibody is a recombinant antibody. In some embodiments the antibody is a single chain (scFv) antibody.

[0014] Also disclosed is an isolated nucleic acid sequence encoding the recombinant antibody disclosed herein. Also disclosed is a vector comprising the disclosed nucleic acid sequence operably linked to an expression control sequence. Also disclosed is a cell comprising the disclosed vector. Also disclosed herein is an antibody drug conjugate (ADC) comprising a chemotherapeutic agent conjugated to an antibody disclosed herein.

[0015] Also disclosed herein is a chimeric antigen receptor (CAR) polypeptide, comprising a SLC2A11-MIF antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region, wherein the SLC2A11-MIF antigen- binding domain is a single-chain variable fragment (scFv) antibody disclosed herein.

[0016] Also disclosed is a CAR-T cell comprising the chimeric antigen receptor (CAR) polypeptide disclosed herein. For example, the cell can be selected from the group consisting of an ĮȕT cell, ȖįT cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.TH Docket No.222117-2430

[0017] Also disclosed is a method of treating a cancer in a subject, the method involving administering to the subject an effective amount of the vaccine, the ADC, or the CAR-T disclosed herein. In some embodiments, the method further involves administering to the subject a checkpoint inhibitor, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

[0018] In some embodiments, the cancer is any cancer expressing SLC2A11-MIF. Examples of cancers include, but are not limited to, hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrixTH Docket No.222117-2430 carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding- Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral- lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.

[0019] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.TH Docket No.222117-2430 BRIEF DESCRIPTION OF FIGURES

[0020] FIGs.1A to 1D show that the SLC2A11-MIF chimeric RNA is present in colorectal cancer cells.

[0021] FIG.2 shows SLC2A11-MIF has two isoforms and does not include SLC2A11 exon 6 and 7.

[0022] FIG.3 shows SLC2A11-MIF creates a novel open reading frame in CRC (SEQ ID NO:30).

[0023] FIGs.4A and 4B show SLC2A11-MIF is a marker for CRC disease.

[0024] FIGs.5A to 5C show SLC2A11-MIF antibody binds protein in WB, IP, and IHC.

[0025] FIGs.6A to 6J show knock-down of SLC2A11-MIF by siRNA slows cell proliferation.

[0026] FIG.7 illustrates a process for isolation and generation of human DCs stimulated with SLC2A11-MIF peptides.

[0027] FIG.8 illustrates the use of antigen-loaded DCs to activate T cells from healthy donors.

[0028] FIGs.9A and 9B show naïve (FIG.9A) and memory (FIG.9B) CD8+T cell response (IFN-Ȗ) using different peptide pools and different T cell donors.

[0029] FIGs.10A and 10B show naïve (FIG.10A) and memory (FIG.10B) CD8+T cell response (IFN-Ȗ) using different peptide pools and different T cell donors.

[0030] FIGs.11A and 11B show quantitative (FIG.11A) and relative (FIG.11B) T cell response (IFN-Ȗ) in naïve CD8+T cells 15 days post-stimulation with peptides PP-1 to PP-13 in two T cell donors.

[0031] FIGs.12A and 12B show quantitative (FIG.11A) and relative (FIG.11B) T cell response (IFN-Ȗ) in naïve CD8+T cells 72 hours post-stimulation with peptides PP-1, PP3-PP-9, and PP-14 in two T cell donors.

[0032] FIGs.13A to 13C show SLC2A11-MIF is present in colorectal cancer and predictive of worse overall survival. In a further analysis using TCGA data of 270 patients, the presence of SLC2A11-MIF predicted worse overall survival, while SLC2A11 WT and MIF WT RNA median expression did not predict differences in survival. N=patients, Events= # of deaths, Median= time of death (days). In those 270 patients, there was no difference between MSI and MSS, and SM did not correlate with MSI instability score.

[0033] FIGs.14A to 14M show SLC2A11-MIF is expressed in early-stage disease and is not associated with any CRC features using data of SLC2A11-MIF expression from 175 normal and cancer paired tissues.TH Docket No.222117-2430

[0034] FIGs.15A to 15R show SLC2A11-MIF is expressed in early-stage disease and is not associated with any CRC features using data of SLC2A11-MIF expression from 175 tumor- adjacent normal and cancer paired tissues.

[0035] FIGs.16A to 16D show SLC2A11-MIF protein is expressed in colorectal cancer and antibody is on-target for SLC2A11-MIF.

[0036] FIGs.17A to 17G show SLC2A11-MIF in other cancers. FIG.17A shows SLC2A11-MIF in ovarian serous carcinoma, prostate adenocarcinoma, esophagus adenocarcinoma, bladder urothelial carcinoma, and lung squamous cell carcinoma. FIGs.17B to 17D show SLC2A11-MIF is detected in TCGA of other cancer types, but is upregulated in tumor compared to tumor-adjacent normal tissue. Figure 17E shows SLC2A11 is completely absent in all RNA-sequencing data of normal tissue in The Genotype-Tissue Expression (GTEx) Portal but is present in cancer tissue RNA-seq in TCGA. Figure 17F shows the proportion of SLC2A11-MIF positive samples in each cancer type compared to the normal tumor-adjacent tissue.

[0037] FIGs.18A to 18J show 14d expansion following in vitro stimulation (IVS) with personalized peptide pools.

[0038] FIG.19A to 19H show IVS at day 21 and 28 with individual peptides to determine which peptides are responsible for IFN-Ȗ secretion.

[0039] FIG.20 shows Real Time Cell Analysis (RTCA) with donor 3 T cell killing of HLA- matched (B7) SW620 CRC cells.

[0040] FIGs.21A to 21F show 2 / 6 Healthy donor CD4+ and CD8+ T cells had a response to SM peptides.

[0041] FIG.22 contains ELISpots showing response to SM peptides.

[0042] FIGs.23A and 23B show healthy donor 6 naïve T cell response with individual peptides.

[0043] FIGs.24A and 24B show healthy donor 9 naïve T cell response with individual peptides.

[0044] FIGs.25A to 25F show 3 / 3 healthy donors had a positive response to class I peptide pools.

[0045] FIGs.26A to 26C show 1 / 3 donors had a positive response to class II peptides.

[0046] FIGs.27A and 27B show RTCA of T cell killing of HLA matched CRC cells. Peptide activated T cells killed HLA-matched HCT116 (FIG.27A) and SW480 cells (FIG.27B).

[0047] FIGs.28A and 28B show donor 9 had a statistically significant positive response to 3 peptides. FIG.28C shows peptide activated T cells killed HLA-matched HCT116 cells.TH Docket No.222117-2430

[0048] FIGs.29A and 29B show CRC patient VCC-009 responds to class II peptides.

[0049] FIGs.30A and 30B show CRC patients VCC-011, VCC-014, and VCC-020 responded to class II peptides.

[0050] FIGs.31A and 31B show no response to class I or II peptides in patients VCC- 027, VCC-028, VCC-033, VCC-034. DETAILED DESCRIPTION

[0051] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0052] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0054] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0055] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components andTH Docket No.222117-2430 features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0056] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0057] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0058] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. Definitions

[0059] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0060] The term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0061] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal orTH Docket No.222117-2430 polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0062] The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fabƍ, F(abƍ)2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0063] The term “antigen binding site” refers to a region of an antibody that specifically binds an epitope on an antigen.

[0064] The term “aptamer” refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity. A “nucleic acid aptamer” is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. A “peptide aptamer” is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.

[0065] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound orTH Docket No.222117-2430 composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0066] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecules is a fusion protein.

[0067] The term “engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen binding site derived from the variable domain of the heavy chain and / or light chain of an antibody and may optionally comprise the entire or part of the variable and / or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY).

[0068] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0069] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0070] The term “Fab fragment” refers to a fragment of an antibody comprising an antigen-binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0071] The term “F(abƍ)2 fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0072] The term “Fc fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain.TH Docket No.222117-2430

[0073] The term “Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.

[0074] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may be transfected into cells, e.g. in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0075] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0076] The term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a singleTH Docket No.222117-2430 linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0077] The term “multivalent antibody” refers to an antibody or engineered antibody comprising more than one antigen recognition site. For example, a “bivalent” antibody has two antigen recognition sites, whereas a “tetravalent” antibody has four antigen recognition sites. The terms “monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, a “monospecific” antibody's antigen recognition sites all bind the same epitope. A “bispecific” antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A “multivalent monospecific” antibody has multiple antigen recognition sites that all bind the same epitope. A “multivalent bispecific” antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

[0078] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0079] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0080] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0081] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.TH Docket No.222117-2430

[0082] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the amino- terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and / or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.

[0083] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0084] The term “single chain variable fragment or scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0085] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0086] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M–1(e.g., 106M–1, 107M–1, 108M–1, 109M–1, 1010M–1, 1011M–1, and 1012M–1or more) with that second molecule.TH Docket No.222117-2430

[0087] The term “specifically deliver” as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule.

[0088] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0089] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0090] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0091] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0092] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid subsitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA)TH Docket No.222117-2430 encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0093] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). Anti-SLC2A11-MIF Antibodies

[0094] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0095] Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ- line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cote et al. and Boerner et al. are also available for the preparation ofTH Docket No.222117-2430 human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991)).

[0096] Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992))

[0097] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed following the method of Winter and co- workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a non human antibody (or a fragment thereof) is a chimeric antibody or fragment (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanizedTH Docket No.222117-2430 antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0098] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0099] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic protein of the present disclosure. Methods for the production of single- chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0100] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VHand two VLregions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0101] Bivalent and bispecific antibodies can be constructed using only antibody variable domains. A fairly efficient and relatively simple method is to make the linker sequence between the VHand VLdomains so short that they cannot fold over and bind one another. Reduction of the linker length to 3-12 residues prevents the monomeric configuration of the scFv molecule and favors intermolecular VH-VL pairings with formation of a 60 kDa non- covalent scFv dimer “diabody”. The diabody format can also be used for generation of recombinant bis-pecific antibodies, which are obtained by the noncovalent association of two single-chain fusion products, consisting of the VH domain from one antibody connected by aTH Docket No.222117-2430 short linker to the VL domain of another antibody. Reducing the linker length still further below three residues can result in the formation of trimers (“triabody”, about 90 kDa) or tetramers (“tetrabody”, about 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson, 2005, Nature Biotechnology, 23:1126-1136. All of such engineered antibodies may be used in the fusion polypeptides provided herein. Tetravalent Tandab® may be prepared substantially as described in WO 1999057150 A3 or US20060233787, which are incorporated by reference for the teaching of methods of making Tandab® molecules.

