Novel TCRS and neoantigens in SRSF2 and ZRSR2 mutated cancers

Abstract

Compositions, kits, and methods that implement neoantigens specific to cancer cells having recurrent mutations in SRSF2 or ZRSR2, wherein the mutations are shared across patients with those mutational genotypes, as well as cognate T cell receptors (TCRs) that specifically recognize those neoantigens.

Description

NOVEL TCRS AND NEO ANTIGENS IN SRSF2 AND ZRSR2 MUTATED CANCERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 575586, filed April 05, 2024, the disclosure of which is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 1896-P96WO_Sequence-Listing.xml. The XML file is 549,924 bytes; was created on April 04, 2025; and is being submitted electronically via Patent Center with the filing of the specification.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with Government support under HL 128239, and CA251138, and CA283364 awarded by National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0004] Following five decades with few approved therapies for acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS), the last five years have brought progress with multiple new FDA-approved therapies. However, despite these advances, the 5-year survival rate for most AML patients is less than 20% and there are few effective therapies for high-risk MDS.

[0005] A challenge to treatment of MDS and AML has been the lack of effective immunotherapies. Despite prior setbacks, the potential for T cell-based immunotherapeutic approaches for treating myeloid leukemias is clear given the curative potential of allogeneic hematopoietic cell transplantation (allo-HCT) for AML and MDS patients, arising from the graft-versus-leukemia (GVL) activity of donor cells.

[0006] To date, chimeric antigen receptor (CAR) T cell approaches for AML have nearly entirely relied on antigens which are shared across myeloid neoplasms and normal hematopoietic stem cells, risking unacceptable on-target but off-tumor toxicities. Given these challenges, identifying additional sources of leukemia-specific neoantigens shared across patients and applicable to multiple HLA alleles could represent a majortherapeutics advance. To this end, cancer-associated mutations in genes encoding RNA splicing factors, which create stereotypical neomorphic changes in splicing consistently across patients were explored to yield novel HLA class-I (HLA-I) displayed peptides.

[0007] A series of oncogenic mutations in RNA splicing factors (in the genes SRSF2, SF3B1. U2AF1, and ZRSR2) are found in up to 50 to 70% of patients with myelodysplastic syndromes (MDS), 50% of chronic myelomonocytic leukemia (CMML), and 20% of acute myeloid leukemia (AML) cases. SRSF2, SF3B1, and U2AF1 are components of the major spliceosome complex and are mainly affected by hotspot, change- of-function mutations. These mutations skew usage of alternative RNA splicing events observed in healthy cells but also create novel RNA isoforms which are reproducibly observed across patients with the same mutations. As such, aberrant mRNA splicing events have the potential to generate mis-splicing-derived neoantigens that are “public,” or shared or in common among patients with the same splicing factor mutation.

[0008] A prior study identified putative aberrant RNA splicing-derived neoantigens in 5F3B7-mutant uveal melanoma; however, validation of such antigens as immunotherapeutic targets through TCR gene transfer was not performed. Moreover, no studies have evaluated mis-splicing-derived neoantigens in myeloid malignancies, where mutations affecting splicing factors occur at the highest frequency.

[0009] There is a long-felt and unmet need for the identification of mis-splicing- derived neoantigens in myeloid leukemias, including with mutations in the RNA splicing factor genes SRSF2 and ZRSR2. These two factors have been focused on in the present disclosure given their high mutation frequencies, associations with adverse outcomes, and unique mechanistic roles in RNA splicing. SRSF2 mutations in particular are especially common across myeloid neoplasms, as they occur in 30-50% patients with CMML, 17- 25% of patients with MDS, 18-20% of patients with AML over the age of 60, and 6-8% of younger patients with AML (< 60 yr).

[0010] For example, in SRSF2, change-of-function mutations are concentrated at the proline residue at position 95 (P95) of the SRSF2 protein and alter the recognition of specific exonic splicing enhancer motifs, leading to differential cassette exon usage. ZRSR2, unlike others, is a component of the minor spliceosome complex involved in the splicing of highly conserved minor introns, and its loss-of-function mutations cause widespread retention of minor introns in many genes.