[0102] The antigen recognition sites or entire variable regions of the engineered antibodies may be derived from one or more parental antibodies directed against any antigen of interest (e.g., chimeric SLC2A11-MIF protein). The parental antibodies can include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using sequences of antibodies or antibody fragments known to be specific for an antigen of interest. Sequences that may be derived from parental antibodies include heavy and / or light chain variable regions and / or CDRs, framework regions or other portions thereof.

[0103] Multivalent, multispecific antibodies may contain a heavy chain comprising two or more variable regions and / or a light chain comprising one or more variable regions wherein at least two of the variable regions recognize different epitopes on the same antigen.

[0104] Candidate engineered antibodies for inclusion in the fusion polypeptides, or the fusion polypeptides themselves, may be screened for activity using a variety of known assays. For example, screening assays to determine binding specificity are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y., 1988, Chapter 6. Antibody Drug Conjugates

[0001] Also disclosed is a composition comprising the disclosed antibody conjugated to an anti-cancer agent. The disclosed antibody can be used to deliver any payload to CRCs in a subject. The payload can be a therapeutic or diagnostic agent. In some embodiments, the payload is an anti-cancer agent that can cause apoptosis or pyroptosis of the targeted tumor cell. In some embodiments, the anti-cancer agent is a small molecule drug. In some embodiments, the anti-cancer agent is monomethyl auristatin E, gemcitabine, or resveratrol. The anti-cancer agent can be a chemotherapy agent, such as drugs that stop DNA building block synthesis (e.g., methotrexate, fluorouracil, hydroxyurea, lurtotecan, mercaptopurine,TH Docket No.222117-2430 pentostatin and pirarubicin), drugs that directly damage DNA (e.g., cisplatin, daunorubicin, doxorubicin, etoposide, teniposide, camptothecin, topotecan, irinotecan, rubitecan, belotecan), drugs that affect mitotic spindle synthesis or breakdown (e.g., vinblastine, vincristine, vinorelbine, vinflunine, vindesine, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, ixabepilone and epithilones), or drugs that disrupt angiogenesis (e.g., anti-VEGF antibody, angiostatin, endostatin, and tumstatin). Alternatively, the anti-cancer agent can be a radiotherapy agent (e.g., 90Y, 125I, 188Re, 111In DTPA, or 131I Sodium iodide).

[0002] Examples of anti-cancer drugs or antineoplastics to be attached to the tumor targeting peptides described herein include, but are not limited to, aclarubicin, altretamine, aminopterin, amrubicin, azacitidine, azathioprine, belotecan, busulfan, camptothecin, capecitabine, carboplatin, carmofur, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, daunorubicin, decitabine, doxorubicin, epirubicin, etoposide, floxuridine, fludarabine, 5-fluorouracil, fluorouracil, gemcitabine, idarubicin, ifosfamide, irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nedaplatin, oxaliplatin, paclitaxel, pemetrexed, pentostatin, pirarubicin, pixantrone, procarbazine, pyrimethamine raltitrexed, rubitecan, satraplatin, streptozocin, thioguanine, triplatin tetranitrate, teniposide, topotecan, tegafur, trimethoprim, uramustine, valrubicin, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, and zorubicin.

[0003] In come embodiments, the disclosed antibody is linked to a vehicle carrier, which is associated with the anti-cancer agent. In one example, the vehicle carrier encapsulates the anti-cancer agent. Vehicle carriers include, but are not limited to, exosome, micelle, liposome (e.g., cationic liposome), nanoparticle, microsphere, or biodegradable polymer. A tumor targeting peptide can be tethered to a vehicle carrier by a variety of linkages (e.g., a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage, or a hydrazine linkage). To improve the association between the antibody and the vehicle carrier, the peptide can be modified by a suitable polymer, such as PEG (peglyated). The detectable label or the anti-cancer agent can be encapsulated within the vehicle via, e.g., association with lipophilic molecules, which can aid in the delivery of the detectable label or the anti-cancer agent to the interior of the vehicle.

[0004] In some embodiments, a tumor targeting antibody described herein is linked to a liposome (as a vehicle carrier) that encapsulates one or more agents of interest (e.g., an anti- cancer agent). Liposome is a vesicle comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase. The aqueous phase typically contains an agent to be delivered to a target site such as a tumor site. Upon reaching the target site, the liposomeTH Docket No.222117-2430 fuses with the plasma membranes of local cells to release the agent into the cytosol. Alternatively, the liposome is endocytosed or otherwise taken in by the cells as the content of a transport vesicle (e.g., an endosome or phagosome). Once in the transport vesicle, the liposome either degrades or fuses with the membrane of the vesicle and releases its contents. Liposome membranes can be constructed so that they become destabilized when the nearby environment becomes acidic (see, e.g., PNAS 84:7851, 1987; Biochemistry 28:908, 1989). Thus, when liposomes enter a target cell, they become destabilized to release their encapsulated contents. This destabilization process is termed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is commonly used to facilitate this process.

[0005] A variety of methods are available for preparing liposomes. See, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng.9:467 (1980), U.S. Pat. Nos.4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91 / 17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al., Chem. Phys. Lip.40:89 (1986); Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vehicles and ether fusion methods, all of which are well known in the art.

[0006] In antibody drug conjugate, the antibody can be conjugated directly to the cytotoxic agent or via a linker. Suitable linkers include, for example, cleavable and non- cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In some embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers hydrolyzable at a pH of less than 5.5, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers.

[0007] Also disclosed is a pharmaceutical composition comprising the tumor targeting antibody and payload disclosed herein in a pharmaceutically acceptable carrier. Also disclosed is a method for treating a CRCs in a subject that involves administering to the subject a therapeutically effective amount of a disclosed pharmaceutical composition.TH Docket No.222117-2430 CAR-T Cells

[0105] Chimeric antigen receptors (CARs) generally incorporate an antigen recognition domain from the single-chain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 20033:35–45). Disclosed herein is a SLC2A11-MIF-specific chimeric antigen receptor (CAR) that can be that can be expressed in immune effector cells to enhance antitumor activity against SLC2A11-MIF-specific CARs.

[0106] The disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain comprises the SLC2A11-MIF- binding region and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain (TD), is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. For example, the endodomain can contain a signaling domain (ISD) and a co-stimulatory signaling region (CSR).

[0107] A “signaling domain (SD)” generally contains immunoreceptor tyrosine-based activation motifs (ITAMs) that activate a signaling cascade when the ITAM is phosphorylated. The term “co-stimulatory signaling region (CSR)” refers to intracellular signaling domains from costimulatory protein receptors, such as CD28, 41BB, and ICOS, that are able to enhance T-cell activation by T-cell receptors.

[0108] In some embodiments, the endodomain contains an SD or a CSR, but not both. In these embodiments, an immune effector cell containing the disclosed CAR is only activated if another CAR (or a T-cell receptor) containing the missing domain also binds its respective antigen.

[0109] In some embodiments, the disclosed CAR is defined by the formula: SP–SM–HG–TM–CSR–SD; or SP–SM–HG–TM–SD–CSR; wherein “SP” represents an optional signal peptide, wherein “SM” represents a SLC2A11-MIF-binding region, wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CSR” represents one or more co-stimulatory signaling regions, wherein “SD” represents a signaling domain, andTH Docket No.222117-2430 wherein “–” represents a peptide bond or linker.

[0110] Additional CAR constructs are described, for example, in Fresnak AD, et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81, which is incorporated by reference in its entirety for the teaching of these CAR models.

[0111] For example, the CAR can be a TRUCK, Universal CAR, Self-driving CAR, Armored CAR, Self-destruct CAR, Conditional CAR, Marked CAR, TenCAR, Dual CAR, or sCAR.

[0112] TRUCKs (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T cell activation. Targeted by CAR specificity, localized production of pro- inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.

[0113] Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0114] Self-driving CARs co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing tumor homing.

[0115] CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.

[0116] A self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small-molecule dimerizer.

[0117] A conditional CAR T cell is by default unresponsive, or switched ‘off’, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.

[0118] Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration ofTH Docket No.222117-2430 the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off- tumor effects.

[0119] A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular co- stimulatory domain(s) and a CD3ȗ domain. TanCAR T cell activation is achieved only when target cells co-express both targets.

[0120] A dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3ȗ domain and the other CAR includes only the co- stimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.

[0121] A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0122] The antigen recognition domain of the disclosed CAR is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.

[0123] The endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Therefore, the endodomain may comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co- receptors. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.

[0124] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, CD3ȗ, CD3į, CD3Ȗ, CD3İ,TH Docket No.222117-2430 CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcȖRIȖ, FcȖRIIIȖ, FcİRIȕ (FCERIB), and FcİRIȖ (FCERIG).

[0125] In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3ȗ) (TCR zeta, GenBank accno. BAG36664.1). T-cell surface glycoprotein CD3 zeta (CD3ȗ) chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.

[0126] First-generation CARs typically had the intracellular domain from the CD3ȗ chain, which is the primary transmitter of signals from endogenous TCRs. Second-generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, third-generation CARs combine multiple signaling domains to further augment potency. T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumor-eradicating efficiency independent of costimulatory receptor / ligand interaction (Imai C, et al. Leukemia 200418:676–84; Maher J, et al. Nat Biotechnol 200220:70–5).

[0127] For example, the endodomain of the CAR can be designed to comprise the CD3ȗ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3ȗ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Thus, while the CAR is exemplified primarily with CD28 as the co-stimulatory signaling element, other costimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0128] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain. The hinge sequence can be anyTH Docket No.222117-2430 suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0129] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL2R beta, IL2R gamma, IL7R Į, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, and PAG / Cbp. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.

[0130] In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.

[0131] In some embodiments, the CAR is a multi-chain CAR, as described in WO2015 / 039523, which is incorporated by reference for this teaching. A multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction. For example, the multi-chain CAR can comprise a part of an FCERI alpha chain and a part of an FCERI beta chain such that the FCERI chains spontaneously dimerize together to form a CAR.

[0132] Also disclosed are immune effector cells that are engineered to express the disclosed CARs (also referred to herein as “CAR-T cells.” These cells are preferably obtainedTH Docket No.222117-2430 from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0133] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dentritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

[0134] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0135] T helper cells (THcells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells canTH Docket No.222117-2430 differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.

[0136] Cytotoxic T cells (TCcells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0137] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0138] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described — naturally occurring Tregcells and adaptive Tregcells.

[0139] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d.

[0140] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T comprise are cytotoxic CD8+T lymphocytes. In some embodiments, the T cells comprise Ȗį T cells, which possess a distinct T-cell receptor (TCR) having one Ȗ chain and one į chain instead of Į and ȕ chains.

[0141] Natural-killer (NK) cells are CD56+CD3–large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 201253:1666–1676). Unlike cytotoxic CD8+T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can also eradicate MHC-I-negative cells (Narni-Mancinelli E, et al. Int Immunol 201123:427–431). NK cells are safer effector cells, as they may avoid the potentiallyTH Docket No.222117-2430 lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 201018:843–851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011365:725–733), and on-target, off-tumor effects. Although NK cells have a well-known role as killers of cancer cells, and NK cell impairment has been extensively documented as crucial for progression of MM (Godfrey J, et al. Leuk Lymphoma 201253:1666–1676; Fauriat C, et al. Leukemia 200620:732–733), the means by which one might enhance NK cell-mediated anti-MM activity has been largely unexplored prior to the disclosed CARs.

[0142] Epstein-Barr virus (EBV)-induced lymphoproliferative diseases (EBV-LPDs) are a significant cause of morbidity and mortality for recipients of allogeneic hematopoietic cell transplantation (HCT), particularly in those who have received certain T-cell reactive Abs to prevent or treat GVHD. Prophylaxis and treatment by the adoptive transfer of EBV-specific T cells and the subsequent long-term restoration of immunity against EBV-associated lymphoproliferation have provided positive outcomes in the management of this uniformly fatal complication of bone marrow transfer. Therefore, in some embodiments, the disclosed immune effector cells are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, this can involve isolating PBMCs from of an autologous or allogenic donor and enriching them for T cells by depletion of monocytes and NK cells. For example, the donor can be an EBV-seropositive donor. These T cells can then be stimulated with autologous EBV- seropostive or transformed lymphocytes. EBV antigens include latent membrane protein (LMP) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A, and LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. These methods are described, for example, in Barker et al., Blood 2010116(23):5045-49; Doubrovina, et al., Blood 2012 119(11):2644-56; Koehne, et al. Blood 200299(5):1730-40; and Smith et al. Cancer Res 2012 72(5):1116-25, which are incorporated by reference for these teachings. Cancer Vaccines Protein Vaccines

[0008] Also disclosed herein is an antigenic peptide comprising at least 4, 5, 6, 7 or 8 contiguous amino acids of the amino acid sequence SEQ ID NO:31. In some embodiment the peptide has at least 4, 5, 6, 7 or 8 contiguous amino acids of a peptide selected from the group consisting of SEQ ID NO: 1 to 21, or a variant thereof, which is at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1 to SEQ ID NO: 21. In some embodiments the peptide or variant thereof has an overall length ofTH Docket No.222117-2430 between 8 and 100, preferably between 8 and 30, and most preferred of between 8 and 14 amino acids.

[0009] In some embodiments, the disclosed peptide binds to class I or II human major histocompatibility complex (MHC) and / or induces T cells cross-reacting with said peptide, or a pharmaceutical acceptable salt thereof, wherein said peptide is not the underlying full-length polypeptide.

[0010] In some embodiments, the peptide is modified and / or includes non-peptide bonds.

[0011] In some embodiments, the peptide is part of a fusion protein. For example, in some embodiments, the peptide is fused to the N-terminal amino acids of the HLA-DR antigen- associated invariant chain (Ii). In some embodiments, the peptide is fused to (or into the sequence of) an antibody, such as, for example, an antibody that is specific for dendritic cells. In some embodiments, the peptide is fused to an adjuvant peptide.

[0012] Also disclosed is a nucleic acid encoding the disclosed peptides, including DNA, cDNA, PNA, RNA, and combinations thereof. Also disclosed is an expression vector capable of expressing and / or expressing these nucleic acids. Also disclosed is a host cell comprising the nucleic acid or expression vector described before. In some embodiments the host cell is an antigen presenting cell, such as a dendritic cell.

[0013] Also disclosed is a method for producing a peptide disclosed that involves culturing the host cell and isolating the peptide from the host cell or its culture medium.

[0014] In some embodiments the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting a sufficient amount of the antigen with an antigen-presenting cell.

[0015] Also disclosed are activated T cells produced using the disclosed peptides, wherein the T cell selectively recognizes a cell that expresses chimeric SLC2A11-MIF.

[0016] Also disclosed is the use of a disclosed peptide, the nucleic acid, expression vector, host cell, or activated T lymphocyte as a medicament or in the manufacture of a medicament for treating a cancer.

[0017] In addition, the peptide or variant may be modified further to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Methods for such an optimization of a peptide sequence are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds.

[0018] In a reverse peptide bond amino acid residues are not joined by peptide (—CO— NH—) linkages but the peptide bond is reversed. Such retro-inverso peptidomimetics may beTH Docket No.222117-2430 made using methods known in the art, for example such as those described in Meziere et al (1997) (Meziere et al., 1997), incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al. (Meziere et al., 1997) show that for MHC binding and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH—CO bonds instead of CO—NH peptide bonds, are much more resistant to proteolysis.

[0019] A non-peptide bond is, for example, —CH2—NH, —CH2S—, —CH2CH2—, — CHőCH—, —COCH2—, —CH(OH)CH2—, and —CH2SO—. U.S. Pat. No.4,897,445 provides a method for the solid phase synthesis of non-peptide bonds (—CH2—NH) in polypeptide chains which involves polypeptides synthesized by standard procedures and the non-peptide bond synthesized by reacting an amino aldehyde and an amino acid in the presence of NaCNBH3.

[0020] Peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and / or carboxy termini, to enhance the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.

[0021] Further, the peptides may be synthesized to alter their steric configuration. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well-known non-naturally occurring amino acid residues. Alterations such as these may serve to increase the stability, bioavailability and / or binding action of the peptides of the invention.

[0022] Similarly, a peptide may be modified chemically by reacting specific amino acids either before or after synthesis of the peptide. Examples for such modifications are well known in the art and are summarized e.g. in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which is incorporated herein by reference. Chemical modification of amino acids includes but is not limited to, modification by acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulphides with other thiol compounds, reaction withTH Docket No.222117-2430 maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH, although without limitation thereto. In this regard, the skilled person is referred to Chapter 15 of Current Protocols In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more extensive methodology relating to chemical modification of proteins.

[0023] Briefly, modification of e.g. arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2- cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. As a result, a large number of reagents are available for the modification of cysteine.

[0024] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward's Reagent K may be used to modify specific glutamic acid residues. N-(3- (dimethylamino)propyl)-Nƍ-ethylcarbodiimide can be used to form intra-molecular crosslinks between a lysine residue and a glutamic acid residue. For example, diethylpyrocarbonate is a reagent for the modification of histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. The reaction of lysine residues and other Į-amino groups is, for example, useful in binding of peptides to surfaces or the cross-linking of proteins / peptides. Lysine is the site of attachment of poly(ethylene)glycol and the major site of modification in the glycosylation of proteins. Methionine residues in proteins can be modified with e.g. iodoacetamide, bromoethylamine, and chloramine T.

[0025] Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking via the formation of dityrosine can be accomplished with hydrogen peroxide / copper ions.

[0026] Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

[0027] Successful modification of therapeutic proteins and peptides with PEG is often associated with an extension of circulatory half-life while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.TH Docket No.222117-2430

[0028] In some embodiments, the peptide is modified or includes non-peptide bonds. Generally, peptides and variants (at least those containing peptide linkages between amino acid residues) may be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and by references as cited therein. Temporary N- amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is done using 20% piperidine in N, N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4ƍ- dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid- phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N, N-dicyclohexyl- carbodiimide / 1hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used include ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Also a combination of solid phase and solution phase methodologies for the synthesis of peptides is possible (see, for example, (Bruckdorfer et al., 2004), and the references as cited therein). Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from e.g. Calbiochem-Novabiochem (Nottingham, UK).

[0029] Purification may be performed by any one, or a combination of, techniques such as re-crystallization, size exclusion chromatography, ion-exchange chromatography,TH Docket No.222117-2430 hydrophobic interaction chromatography and (usually) reverse-phase high performance liquid chromatography using e.g. acetonitrile / water gradient separation. RNA Vaccines

[0030] Also disclosed herein is a ribonucleic acid (RNA) cancer vaccine of an RNA (e.g., messenger RNA (mRNA)) encoding at least 4, 5, 6, 7 or 8 contiguous amino acids of the amino acid sequence SEQ ID NO:31. In some embodiment the RNA encodes at least 4, 5, 6, 7 or 8 contiguous amino acids of a peptide selected from the group consisting of SEQ ID NO: 1 to 21, or a variant thereof, which is at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1 to SEQ ID NO: 21.

[0031] In some embodiments, the RNA is a modified RNA. The RNA vaccines of the present disclosure may be used to induce a balanced immune response against SLC2A11-MIF- expressing cancers.

[0032] The RNA vaccines may be utilized in various settings depending on the prevalence of the cancer or the degree or level of unmet medical need. The RNA vaccines may be utilized to treat and / or prevent a SLC2A11-MIF-expressing cancer of various stages or degrees of metastasis.

[0033] The RNA vaccines may include a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least 4, 5, 6, 7 or 8 contiguous amino acids of the amino acid sequence SEQ ID NO:31 (e.g., an immunogenic fragment capable of inducing an immune response to cancer). Other embodiments include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding two or more antigens or epitopes capable of inducing an immune response to cancer.

[0034] In some embodiments the RNA is formulated in a lipid nanoparticle. For example, in some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% neutral lipid:25-55% sterol; and 0.5-15% PEG-modified lipid, optionally wherein the ionizable amino lipid is a cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of about 50% compound 25:about 10% DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG. In some embodiments, the ionizable amino lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1- yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH value.TH Docket No.222117-2430

[0035] In some embodiments the mRNA includes at least one chemical modification. The chemical modification may be selected from the group consisting of pseudouridine, N1- methylpseudouridine, 2-thiouridine, 4ƍ-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2ƍ- O-methyl uridine. In some embodiments, an mRNA of the disclosure encoding an immune potentiator can comprises one or more modified nucleobases.