[0011] Mutations in the RNA splicing factors induce aberrant splicing events that are highly recurrent across patients and cancer types, and some of these events have been demonstrated to drive disease pathogenesis. Without wishing to be bound by any particular theory, one can hypothesize that cancer cells bearing these spliceosomal mutations generate immunogenic tumor-specific and / or tumor-associated antigens that are derived from misspliced mRNA isoforms. By identifying and functionally characterizing these tumor antigens, novel immune interventions in MDS, CMML, and AML patient populations, such as T-cell receptor (TCR) therapeutics targeting these tumor antigens, can be implemented. Given the recurrent nature of mis-splicing events associated with spliceosomal mutations, mis-splicing-derived tumor antigens are likely to be shared across patients, and the resulting TCR therapeutics would be broadly applicable to cancers with relevant genotypes.

[0012] Spliceosomal gene mutations are also common in clonal hematopoiesis (CH), which is characterized by the premalignant clonal expansion of mutated hematopoietic stem and progenitor cells. These mutations, especially those in SRSF2 and U2AF1, confer a higher risk of developing overt MDS, CMML, and AML. By identifying mis-splicing-derived tumor antigens, safe and effective immune-based cancer prevention strategies for CH patient populations, such as vaccines based on these tumor antigens, can be implemented.

[0013] Accordingly, there is a need for improved compositions and methods for treatment and prevention of cancers. The present disclosure addresses these and other long felt needs unmet in the art.SUMMARY

[0014] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0015] In an aspect, the disclosure provides a neoantigen comprising a polypeptide sequence associated with an oncogenic mutation in one or more RNA splicing factors.

[0016] In embodiments, the one or more RNA splicing factors comprises SRSF2, SF3B1, U2AF1, ZRSR2, or any combination thereof.

[0017] In embodiments, the polypeptide sequence is selected from the group consisting of: SEQ ID NOs: l-76.

[0018] In embodiments, the one or more RNA splicing factors comprises SRSF2.

[0019] In embodiments, the oncogenic mutation comprises a P95 mutation of SRSF2 as a change-of-function mutation.

[0020] In embodiments, the P95 mutation of SRSF2 comprises a P95H mutation, a deletion of the contiguous amino acid sequence from P95 to R102, or a P95-R insertion o SRSF2.

[0021] In embodiments, the neoantigen comprises the polypeptide sequence of SEQ ID NO:6, SEQ ID NO:31, or SEQ ID NO:32.

[0022] In embodiments, the one or more RNA splicing factors comprises ZRSR2.

[0023] In embodiments, the oncogenic mutation comprises a loss-of-function mutation of ZRSR2.

[0024] In embodiments, the neoantigen comprises the polypeptide sequence of SEQ ID NO: 7, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or any combination thereof.

[0025] In embodiments, the neoantigen comprises a polypeptide sequence associated with a mis-spliced CLK3 RNA transcript.

[0026] In another aspect, the disclosure provides a vaccine comprising a neoantigen.

[0027] In embodiments, the vaccine further comprises a pharmaceutically acceptable carrier.

[0028] In another aspect, the disclosure provides a method for vaccinating a subject against one or more cancers, the method comprising administering an effective amount of the vaccine to the subject.

[0029] In another aspect, the disclosure provides a method for treating a subject suspected to develop one or more cancers, the method comprising administering an effective amount of the vaccine to the subject.

[0030] In another aspect, the disclosure provides a use of a neoantigen in the preparation of a medicament or a vaccine, wherein the medicament or the vaccine is for administration to a subject for treatment or prevention of one or more cancers.

[0031] In embodiments, the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.

[0032] In another aspect, the disclosure provides a T cell receptor (TCR) or TCR- like protein comprising an antigen binding domain configured to bind to the neoantigen.

[0033] In another aspect, the disclosure provides a T cell receptor (TCR) or TCR- like protein configured to bind an antigen that comprises a polypeptide sequence associated with a mis-spliced CLK3 RNA transcript.

[0034] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:79; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:80; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:81; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 83; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:84; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:85.

[0035] In embodiments, the beta chain comprises: a beta variable polypeptide sequence comprising SEQ ID NO:78.

[0036] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence comprising SEQ ID NO:82.

[0037] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:86.

[0038] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:87.

[0039] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:77.

[0040] In embodiments, the protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:90; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:91; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:92; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:94; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:95; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:96.

[0041] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:89.

[0042] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 93.

[0043] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:97.

[0044] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 98.