[0036] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), including but not limited to chemical modification, that are useful in the compositions, methods and synthetic processes of the present disclosure include, but are not limited to the following: uniformly nucleotides, nucleosides, and nucleobases: 2-methylthio-N6- (cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine; N6-threonylcarbamoyladenosine; 1,2ƍ-O-dimethyladenosine; 1- methyladenosine; 2ƍ-O-methyladenosine; 2ƍ-O-ribosyladenosine (phosphate); 2- methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2ƍ-O-methyladenosine; 2ƍ-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2ƍ-O-dimethyladenosine; N6,2ƍ-O-dimethyladenosine; N6,N6,2ƍ-O-trimethyladenosine; N6,N6-dimethyladenosine; N6- acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6- threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7- deaza-adenosine; N1-methyl-adenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy- isopentenyl-adenosine; Į-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2- (aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2ƍ-Amino-2ƍ-deoxy- ATP; 2ƍ-Azido-2ƍ-deoxy-ATP; 2ƍ-Deoxy-2ƍ-a-aminoadenosine TP; 2ƍ-Deoxy-2ƍ-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8- (alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8- (hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7- methyladenine; 1-Deazaadenosine TP; 2ƍFluoro-N6-Bz-deoxyadenosine TP; 2ƍ-OMe-2-Amino-TH Docket No.222117-2430 ATP; 2ƍO-methyl-N6-Bz-deoxyadenosine TP; 2ƍ-a-Ethynyladenosine TP; 2-aminoadenine; 2- Aminoadenosine TP; 2-Amino-ATP; 2ƍ-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2ƍ-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2ƍ-b-Trifluoromethyladenosine TP; 2- Chloroadenosine TP; 2ƍ-Deoxy-2ƍ,2ƍ-difluoroadenosine TP; 2ƍ-Deoxy-2ƍ-a-mercaptoadenosine TP; 2ƍ-Deoxy-2ƍ-a-thiomethoxyadenosine TP; 2ƍ-Deoxy-2ƍ-b-aminoadenosine TP; 2ƍ-Deoxy-2ƍ-b- azidoadenosine TP; 2ƍ-Deoxy-2ƍ-b-bromoadenosine TP; 2ƍ-Deoxy-2ƍ-b-chloroadenosine TP; 2ƍ- Deoxy-2ƍ-b-fluoroadenosine TP; 2ƍ-Deoxy-2ƍ-b-iodoadenosine TP; 2ƍ-Deoxy-2ƍ-b- mercaptoadenosine TP; 2ƍ-Deoxy-2ƍ-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2- lodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2- Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4ƍ- Azidoadenosine TP; 4ƍ-Carbocyclic adenosine TP; 4ƍ-Ethynyladenosine TP; 5ƍ-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2- aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2- thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4- acetylcytidine; 2ƍ-O-methylcytidine; 2ƍ-O-methylcytidine; 5,2ƍ-O-dimethylcytidine; 5-formyl-2ƍ-O- methylcytidine; Lysidine; N4,2ƍ-O-dimethylcytidine; N4-acetyl-2ƍ-O-methylcytidine; N4- methylcytidine; N4,N4-Dimethyl-2ƍ-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo- iso-cytidine; pyrrolo-cytidine; Į-thio-cytidine; 2-(thio)cytosine; 2ƍ-Amino-2ƍ-deoxy-CTP; 2ƍ-Azido- 2ƍ-deoxy-CTP; 2ƍ-Deoxy-2ƍ-a-aminocytidine TP; 2ƍ-Deoxy-2ƍ-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3- (methyl)cytidine; 4,2ƍ-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5- (halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo- cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy- 5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl- pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4- thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl- zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2ƍ- anhydro-cytidine TP hydrochloride; 2ƍFluor-N4-Bz-cytidine TP; 2ƍFluoro-N4-Acetyl-cytidine TP; 2ƍ-O-Methyl-N4-Acetyl-cytidine TP; 2ƍO-methyl-N4-Bz-cytidine TP; 2ƍ-a-Ethynylcytidine TP; 2ƍ-a- Trifluoromethylcytidine TP; 2ƍ-b-Ethynylcytidine TP; 2ƍ-b-Trifluoromethyl cytidine TP; 2ƍ-Deoxy-TH Docket No.222117-2430 2ƍ,2ƍ-difluorocytidine TP; 2ƍ-Deoxy-2ƍ-a-mercaptocytidine TP; 2ƍ-Deoxy-2ƍ-a-thiomethoxycytidine TP; 2ƍ-Deoxy-2ƍ-b-aminocytidine TP; 2ƍ-Deoxy-2ƍ-b-azidocytidine TP; 2ƍ-Deoxy-2ƍ-b- bromocytidine TP; 2ƍ-Deoxy-2ƍ-b-chlorocytidine TP; 2ƍ-Deoxy-2ƍ-b-fluorocytidine TP; 2ƍ-Deoxy-2ƍ- b-iodocytidine TP; 2ƍ-Deoxy-2ƍ-b-mercaptocytidine TP; 2ƍ-Deoxy-2ƍ-b-thiomethoxycytidine TP; 2ƍ- O-Methyl-5-(1-propynyl)cytidine TP; 3ƍ-Ethynylcytidine TP; 4ƍ-Azidocytidine TP; 4ƍ-Carbocyclic cytidine TP; 4ƍ-Ethynyl cytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2- thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5- Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5ƍ-Homo-cytidine TP; 5-Methoxycytidine TP; 5- Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2ƍ-O-dimethylguanosine; N2-methylguanosine; Wyosine; 1,2ƍ-O- dimethylguanosine; 1-methylguanosine; 2ƍ-O-methylguanosine; 2ƍ-O-ribosylguanosine (phosphate); 2ƍ-O-methylguanosine; 2ƍ-O-ribosylguanosine (phosphate); 7-aminomethyl-7- deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7- dimethylguanosine; N2,N2,2ƍ-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2- dimethylguanosine; N2,7,2ƍ-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo- guanosine; N1-methyl-guanosine; Į-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2ƍ- Amino-2ƍ-deoxy-GTP; 2ƍ-Azido-2ƍ-deoxy-GTP; 2ƍ-Deoxy-2ƍ-a-aminoguanosine TP; 2ƍ-Deoxy-2ƍ-a- azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl- guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7- (deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy- guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl- guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio- guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2ƍFluoro-N2-isobutyl-guanosine TP; 2ƍO- methyl-N2-isobutyl-guanosine TP; 2ƍ-a-Ethynylguanosine TP; 2ƍ-a-Trifluoromethylguanosine TP; 2ƍ-b-Ethynylguanosine TP; 2ƍ-b-Trifluoromethylguanosine TP; 2ƍ-Deoxy-2ƍ,2ƍ-difluoroguanosine TP; 2ƍ-Deoxy-2ƍ-a-mercaptoguanosine TP; 2ƍ-Deoxy-2ƍ-a-thiomethoxyguanosine TP; 2ƍ-Deoxy- 2ƍ-b-aminoguanosine TP; 2ƍ-Deoxy-2ƍ-b-azidoguanosine TP; 2ƍ-Deoxy-2ƍ-b-bromoguanosine TP; 2ƍ-Deoxy-2ƍ-b-chloroguanosine TP; 2ƍ-Deoxy-2ƍ-b-fluoroguanosine TP; 2ƍ-Deoxy-2ƍ-b- iodoguanosine TP; 2ƍ-Deoxy-2ƍ-b-mercaptoguanosine TP; 2ƍ-Deoxy-2ƍ-b-thiomethoxyguanosine TP; 4ƍ-Azidoguanosine TP; 4ƍ-Carbocyclic guanosine TP; 4ƍ-Ethynylguanosine TP; 5ƍ-Homo- guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-TH Docket No.222117-2430 methylinosine; Inosine; 1,2ƍ-O-dimethylinosine; 2ƍ-O-methylinosine; 7-methylinosine; 2ƍ-O- methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2ƍ-O-methyluridine; 2- thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5- taurinomethyl-2-thiouridine; 5-taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino- 3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1- methylpseduouridine; 1-ethyl-pseudouridine; 2ƍ-O-methyluridine; 2ƍ-O-methylpseudouridine; 2ƍ- O-methyluridine; 2-thio-2ƍ-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2ƍ-O- dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5- (carboxyhydroxymethyl)uridine methyl ester; 5,2ƍ-O-dimethyluridine; 5,6-dihydro-uridine; 5- aminomethyl-2-thiouridine; 5-carbamoylmethyl-2ƍ-O-methyluridine; 5-carbamoylmethyluridine; 5- carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5- carboxymethylaminomethyl-2ƍ-O-methyluridine; 5-carboxymethyl aminomethyl-2-thiouridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2ƍ- O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5- methyluridine,), 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5- Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3- Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso- Pentenylaminomethyl)-2ƍ-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5- propynyl uracil; Į-thio-uridine; 1 (aminoalkylamino-carb onylethylenyl)-2(thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminoalkylaminocarbonyl ethylenyl)-4 (thio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarb onylethylenyl)-2(thio)-pseudouracil; 1 (aminocarbonylethylenyl)-2,4- (dithio)pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted 2,4- (dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; 1- (aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; 1-Methyl-3-(3-amino-3- carboxypropyl) pseudouridine TP; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl- pseudo-UTP; 1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2ƍ deoxy uridine; 2ƍ fluorouridine; 2- (thio)uracil; 2,4-(dithio)psuedouracil; 2ƍ methyl, 2ƍamino, 2ƍazido, 2ƍfluro-guanosine; 2ƍ-Amino-2ƍ- deoxy-UTP; 2ƍ-Azido-2ƍ-deoxy-UTP; 2ƍ-Azido-deoxyuridine TP; 2ƍ-O-methylpseudouridine; 2ƍTH Docket No.222117-2430 deoxy uridine; 2ƍ fluorouridine; 2ƍ-Deoxy-2ƍ-a-aminouridine TP; 2ƍ-Deoxy-2ƍ-a-azidouridine TP; 2- methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4- (thio)pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5 (2- aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5- (alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5- (dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5- (methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5- (methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1- carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1- taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl- pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio- pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl- pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (+)1-(2- Hydroxypropyl)pseudouridine TP; (2R)-1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2- Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo- vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1-(2,2,2- Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2- Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl- benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2- carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; 1- (3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-TH Docket No.222117-2430 Amino-propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4- carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 1- (4-Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4- Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4- Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4- Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4- Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4- Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4- Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP; 1- (4-Thiomethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4- Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino- hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}- ethoxy)-propionyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)- pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl- pseudo-UTP; 1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo- UTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1- Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1- Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1- Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1- Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; 1- Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1- Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6- (2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4- thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino- pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP; 1-Methyl-6-butyl- pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6- dimethylamino-pseudo-UTP; 1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate- pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl- pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP; 1-Methyl-6-hydroxy-pseudo-UTP; 1- Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-TH Docket No.222117-2430 UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP; 1-Methyl-6-propyl- pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6-trifluoromethoxy-pseudo-UTP; 1- Methyl-6-trifluoromethyl-pseudo-UTP; 1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo- UTP; 1-Phenyl-pseudo-UTP; 1-Pivaloylpseudouridine TP; 1-Propargylpseudouridine TP; 1- Propyl-pseudo-UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1- Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2ƍ- anhydro-uridine TP; 2ƍ-bromo-deoxyuridine TP; 2ƍ-F-5-Methyl-2ƍ-deoxy-UTP; 2ƍ-OMe-5-Me-UTP; 2ƍ-OMe-pseudo-UTP; 2ƍ-a-Ethynyluridine TP; 2ƍ-a-Trifluoromethyluridine TP; 2ƍ-b-Ethynyluridine TP; 2ƍ-b-Trifluoromethyluridine TP; 2ƍ-Deoxy-2ƍ,2ƍ-difluorouridine TP; 2ƍ-Deoxy-2ƍ-a- mercaptouridine TP; 2ƍ-Deoxy-2ƍ-a-thiomethoxyuridine TP; 2ƍ-Deoxy-2ƍ-b-aminouridine TP; 2ƍ- Deoxy-2ƍ-b-azidouridine TP; 2ƍ-Deoxy-2ƍ-b-bromouridine TP; 2ƍ-Deoxy-2ƍ-b-chlorouridine TP; 2ƍ- Deoxy-2ƍ-b-fluorouridine TP; 2ƍ-Deoxy-2ƍ-b-iodouridine TP; 2ƍ-Deoxy-2ƍ-b-mercaptouridine TP; 2ƍ- Deoxy-2ƍ-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2ƍ-O-Methyl-5- (1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4ƍ-Azidouridine TP; 4ƍ-Carbocyclic uridine TP; 4ƍ- Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5- Dimethylaminouridine TP; 5ƍ-Homo-uridine TP; 5-iodo-2ƍ-fluoro-deoxyuridine TP; 5- Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4- Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6- Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6- Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate- pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6- Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl- pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6- Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6- Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)- ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}- ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)- ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-TH Docket No.222117-2430 pentanoic acid; Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid; Pseudo- UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4- demethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2- (oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)- naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2ƍ methyl, 2ƍamino, 2ƍazido, 2ƍfluro- cytidine; 2ƍ methyl, 2ƍamino, 2ƍazido, 2ƍfluro-adenine; 2ƍmethyl, 2ƍamino, 2ƍazido, 2ƍfluro-uridine; 2ƍ-amino-2ƍ-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2ƍ-azido-2ƍ-deoxyribose; 2ƍfluoro-2ƍ-deoxyribose; 2ƍ-fluoro-modified bases; 2ƍ-O-methyl-ribose; 2-oxo-7- aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)- 7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4- (methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6- (methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7- (aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2- (thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7- (aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2- (oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenoxazinl-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7- (guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7- (guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidiniumalkyl-hydroxy)- 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)- phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7- substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6- substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on- 3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-TH Docket No.222117-2430 amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5ƍ-TP; 2- thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2ƍ-OH-ara-adenosine TP; 2ƍ-OH-ara-cytidine TP; 2ƍ-OH-ara-uridine TP; 2ƍ-OH-ara-guanosine TP; 5-(2- carbomethoxyvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP.