[0045] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:88.

[0046] In embodiments, the protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 101; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 102; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 103; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 105; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 106; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 107.

[0047] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 100.

[0048] In embodiments, the TCR or TCR-like protein comprises: an alpha variable polypeptide sequence of SEQ ID NO: 104.

[0049] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 108.

[0050] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 109.

[0051] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:99.

[0052] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 112; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 113; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 114; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 116; an alpha variable CDR2 polypeptide sequencecomprising SEQ ID NO: 117; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 118.

[0053] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 111.

[0054] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 115.

[0055] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 119.

[0056] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 120.

[0057] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 110.

[0058] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 123; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 124; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 125; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 127; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 128; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 129.

[0059] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 122.

[0060] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 126.

[0061] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 130.

[0062] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 131.

[0063] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 121.

[0064] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 134; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 135; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 136; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 138; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 139; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 140.

[0065] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 133.

[0066] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 137.

[0067] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 141.

[0068] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 142.

[0069] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 132.

[0070] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 145; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 146; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 147; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 149; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 150; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 151.

[0071] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 144.

[0072] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 148.

[0073] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 152.

[0074] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 153.

[0075] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 143.

[0076] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 156; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 157; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 158; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 160; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 161; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 162.

[0077] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 155.

[0078] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 159.

[0079] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 163.

[0080] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 164.

[0081] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 154.

[0082] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 167; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 168; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 169; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 171; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 172; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 173.

[0083] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 166.

[0084] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 170.

[0085] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 174.

[0086] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 175.

[0087] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 165.

[0088] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 178; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 179; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 180; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 182; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 183; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 184.

[0089] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 177.

[0090] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 181.

[0091] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 185.

[0092] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 186.

[0093] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 186.

[0094] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 189; a beta variable CDR2 polypeptide sequencecomprising SEQ ID NO: 190; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 191; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 193; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 194; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 195.

[0095] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 188.

[0096] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 192.

[0097] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 196.

[0098] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 197.

[0099] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 187.

[0100] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:200; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:201; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:202; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:204; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:205; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:206.

[0101] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 199.

[0102] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:203.

[0103] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:207.

[0104] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:208.

[0105] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 198.

[0106] In embodiments, the TCR or TCR-like protein comprises a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:211; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:212; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:213; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:215; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:216; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:217.

[0107] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:210.

[0108] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:214.

[0109] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:218.

[0110] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:219.[OHl] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:209.

[0112] In another aspect, the disclosure provides a T cell receptor (TCR) or TCR- like protein configured to bind an antigen that comprises a polypeptide sequence associated with a mis-spliced RHOT2 RNA transcript, the TCR or TCR-like protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:482; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:483; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:484; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:486; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:487; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:488.

[0113] In embodiments, the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:481.

[0114] In embodiments, the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:485.

[0115] In embodiments, the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:489.

[0116] In embodiments, the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:490.

[0117] In embodiments, the TCR or TCR-like protein comprises a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:480.

[0118] In embodiments, the TCR or TCR-like protein is a TCR-like antibody or a TCR-like bispecific antibody.

[0119] In another aspect, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the TCR or TCR-like protein.

[0120] In another aspect, the disclosure provides a nucleic acid vector or nucleic acid vaccine comprising the polynucleotide sequence.

[0121] In another aspect, the disclosure provides a composition comprising the TCR or TCR-like protein, the nucleic acid, the nucleic acid vector or nucleic acid vaccine, or any combination thereof.

[0122] In another aspect, the disclosure provides an immune cell comprising the nucleic acid.

[0123] In embodiments, the immune cell is a T cell.

[0124] In embodiments, the T cell is transgenic and comprises a polynucleotide sequence encoding the TCR or TCR-like protein of any one of Embodiments 19-104 stably integrated within a genome of the T cell.

[0125] In another aspect, the disclosure provides a composition for immune therapy by administration of the composition to a subject for treatment of one or more cancers in the subject, the composition comprising the immune cell.

[0126] In embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0127] In another aspect, the disclosure provides a method for treatment of one or more cancers in a subject in need thereof, the method comprising administering, to thesubject, an effective amount of: the TCR or TCR-like protein; the nucleic acid; the nucleic acid vector or nucleic acid vaccine; the immune cell; the composition; or any combination thereof.

[0128] In another aspect, the disclosure provides a use of an agent in the preparation of a medicament, wherein the medicament is for administration to a subject for treatment or prevention of one or more cancers and the agent comprises: the TCR or TCR- like protein; the nucleic acid; the nucleic acid vector or nucleic acid vaccine; the immune cell; the composition; or any combination thereof.

[0129] In embodiments of the method or the use, the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.DESCRIPTION OF THE DRAWINGS

[0130] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0131] FIGs 1A-1E show prediction of RNA mis-splicing-derived neoantigens and confirmation of HLA-I binding in splicing factor mutant myeloid leukemias, according to aspects of the disclosure. FIG. 1A shows a heatmap illustrating isoform PSI (percent spliced-in) values for splicing events differentially spliced between SRSF2 P95-mutant (n=54) and splicing factor wild-type myeloid malignancies (n=325) across the Beat AML cohort, Body Map 2.0 panel of healthy tissues (n=14), and healthy bone marrow (n=448). Each row represents a patient transcriptome, and columns represent isoforms. Groups of isoforms exhibiting specificity for 57?5F2-mutant cells are below heatmap. FIG. IB shows results as in FIG. 1A, but for ZF57?2-mutant malignancies (n=33). FIG. 1C shows, at top: relative frequency of CCNG and GGNG (wherein N=any nucleotide) motifs in cassette exons promoted versus repressed by SRSF2 mutations from FIG. 1A. Bottom: identical analysis but for the Leucegene AML cohort. Shading, 95% confidence interval by bootstrapping. Schematic illustrates a metagene containing the differentially spliced cassette exon. Horizontal axis, genomic coordinates defined with respect to 5' and 3' splice sites, where 0 is the splice site. Vertical axis, relative frequency of the motifs over genomic loci containing cassette exons promoted versus repressed by SRSF2 mutations (log scale). FIG. ID shows quantification of differential splicing of minor (U12-type) introns fromFIG. IB. Each point corresponds to a single intron and illustrates percentage of mRNA in which the intron is spliced out. Dots show introns with significantly increased retention in Z A C-mutant versus WT cells, defined as an absolute change in retention of >10% or absolute log fold-change of >2 with associated P < 0.05. FIG. IE shows median fluorescence intensity (MFI) values of HLA-ABC surface expression from T2 HLA-A2 stabilization experiments. Shaded area corresponds to peptides with significant HLA-I binding. Mean+SEM. P-values, unpaired Student’s t test. Upper left text, HLA-A*02:01 MART-1 peptide positive controls (“ELA”: ELAGIGILTV (SEQ ID NO:507), “EAA”: EAAGIGILTV (SEQ ID NO:508)). Negative control 10-mer peptides are predicted nonbinders to HLA-A*02:01.

[0132] FIGs 2A-2J shows immunogenicity of candidate SRSF2 mutant-induced neoantigens and identification of neoantigen-reactive T cells, according to aspects of the disclosure. FIG. 2A shows, at left y-axis: Fold-change in percent of IFNy+and / or TNFa+CD8+T cells upon candidate peptide re-stimulation (normalized to DMSO), tested across n=14 healthy donor PBMCs. FIG. 2A shows, at right y-axis: Fold-change in IFNy ELISpot spot forming units upon restimulation with the test peptide (versus DMSO). Each dot represents mean value across n=3 technical replicates for a single healthy donor. Text indicates HLA-A*02:01 MART-1 positive control peptides. Negative control peptide is a predicted nonbinder to HLA-A*02:01. P-values calculated for each candidate peptide (versus negl0mer_4 peptide) using Wilcoxon signed-rank test (p.adj *<0.05). Upper and lower asterisks: peptides with statistical significance from ICS and IFNy ELISpot, respectively. FIG. 2B shows box plot quantifications of PSI values of the RHOT2 intron retention event in the BeatAML, Leucegene, and AML TCGA cohorts. Normal tissue controls from Body Map 2.0. Horizontal line, median; box, interquartile range; whiskers, minimum and maximum values within 1.5X of the interquartile range; dots outside whiskers, outliers. P-values, one-sided Mann-Whitney U test. FIG. 2C shows MS2 spectra of RHOT2 peptide #5 eluted from HLA-A*02:01+ SRSF2P95H / WTK562 cells. Peaks represent b ions (left) and y ions (right). FIG. 2D shows ICS FACS plots from FIG. 2A demonstrating IFNy+and / or TNFa+CD8+T cells upon restimulation with RHOT2 peptide #5 or DMSO. FIG. 2E shows IFNy ELISpot images from PBMCs upon restimulation with RHOT2 peptide #5 or DMSO. FIG. 2F shows FACS plots of RHOT2 peptide #5-primed CD8+T cells from two donors, stained with dual color RHOT2 peptide #5 -HL A- A* 02:01 dextramers. FIG. 2G shows results as in FIG. 2B but for the exon inclusion event incl6orf70. FIG. 2H shows results as in FIG. 2C but for cl6orf70 peptide. FIG. 21 shows results as in FIG. 2D but for cl6orf70 peptide. FIG. 2 J shows results as in FIG. 2F but for cl6orf70 peptide.