[0037] In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0038] In some embodiments, the mRNA comprises at least one chemically modified nucleoside. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine (^), 2-thiouridine (s2U), 4ƍ-thiouridine, 5- methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2ƍ-O- methyl uridine, 1-methyl-pseudouridine (m1^), 1-ethyl-pseudouridine (e1^), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), Į-thio-guanosine, Į-thio-adenosine, 5-cyano uridine, 4ƍ-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl- adenosine (m6A), and 2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7- aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2- geranylthiouridine, 2-lysidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)- 2ƍ-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2- selenouridine, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2- thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5- carboxymethyl aminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5- methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwyosine, 7- aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethyl cytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine, methylated undermodified hydroxywybutosine, N4,N4,2ƍ-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-TH Docket No.222117-2430 thiouridine, geranylated 5-carboxymethyl aminomethyl-2-thiouridine, Qbase, preQ0base, preQlbase, and two or more combinations thereof. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, 1- methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0039] In some embodiments, the mRNA is a uracil-modified sequence comprising an ORF encoding one or more cancer epitope polypeptides, wherein the mRNA comprises a chemically modified nucleobase, e.g., 5-methoxyuracil. In certain aspects of the invention, when the 5-methoxyuracil base is connected to a ribose sugar, as it is in polynucleotides, the resulting modified nucleoside or nucleotide is referred to as 5-methoxyuridine. In some embodiments, uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% 5-methoxyuracil. In one embodiment, uracil in the polynucleotide is at least 95% 5-methoxyuracil. In another embodiment, uracil in the polynucleotide is 100% 5-methoxyuracil. DC-based therapies

[0143] Also disclosed is a dendritic cell (DC) vaccine involving DCs, e.g. autologous DCs, loaded with one or more antigenic peptides disclosed herein. The cells can be loaded with tumor antigen in a manner that allows the cells to present the antigen to the host immune system at an appropriate time. The cells can be genetically modified with mRNA encoding the disclosed antigens or pulsed with the antigen, e.g. near the time of administration. Alternatively, the cells can be transduced at any stage in the differentiation pathway with a recombinant gene that causes the disclosed antigen to be expressed in the end-stage dendritic cell. As an option, the antigen can be expressed under control of an inducible promoter. This allows the kinetics of antigen pulsing to be mimicked by combining with the compound that induces the promoter near the time of administration, thereby initiating antigen presentation.

[0144] Dendritic cells loaded with antigen can then be administered to a patient having a tumor in order to elicit an immune response (ideally cytotoxic CD8+ T lymphocytes with CD4+ help). The effect of early stage dendritic cells can be enhanced by treating the injection site with an adjuvant that promotes maturation, such as imiquimod or polyarginine. If necessary, reactivity against the histocompatibility type of the cells can be decreased by pretreating the patient with toleragenic dendritic cells made from the same cell line. The antigen-loadedTH Docket No.222117-2430 dendritic cells are then administered to the patient in a series that initiates an immunological or therapeutic response. Once initiated, the response can be maintained or boosted by further periodic administration of the loaded dendritic cells, or with the tumor antigen in another form (such as a peptide vaccine, or a viral or plasmid vector). Pharmaceutical composition

[0040] Also disclosed is a pharmaceutical composition comprising a disclosed molecule in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0041] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0042] Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additivesTH Docket No.222117-2430 may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0043] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0044] The disclosed composition may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate the immune response (e.g., immune responses mediated by CD8-positive T cells and helper-T (TH) cells to an antigen, and would thus be considered useful in the medicament of the present invention. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP- 870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL- 2, IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactid co-glycolid) [PLG]-based and dextran microparticles, talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Allison and Krummel, 1995). Also cytokines may be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T- lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).TH Docket No.222117-2430

[0045] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly it enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co- administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak. They also accelerate the immune response and enable the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments (Krieg, 2006). U.S. Pat. No.6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany) which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and / or TLR 9 may also be used.

[0046] Other examples for useful adjuvants include, but are not limited to chemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such as Poly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan without undue experimentation.TH Docket No.222117-2430

[0047] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivates, poly-(I:C) and derivates, RNA, sildenafil, and particulate formulations with PLG or virosomes.

[0048] In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.

[0049] In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinations thereof. Methods of Treatment

[0050] Also disclosed is a method for treating a cancer in a subject by administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition. Pharmaceutical compositions (i.e., the vaccine or immunogenic composition) comprising a peptide as herein described may be administered to an individual already suffering from cancer. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the tumor antigen and to cure or at least partially arrest symptoms and / or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 ^g to about 50,000 ^g of peptide for a 70 kg patient, followed by boosting dosages or from about 1.0 ^g to about 10,000 ^g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. It must be kept in mind that the peptide and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations, especially when the cancer hasTH Docket No.222117-2430 metastasized. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptide, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

[0051] The disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.

[0052] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0053] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical daily dosage of theTH Docket No.222117-2430 disclosed composition used alone might range from about 1 μg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

[0054] In some embodiments, the molecule is administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1^g to about 100 mg per kg of body weight, from about 1 ^g to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of molecule containing lenalidomide administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 ^g, 10 ^g, 100 ^g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.

[0055] The peptides as herein described may also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing the half-life of the peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic / immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng.9;467 (1980 ), USA U.S. Patent Nos.4,235,871 , 4501728 USA 4,501,728 , 4,837,028 , and 5,019,369.

[0056] Suitable vaccines or immunogenic compositions may also be in the form of DNA or RNA relating to neoantigenic peptides as described herein. For example, DNA or RNA encoding one or more neoantigenic peptides or proteins derived therefrom may be used as the vaccine, for example by direct injection to a subject. For example, DNA or RNA encoding at least 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 neoantigenic peptides or proteins derived therefrom.TH Docket No.222117-2430

[0057] A number of methods are conveniently used to deliver the nucleic acids to the patient. For instance, the nucleic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 1465-1468 (1990 ) as well as USAU.S. Patent Nos.5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Patent No.5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.

[0058] The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in 9618372WOAWO 96 / 18372; 9324640WOAWO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988 ); 5279833USARose U.S. Pat No.5,279,833 ; 9106309WOAWO 91 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987 ).

[0059] Delivery systems may optionally include cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, or any combination thereof. Cell penetrating peptides include TAT peptide, herpes simplex virus VP22, transportan, Antp. Liposomes may be used as a delivery system. Listeria vaccines or electroporation may also be used.

[0060] The one or more neoantigenic peptides may also be delivered via a bacterial or viral vector containing DNA or RNA sequences which encode one or more neoantigenic peptides. The DNA or RNA may be delivered as a vector itself or within attenuated bacteria virus or live attenuated virus, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptide of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No.4,722,848 ,. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991 )). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art from the description herein.