[0133] FIG. 3A-3G show discovery and validation of SRSF2 mutant-induced CLK3 neoantigen, according to aspects of the disclosure. FIG. 3A shows IFNy ELISpot images of two healthy donor PBMCs in vitro primed and re-stimulated with CLK3 peptide or DMSO. FIG. 3B shows fold-change in percent of IFNy+and / or TNFa+CD8+T cells restimulated with CLK3 peptide or DMSO, across six healthy donors. n=3 technical replicates. Mean+SEM. P-values, unpaired Student’s t test (p.adj **<0.01, ***<0.001, ****<0.0001). FIG. 3C shows ICS FACS plots in two donors from FIG. 3B. FIG. 3D shows CLK3 exon 4 skipping RNA-seq coverage plots in SRSF2P95-mutant (n=34) and splicing factor wild-type (n=357) patients from the Leucegene cohort and normal bone marrow samples (n=4). FIG. 3E shows quantification of PSI of CLK3 exon 4 skipping event in AML TCGA, BeatAML, and Boultwood MDS datasets. Normal tissue controls from Body Map 2.0. Horizontal line, median; box, interquartile range; whiskers, minimum and maximum values within 1.5X of the interquartile range; dots outside whiskers, outliers. P- values, one-sided Mann-Whitney U test. FIG. 3F shows RT-PCR of CLK3 exon 4 skipping event in isogenic K562 cells and SRSF2P95H-mutant KO52 cells treated with SMG1 inhibitor. FIG. 3G shows MS2 spectra of CLK3 peptide eluted from HLA-A*02:01+SRSF2P95H / WTknockin K562 cells (top) and synthetic peptide (bottom). Peaks represent b ions (e.g., b6, b5) and y ions (e.g., y2, y3). FIG. 3H shows FACS plots of CLK3 peptide- primed CD8+T cells from two donors, stained with dual color CLK3 peptide-HLA- A* 02:01 dextramers.

[0134] FIGs 4A-4H show discovery and characterization of TCRs recognizing the CLK3 neoantigen, according to aspects of the disclosure. FIG. 4A shows a schema of an assay to isolate CLK3 neoantigen-reactive TCRs from HLA-A*02:01+healthy donors. FIG. 4B shows proportion of total cells with the indicated TCR clonotype in dextramer+(right bars) versus dextramer (left bars) cells. Arrows, TCR clonotypes enriched in dextramer+cells. FIG. 4C shows FACS plots of CLK3 peptide-HLA-A*02:01 dextramer staining of CD8+T cells transduced with the isolated TCRs. Percentages denote dextramer- double-positive cells. FIG. 4D shows a schema of an experiment testing antigen reactivity of isolated TCRs. T cells transduced with each TCR were co-cultured with K562 cells loaded with CLK3 peptide or electroporated with CLK3 mis-spliced mRNA. FIG. 4Eshows, at top: IFNy and TNFa ICS FACS plots of CD8+T cells transduced with CLK3 TCR clonotype 3 and exposed to K562 cells loaded with increasing concentration of CLK3 peptide. FIG. 4E shows, at bottom: Frequency of IFNy+and / or TNFa+CD8+T cells expressing four distinct CLK3 TCRs or CMV TCR. FIG. 4F shows half-maximal effective concentration (EC50) of the CLK3 peptide for each TCR based on TNFa and IFNy ICS from FIG. 4E. FIG. 4G shows IFNy and TNFa ICS FACS plots of CD8+or CD4+T cells transduced with CLK3 TCR clonotype 2 or 3 and exposed to K562 cells electroporated with CLK3 wild-type or mis-spliced mRNA. FIG. 4H shows percent lysis of HLA- A*02:01+K562 cells (left) or HLA-A*03:01+MV4;11 cells (right) expressing CLK3 mRNA and exposed to CD8+T cells transduced with CLK3 TCR clonotype 3. Mean+SEM.