[0061] An appropriate mean of administering nucleic acids encoding the peptides as herein described involves the use of minigene constructs encoding multiple epitopes. To create a DNA sequence encoding the selected CTL epitopes (minigene) for expression in human cells,TH Docket No.222117-2430 the amino acid sequences of the epitopes are reverse translated. A human codon usage table is used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences are directly adjoined, creating a continuous polypeptide sequence. To optimize expression and / or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequence that could be reverse translated and included in the minigene sequence include: helper T lymphocyte, epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal. In addition, MHC presentation of CTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.

[0062] Vaccines or immunogenic compositions comprising peptides may be administered in combination with vaccines or immunogenic compositions comprising polynucleotide encoding the peptides. For example, administration of peptide vaccine and DNA vaccine may be alternated in a prime-boost protocol. For example, priming with a peptide immunogenic composition and boosting with a DNA immunogenic composition is contemplated, as is priming with a DNA immunogenic composition and boosting with a peptide immunogenic composition.

[0063] In some embodiments, the cancer may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus. For example, the tumors or cancers can be leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus,TH Docket No.222117-2430 ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above. Embodiments

[0064] Embodiment 1. A method for diagnosing a SLC2A11-MIF cancer in a subject, comprising assaying a sample from the subject for a chimeric RNA comprising a fusion or splicing of SLC2A11 and MIF genes or transcripts or for a chimeric protein comprising a fusion of SLC2A11 and MIF proteins.

[0065] Embodiment 2. The method of embodiment 1, comprising assaying a sample from the subject for a chimeric RNA encoding the amino acid sequence SEQ ID NO:30 or for a peptide having the amino acid sequence SEQ ID NO:30.

[0066] Embodiment 3. The method of embodiment 1 or 2, further comprising treating the subject with an antibody drug conjugate that selectively binds chimeric SLC2A11-MIF protein.

[0067] Embodiment 4. The method of embodiment 1 or 2, further comprising treating the subject with a vaccine comprising an antigenic fragment of CTPTPPPCSGRQECRKRRSSTRSSGLGAASCSRRLLVTSRCTWSRTSSWPSAAPASRARSA ACTASARSAARRTAPTASCCAACWPSACASARTGSTSTITT (SEQ ID NO:31).

[0068] Embodiment 5. An antigenic peptide comprising at least 4, 5, 6, 7 or 8 contiguous amino acids of the amino acid sequence SEQ ID NO:31, wherein the peptide is in the form of a pharmaceutically acceptable salt, wherein the peptide is acylated or pegylated, wherein the peptide further comprises heterologous amino acids, or any combination thereof.

[0069] Embodiment 6. The peptide of embodiment 5, wherein the peptide comprises at least 4, 5, 6, 7 or 8 contiguous amino acids of any one of SEQ ID NO: 1-21.

[0070] Embodiment 7. The peptide of embodiment 5 or 6, wherein the pharmaceutically acceptable salt is a chloride salt, acetate salt, or trifluoro-acetate salt.

[0071] Embodiment 8. The peptide of any one of embodiments 5 to 7, wherein the heterologous amino acids comprise an adjuvant peptide.

[0072] Embodiment 9. A vector comprising a polynucleotide encoding one or more of the peptides as defined in any one of embodiments 5 to 8 operably linked to a heterologous expression control sequence.

[0073] Embodiment 10. A population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides as defined in any one of claims 5-9 or transfected with the vector of embodiment 9TH Docket No.222117-2430

[0074] Embodiment 11. A colorectal cancer vaccine comprising one or more antigenic peptides as defined in any one of embodiments 5 to 8 in a physiologically acceptable buffer, carrier, or excipient.

[0075] Embodiment 12. The vaccine of embodiment 11, further comprising an adjuvant or immunostimulant.

[0076] Embodiment 13. The vaccine of embodiment 12, wherein the adjuvant is selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0077] Embodiment 14. An antibody that binds SLC2A11-MIF comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; wherein the CDR1 sequence of the VHdomain comprises the amino acid sequence SYGVH (SEQ ID NO:22); the CDR2 sequence of the VHdomain comprises the amino acid sequence LIWRGSTNYNAAFMS (SEQ ID NO:23); the CDR3 sequence of the VHdomain comprises the amino acid sequence NFYGDYDAMDY (SEQ ID NO:24); the CDR1 sequence of the Vldomain comprises the amino acid sequence KSTKSLLNSDGFTYLD (SEQ ID NO:25); the CDR2 sequence of the VLdomain comprises the amino acid sequence LVSNRFS (SEQ ID NO:26); and the CDR3 sequence of the VLdomain comprises the amino acid sequence FQSNYLPYT (SEQ ID NO:27).

[0078] Embodiment 15. The antibody of embodiment 14, wherein the VHdomain comprises the amino acid sequence QVQLKQSGPSLVQPSQSLSITCTVSGFSLTSYGVHWVRSPGKGLEWLGLIWGRGSTNYNAAF MSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKNFYGDYDAMDYWGQGTSVTVSS SEQ ID NO:28 and wherein the VLdomain comprises the amino acid sequence DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPHLLIYLVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPYTFGGGTKLEIK SEQ ID NO:29.

[0079] Embodiment 16. The antibody of embodiment 14 or 15, wherein the antibody is a recombinant antibody.

[0080] Embodiment 17. The antibody of embodiment 16, wherein the antibody is a single chain (scFv) antibody.

[0081] Embodiment 18. An isolated nucleic acid sequence encoding the recombinant antibody of any one of embodiments 14 to 18.TH Docket No.222117-2430

[0082] Embodiment 19. A vector comprising the isolated nucleic acid sequence of embodiment 18.

[0083] Embodiment 20. A cell comprising the vector of embodiment 19.

[0084] Embodiment 21. An antibody drug conjugate (ADC) comprising a chemotherapeutic agent conjugated to the antibody of any one of embodiments 14 to 17.

[0085] Embodiment 22. A chimeric antigen receptor (CAR) polypeptide, comprising a SLC2A11-MIF antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region; wherein the SLC2A11-MIF antigen-binding domain is a single-chain variable fragment (scFv) comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; wherein the CDR1 sequence of the VHdomain comprises the amino acid sequence SYGVH (SEQ ID NO:22); the CDR2 sequence of the VHdomain comprises the amino acid sequence LIWRGSTNYNAAFMS (SEQ ID NO:23); the CDR3 sequence of the VHdomain comprises the amino acid sequence NFYGDYDAMDY (SEQ ID NO:24); the CDR1 sequence of the VLdomain comprises the amino acid sequence KSTKSLLNSDGFTYLD (SEQ ID NO:25); the CDR2 sequence of the VLdomain comprises the amino acid sequence LVSNRFS (SEQ ID NO:26); and the CDR3 sequence of the VLdomain comprises the amino acid sequence FQSNYLPYT (SEQ ID NO:27).

[0086] Embodiment 23. The CAR polypeptide of embodiment 22, wherein the VHdomain comprises the amino acid sequence SEQ ID NO:28 and wherein the VLdomain comprises the amino acid sequence SEQ ID NO:29.

[0087] Embodiment 24. A CAR-T cell comprising the chimeric antigen receptor (CAR) polypeptide of embodiment 22 or 23.

[0088] Embodiment 25. The CAR-T cell of embodiment 24, wherein the cell is selected from the group consisting of an ĮȕT cell, ȖįT cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

[0089] Embodiment 26. A method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of the vaccine of any one of claims 11 to 13, the ADC of claim 21, or the CAR-T cell of embodiment 24 or 25.

[0090] Embodiment 27. The method of embodiment 26, further comprising administering to the subject a checkpoint inhibitor.TH Docket No.222117-2430

[0091] Embodiment 28. The method of embodiment 27, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

[0092] Embodiment 29. The method of any one of embodiments 26 to 28, wherein the cancer is a colorectal cancer.

[0093] Embodiment 30. The method of any one of embodiments 26 to 28, further comprising assaying a sample from the subject for a chimeric RNA comprising a fusion or splicing of SLC2A11 and MIF genes or transcripts or for a chimeric protein comprising a fusion of SLC2A11 and MIF proteins.

[0145] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES Example 1:

[0146] Using RNA-Sequencing data from The Cancer Genome Atlas (TCGA) database, a chimeric RNA SLC2A11-MIF was identified that is present in nearly 50% of CRC samples but absent in non-cancer colon tissue. This chimeric RNA results from cis-splicing between adjacent genes, producing a novel chimeric protein. This protein includes a splice variant of the 5’ gene, which alters the reading frame of the 3’ gene, making a completely novel peptide sequence which may be immunogenic. The protein is predicted to be a transmembrane protein with the novel peptide sequence on the extracellular surface, making it an ideal target for antibody therapeutics. Methods

[0147] An antibody was generated to the chimeric peptide sequence and have validated its use for immunoblotting, immunoprecipitation, and immunohistochemistry. NetMHCpan4.1 was used to predict binding of peptides from the novel protein to common HLA alleles. The immunogenicity of peptides was tested by presenting monocyte-derived dendritic cells with peptides and co-culturing peptide-loaded DCs with autologous T cells from healthy donors. Junction-specific siRNAs was used to knockdown SLC2A11-MIF in CRC cell lines. Results

[0148] The novel SLC2A11-MIF protein is expressed in patient CRC samples. Interestingly, the protein localizes in a speckled nuclear pattern in a subset of patients with adenocarcinoma but is localized to the membrane and cytoplasm of adenomas. Co-culture withTH Docket No.222117-2430 peptides from SLC2A11-MIF increases autologous T cell proliferation and IFN-Ȗ secretion. Overexpression of this chimeric RNA resulted in increased cell proliferation, while chimeric junction-specific siRNA-mediated knockdown led to a reduction in cell proliferation.

[0149] FIGs.1A to 1D show that the SLC2A11-MIF chimeric RNA is present in colorectal cancer cell lines using primers flanking the junction sequence. FIG.1A shows SLC2A11-MIF chimeric RNA is expressed in numerous CRC cell lines compared to the normal colon epithelial cell line CCD 841 CoN. SLC2A11-MIF was expressed highest in HCT116 cells. FIG.1B shows that the chimeric RNA is expressed in cancer compared to normal samples, however FIG.1C shows that neither parental genes shows a change in expression from normal to cancer cells. FIG.1D shows there was significant expression of this chimeric RNA in various other types of cancer cell lines, including bladder, breast, prostate and liver cancer cell lines.

[0150] FIG.2 shows primers were designed at the first exon of three SLC2A11 isoforms (designated A,B,C) to identify the isoform of SLC2A11 responsible for the chimeric RNA. The slc2a11 gene has three isoforms, varying primarily by the first exon. The chimeric junction begins at exon 2 of the MIF gene, so the reverse primer was in exon 3 of MIF. PCR was performed with HCT116 cells and then sequenced the bands. The sequence of the band in the first lane contained exons 1-5, and exon 8 of SLC2A11 and exons e2-3 of MIF. The overall trend was that exon 6 and 7 were spliced out of the chimeric RNA, joining exon 5 to exon 8 of SLC2A11 with exons 2 and 3 of MIF.

[0151] FIG.3 shows the chimeric RNA was predicted to be an in-frame chimeric protein. However, the isoform predominately present in CRC is missing exon 6 and 7, but contains exon 8 which alters the reading frame of exon 8 of the SLC2A11 gene and 2 and 3 of the MIF gene, creating a completely novel peptide sequence that has not previously been identified. MIF exon 1 contains a stop codon that would prevent this isoform from normally occurring. The illustrated SLC2A11-MIF protein sequence is SEQ ID NO:30.

[0152] FIGs.4A and 4B and Table 1 show SLC2A11-MIF is a marker for CRC disease.TH Docket No.222117-2430

[0153] SLC2A11 is a transmembrane sugar transport molecule, responsible for the transport of glucose and fructose across the cell membrane and the MIF (macrophage migration inhibitory factor) gene encodes a pro-inflammatory cytokine that regulates innate immunity. SLC2A11-MIF was predicted to be a transmembrane protein, with the novel peptide sequence on the external cell surface using the PHOBIUS protein localization prediction software. This is important because it allows for the possibility of developing antibody drug conjugates or antibody therapies against the protein in cancer, because the novel open reading frame would not be present in normal tissue.

[0154] Because the reading frame is altered and creates a novel open reading frame, current antibodies cannot be used for either gene to test for presence of the chimeric protein. Therefore an antibody was made to the novel peptide sequence after the frame shift.

[0155] 293T cells were transfected with GFP- and Flag-tagged SLC2A11-MIF construct and probed with the anti-SLC2A11-MIF (anti-SM) antibody. The anti-SM antibody recognized a protein near the expected size of 30kDa, and the flag antibody produced a band at a similar size (FIG.5A).

[0156] In addition, 293T cells expressing a myc-tagged SLC2A11-MIF protein were immunoprecipitated with the myc tag and immunoblotted for the anti-SM antibody (FIG.5B). The reciprocal IP was also done where the SM antibody was used to immunoprecipitate the SM antibody which was then immunoblotted with myc (FIG.5B). In both cases, there were consistent bands pulled down at the expected size.

[0157] GFP-tagged SLC2A11-MIF was also overexpressed in 293T cells, which were then evaluated by immunohistochemistry. There was similar staining patterns between GFP and anti-SM antibodies (FIG.5C).

[0158] The anti-SM antibody was used to show that SLC2A11-MIF is expressed in CRC patient samples. SLC2A11-MIF intensity and localization differ with progression of colon cancer. For example, SLC2A11-MIF intensity was higher in adenomas less than 2 cm and less intense in adenomas greater than 2 cm. SLC2A11-MIF was also detected in lymph node metastases. SLC2A11-MIF was shown by antibody to be present in ovarian serous carcinoma, prostateTH Docket No.222117-2430 adenocarcinoma, esophagus adenocarcinoma, colorectal adenocarcinoma, bladder urothelial carcinoma, and lung squamous cell carcinoma.

[0159] An siRNA was produced that could knockdown SLC2A11-MIF in 4 out of 6 CRC cell lines (FIGs.6A-6J).

[0160] FIG.7 illustrates a process for isolation and generation of human DCs stimulated with SLC2A11-MIF peptides, and FIG.8 illustrates the use of antigen-loaded DCs to activate autologous T cells from healthy donors.

[0161] NetMHCpan-4.1 prediction server was to identify peptides that are predicted to bind to common HLA alleles on CD8 T Cells.9mer peptides were predicted to bind to HLA-A1, A2, A3, B7, B8, B44 (Table 2).

[0162] FIGs.9A and 9B show naïve (FIG.9A) and memory (FIG.9B) CD8+T cell response (IFN-Ȗ) using different peptide pools and different T cell donors.TH Docket No.222117-2430

[0163] FIGs.10A and 10B show naïve (FIG.10A) and memory (FIG.10B) CD8+T cell response (IFN-Ȗ) using different peptide pools and different T cell donors.

[0164] FIGs.11A and 11B show quantitative (FIG.11A) and relative (FIG.11B) T cell response (IFN-Ȗ) in naïve CD8+T cells 15 days post-stimulation with peptides PP-1 to PP-13 in two T cell donors.

[0165] FIGs.12A and 12B show quantitative (FIG.11A) and relative (FIG.11B) T cell response (IFN-Ȗ) in naïve CD8+T cells 72 hours post-stimulation with peptides PP-1, PP3-PP-9, and PP-14 in two T cell donors. Conclusions

[0166] Chimeric RNA SLC2A11-MIF is expressed in a large proportion of colorectal cancers. This protein-coding RNA contains a splicing event which shifts the reading frame, making a completely novel peptide sequence which is not expressed in normal tissue. Example 2:

[0167] RNA-seq data, patient survival, disease stage, and MSI status in colorectal cancer in the TCGA was used to create a Kaplan Meier curve of Overall Survival for patients with SLC2A11-MIF present. Those expressing SM fusion RNA have worse overall survival (Fig. 13). Median SLC2A11 and MIF WT expression was not predictive of overall survival (Fig.13). SM is detected as early as stage I disease, and increased from stage I to stage III, but is detected in all 4 disease stages (Fig.13). SM is present in both MSI and MSS patient tumors, and does not correlate with MSI status.

[0168] RNA was collected from both tumor and tumor-adjacent normal tissue from 175 colorectal cancer patients. Figure 14A shows descriptive characteristics of these samples. Figure 14B represents the percent positive cases in tumor-adjacent normal and tumor tissue, where we found SM is expressed in greater percentage of cases of tumor compared to the normal tissue. Figures 14C-14E represent the relative expression of SM, SLC2A11 WT, and MIF WT in tumor and adjacent normal tissue. Data is relative to HCT116 cell line, to account for data variability across experimental plates. Figures 14F and 14G show that SLC2A11 WT and MIF WT expression do not correlative with SM chimeric RNA expression. Figures 14H-14M show that SM is expressed across all disease characteristics, with expression in both early and late-stage disease and in both early-onset and late-onset disease. It also shows no SM expression correlation with sex, race, MSI status, or disease stage.

[0169] Figures 15A to 15R show data of SM, SLC2A11 WT, and MIF WT expression in both tumor-adjacent normal and tumor tissue for all characteristics.TH Docket No.222117-2430

[0170] Figures 16A and 16B are western blots of HCT116 cells overexpressing and knocking down SM. This shows that SM antibody is on-target, and that expression is endogenously detected in colorectal cancer. Figure 15C shows IHC with SM antibody of a TMA containing progressing colorectal cancer disease. SM protein is not present in non-cancer normal tissue or tumor-adjacent normal tissue, but is present throughout disease progression as early as in small adenomas, through invasive adenocarcinomas, and in distant site metastasis. Figure 16D shows that SM antibody is specific for SM protein, with IHC staining being lost in HCT116 CRC cells transfected with a chimeric junction specific siRNA to SM.

[0171] Figure 17A is a pan-cancer TMA that shows IHC staining of SM is observed throughout other cancer types, supporting SM expression in ovarian serous carcinoma, prostate adenocarcinoma, esophageal adenocarcinoma, bladder urothelial carcinoma, and lung squamous cell carcinoma. Figures 17B-17G are data from TCGA showing that SM is detected in various other cancers, but is detected at higher frequency in tumor compared to tumor- adjacent normal tissue RNA seq and that SM is not detected in any normal physiological tissue from non-cancer patients in GTEx data.

[0172] Figures 18A to 18J are IFN-Ȗ secretion ELISA results from donor naïve and memory CD8+T cells after expansion for 14d and restimulation. Dendritic cells were generated by differentiation from monocytes and were pulsed with peptides to CEF (CMV, EBV, Influenza) or SM peptide pools that were predicted to bind to each donor’s HLA type. Unbound peptides were washed out of culture, and dendritic cells were cultured with autologous CD8+ Naïve and Memory T cells which were sorted by magnetic bead selection. Donor 3 had a response to SM peptides in the memory T cell population and Donor 9 had a positive response to SM peptides in the Naïve T cell population.

[0173] Figures 19A 19H are IFN-J secretion ELISA results from donor naïve and memory CD8+T cells after expansion for 21d or 28d and restimulation. Dendritic cells were generated by differentiation from monocytes and were pulsed with peptides to CEF (CMV, EBV, Influenza) or SM peptides that were predicted to bind to each donor’s HLA type. Unbound peptides were washed out of culture, and dendritic cells were cultured with autologous CD8+ Naïve and Memory T cells which were sorted by magnetic bead selection. Donor 3 did not have a response to SM peptides after 21-28d of culture. Donor 9 had a positive response to single SM peptides in the Naïve and memory T cell population after 28d of culture.

[0174] Figure 20 shows Real Time Cell Analysis (RTCA) results of SW620 CRC cells measured by xCELLigence instrument. SW620 cells that were cultured with HLA-matched (B7) donor 3 T cells that had been expanded with SM peptides were able to kill SW620 cells, while TTH Docket No.222117-2430 cells that were unpulsed did not kill SW620 cells. SW620 cells express SM endogenously and this supports and antigen-specific T cell response.

[0175] Figures 21A to 21F are IFN-J secretion ELISA results from Donor CD3+T cells after expansion for 14d and restimulation. Dendritic cells were generated by differentiation from monocytes and were pulsed with peptides to CEF (CMV, EBV, Influenza) or class I and class II SM peptide pools that were predicted to bind to each donor’s class I HLA type. Unbound peptides were washed out of culture, and dendritic cells were cultured with autologous CD3+ T cells which were sorted by magnetic bead selection. Donor 1 had a response to both class I and class II SM peptide pools. Donor 6 and Donor 9 had a response to class I SM peptide pools. Overall, 3 / 6 donors had a response to SM peptides.

[0176] Figure 22 contains IFN-Ȗ ELISpot results obtained from Donor CD8+Naïve T cells after activation at day 0 with dendritic cells pulsed with peptides to CEF (CMV, EBV, Influenza) or class I SM peptide pools that were predicted to bind to each donor’s class I HLA type, and restimulation at day 14 during ELISpot. Both donor 6 and 9 had positive responses to SM peptides.

[0177] Figure 23A contains IFN-Ȗ ELISpot results obtained from Donor 6 CD8+Naïve T cells. T cells were activated at day 0 with dendritic cells pulsed with peptides to CEF (CMV, EBV, Influenza) or class I SM peptide pools that were predicted to bind to each donor’s class I HLA type. At day 21, T cells were restimulated with single peptides. Donor 6 had a response to Peptide 9, Peptide 10, Peptide 11, and Peptide 14. Figure 23B shows quantification of the positive spots observed in Figure 23A.

[0178] Figure 24A contains IFN-Ȗ ELISpot results obtained from Donor 9 CD8+Naïve T cells. T cells were activated at day 0 with dendritic cells pulsed with peptides to CEF (CMV, EBV, Influenza) or class I SM peptide pools that were predicted to bind to each donor’s class I HLA type. At day 21, T cells were restimulated with single peptides. Donor 9 had a visible positive response to Peptide 5, Peptide 7, Peptide 9, Peptide 12, and Peptide 16. Figure 24B shows quantification of the positive spots observed in Figure 24A. A statistically significant response was observed to Peptide 12.

[0179] Figures 25A to 25C are IFN-Ȗ ELISpot results obtained from Donor PBMCs. All PBMCs were stimulated at day 0 with class I SM peptides. After 14d of expansion, antigen- specific T cells were plated without restimulation (media) or were restimulated with GAG negative control, or SM peptide pools. All three donors had a positive to SM peptides compared to Media and GAG negative controls. Donor 3 had a positive response to SM-Pool-1 and SM-TH Docket No.222117-2430 Pool-17. Donor 6 had a positive response to SM-Pool-1. Donor 9 had a positive response to SM-Pool-1 and SM-Pool-17.

[0180] Figures 26A to 26C are IFN-Ȗ ELISpot results obtained from Donor PBMCs. All PBMCs were stimulated at day 0 with class II SM peptides. After 14d of expansion, antigen- specific T cells were plated without restimulation (media) or were restimulated with GAG negative control, or SM peptide pool. Donor 6 had a positive response to SM class II peptides compared to media and GAG negative controls.

[0181] Figure 27A is a growth curve of HCT116 measured by RTCA. Hct116 cells were effectively killed by Donor 3 HLA-matched T cells expanded following activation with SM peptides. Figure 27B is a growth curve of SW480 CRC cells measured by RTCA. SW480 cells were effectively killed by Donor 6 HLA-matched T cells expanded following activation with SM class II peptides, but not class I peptides.

[0182] Figure 28 shows IFN-J ELISpot results obtained from Donor 9 PBMCs. All PBMCs were stimulated at day 0 with class I SM peptides. After 16d of expansion, antigen- specific T cells were plated without restimulation (media) or were restimulated with GAG negative control, or SM individual peptides. Donor 9 had a positive response to Peptide 4, Peptide 9, and Peptide 21. Figure 28D shows quantification of ELIspots. Figure 28E is a growth curve of HCT116 measured by RTCA. Hct116 cells were effectively killed by Donor 9 HLA- matched T cells expanded following activation with both class I and class II SM peptides.

[0183] Figure 29A shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class I SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) or were restimulated with GAG negative control or SM peptides. Figure 29B shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class II SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) or were restimulated with GAG negative control or SM peptides. Colorectal cancer patient VCC- 009 had a positive response to class II peptides.

[0184] Figure 30A shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class I SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) or were restimulated with GAG negative control or SM peptides. Figure 30B shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class II SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) orTH Docket No.222117-2430 were restimulated with GAG negative control or SM peptides. Colorectal cancer patient VCC- 011, VCC-014, and VCC-020 had a positive response to class II peptides.

[0185] Figure 31A shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class I SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) or were restimulated with GAG negative control or SM peptides. Figure 31B shows IFN-Ȗ ELISpot results obtained from colorectal cancer patient PBMCs. All PBMCs were stimulated at day 0 with class II SM peptides. After 14d of expansion, antigen-specific T cells were plated without restimulation (media) or were restimulated with GAG negative control or SM peptides.TH Docket No.222117-2430TH Docket No.222117-2430TH Docket No.222117-2430

[0186] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0187] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

TH Docket No.222117-2430 CLAIMS 1. A method for diagnosing a SLC2A11-MIF cancer in a subject, comprising assaying a sample from the subject for a chimeric RNA comprising a fusion or splicing of SLC2A11 and MIF genes or transcripts or for a chimeric protein comprising a fusion of SLC2A11 and MIF proteins.

2. The method of claim 1, comprising assaying a sample from the subject for a chimeric RNA encoding the amino acid sequence SEQ ID NO:30 or for a peptide having the amino acid sequence SEQ ID NO:

30.

3. The method of claim 1, further comprising treating the subject with an antibody drug conjugate that selectively binds chimeric SLC2A11-MIF protein.

4. The method of claim 1, further comprising treating the subject with a vaccine comprising an antigenic fragment of CTPTPPPCSGRQECRKRRSSTRSSGLGAASCSRRLLVTSRCTWSRTSSWPSAAPASRARSA ACTASARSAARRTAPTASCCAACWPSACASARTGSTSTITT (SEQ ID NO:31).

5. An antigenic peptide comprising at least 4, 5, 6, 7 or 8 contiguous amino acids of the amino acid sequence SEQ ID NO:31, wherein the peptide is in the form of a pharmaceutically acceptable salt, wherein the peptide is acylated or pegylated, wherein the peptide further comprises heterologous amino acids, or any combination thereof.

6. The peptide of claim 5, wherein the peptide comprises at least 4, 5, 6, 7 or 8 contiguous amino acids of any one of SEQ ID NO: 1-21.

7. The peptide of claim 5, wherein the pharmaceutically acceptable salt is a chloride salt, acetate salt, or trifluoro-acetate salt.

8. The peptide of claim 5, wherein the heterologous amino acids comprise an adjuvant peptide.

9. A vector comprising a polynucleotide encoding one or more of the peptides as defined in any one of claims 5 to 8 operably linked to a heterologous expression control sequence.

10. A population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides as defined in claim 5.

11. A colorectal cancer vaccine comprising one or more antigenic peptides as defined in claim 5 in a physiologically acceptable buffer, carrier, or excipient.

12. The vaccine of claim 11, further comprising an adjuvant or immunostimulant.

13. The vaccine of claim 12, wherein the adjuvant is selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C)TH Docket No.222117-2430 and derivatives, RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

14. An antibody that binds SLC2A11-MIF comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; wherein the CDR1 sequence of the VHdomain comprises the amino acid sequence SYGVH (SEQ ID NO:22); the CDR2 sequence of the VHdomain comprises the amino acid sequence LIWRGSTNYNAAFMS (SEQ ID NO:23); the CDR3 sequence of the VHdomain comprises the amino acid sequence NFYGDYDAMDY (SEQ ID NO:24); the CDR1 sequence of the Vldomain comprises the amino acid sequence KSTKSLLNSDGFTYLD (SEQ ID NO:25); the CDR2 sequence of the VLdomain comprises the amino acid sequence LVSNRFS (SEQ ID NO:26); and the CDR3 sequence of the VLdomain comprises the amino acid sequence FQSNYLPYT (SEQ ID NO:27).

15. The antibody of claim 14, wherein the VHdomain comprises the amino acid sequence QVQLKQSGPSLVQPSQSLSITCTVSGFSLTSYGVHWVRSPGKGLEWLGLIWGRGSTNYNAAF MSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKNFYGDYDAMDYWGQGTSVTVSS SEQ ID NO:28 and wherein the VLdomain comprises the amino acid sequence DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPHLLIYLVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPYTFGGGTKLEIK SEQ ID NO:

29.

16. The antibody of claim 14, wherein the antibody is a recombinant antibody.

17. The antibody of claim 16, wherein the antibody is a single chain (scFv) antibody.

18. An isolated nucleic acid sequence encoding the recombinant antibody of claim 14.

19. A vector comprising the isolated nucleic acid sequence of claim 18.

20. A cell comprising the vector of claim 19.

21. An antibody drug conjugate (ADC) comprising a chemotherapeutic agent conjugated to the antibody of claim 14.

22. A chimeric antigen receptor (CAR) polypeptide, comprising a SLC2A11-MIF antigen- binding domain, a transmembrane domain, an intracellular signaling domain, and a co- stimulatory signaling region; wherein the SLC2A11-MIF antigen-binding domain is a single- chain variable fragment (scFv) comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; wherein the CDR1 sequence of the VHdomain comprises the amino acid sequence SYGVH (SEQ ID NO:22); the CDR2 sequence of the VHdomain comprises the amino acid sequence LIWRGSTNYNAAFMS (SEQ ID NO:23); the CDR3 sequence of the VHdomain comprises the amino acid sequence NFYGDYDAMDY (SEQ ID NO:24); the CDR1 sequence ofTH Docket No.222117-2430 the VLdomain comprises the amino acid sequence KSTKSLLNSDGFTYLD (SEQ ID NO:25); the CDR2 sequence of the VLdomain comprises the amino acid sequence LVSNRFS (SEQ ID NO:26); and the CDR3 sequence of the VLdomain comprises the amino acid sequence FQSNYLPYT (SEQ ID NO:27).

23. The CAR polypeptide of claim 22, wherein the VHdomain comprises the amino acid sequence SEQ ID NO:28 and wherein the VLdomain comprises the amino acid sequence SEQ ID NO:

29.

24. A CAR-T cell comprising the chimeric antigen receptor (CAR) polypeptide of claim 22.

25. The CAR-T cell of claim 24, wherein the cell is selected from the group consisting of an ĮȕT cell, ȖįT cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

26. A method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of the vaccine of claim 11.

27. The method of claim 26, further comprising administering to the subject a checkpoint inhibitor.

28. The method of claim 27, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

29. The method of claim 26, wherein the cancer is a colorectal cancer.

30. The method of claim 26, further comprising assaying a sample from the subject for a chimeric RNA comprising a fusion or splicing of SLC2A11 and MIF genes or transcripts or for a chimeric protein comprising a fusion of SLC2A11 and MIF proteins.

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