[0135] FIGs 5A-5I show anti-tumor efficacy of CLK3 neoantigen-reactive TCR- T cells against SRSF2 mutant AML cells, according to aspects of the disclosure. FIG. 5A shows a normalized number of HLA-A*02:01+eGFP+KO52 cells over time in co-culture with CD8+T cells expressing indicated CLK3 TCRs or CMV TCR. E:T ratio of 4: 1. Mean+SEM. P-values, one-way ANOVA (p.adj ****<0.0001). FIG. 5B shows a 24-hour co-culture of CD8+ CLK3 TCR.9' or TCR11'T cells with KO52 cells at 1 : 1 E:T ratio. TCR- T cell activation assessed by CD69 and 4-1BB expression in CD8+mTCR+(mouse TCR) cells, and AML cell killing by activated caspase 3 / 7 and 7-AAD expression in CD8- cells. FIG. 5C shows percent lysis of firefly luciferase-transduced leukemia cells co-cultured with CD8+CLK3 TCR9-T cells across E:T ratios. AML cell lines used include SRSF2 mutant KO52 cells with or without HLA-A2 and HLA-A2+ SRSF2 wild-type THP-1 and MV4;11 cells. Mean+SEM. P-values, unpaired Student’s t test (p.adj **<0.01, ***<0.001, ****<0.0001). FIG. 5D shows CD69 and 4-1BB expression in CLK3 or CMV TCR-T cells (labeled with CellTrace dye), upon co-culture with BM MNCs or PBMCs from HLA- A*02:01+SRSF2 mutant AML patients at 1 : 1 E:T ratio for 24 hours. Primary cells from HLA-A*02:01+SRSF2 wild-type patients or normal donors were negative controls. FIG. 5E shows frequency of CD69+and 4-1BB+ TCR-T cells upon co-culture with HLA- A*02:01+patient samples. Mean+SEM. P-values, unpaired Student’s t test (p.adj ****<0.0001). FIG. 5F shows identification of peptide recognition motif for CLK3 TCR9 (top) and TCR11 (bottom) using alanine scanning. Frequency of TNFa+and / or IFNy+TCR-T cells upon 1 : 1 E:T co-culture with peptide-loaded K562 cells (1 pg / ml). % maximum response relative to unsubstituted CLK3 peptide (y-axis). n=3 technical replicates. FIG. 5G shows a schema of in vivo experiments to test therapeutic efficacy ofTCR-T cells expressing CLK3 TCR9 or TCR11. FIG. 5H shows BLI images of animals treated with CLK3 TCR11-T cells, or either PBS or CMV TCR-T cells. FIG. 51 shows box- and-whisker plots of region of interest (ROI) BLI signals in mice from FIG. 5G. Bar, median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. P-value, unpaired Student’s t-test (p.adj *<0.05, **<0.01, ***<0.001, ****<0.0001). See also FIG. 8.

[0136] FIGs 6A-6I show characterization of neoantigen-reactive CD8+T cells in splicing factor mutant leukemia patients, according to aspects of the disclosure. FIG. 6A shows a schema of an experiment to evaluate antigen-reactive CD8+T cells in leukemia patients using 50 distinct DNA-barcoded p / HLA-I-dextramers. FIG. 6B shows a UMAP of CD8+T cells from five HLA-A*02:01+SRSF2 mutant patients. Clusters denoted by shades and labelled with inferred cell states. FIG. 6C shows results as in FIG. 6B but indicating fraction of total cells within each cluster for neoantigen-directed CD8+T cells, viral- antigen directed CD8+T cells, and CD8+T cells without dextramer barcode specificity. Asterisk, NF-kB signaling low effector memory CD8+T cell cluster populated by neoantigen-directed CD8+T cells. FIG. 6D shows a fraction of CD8+T cells in each cell state cluster from FIG. 6B based on antigen reactivity. FIG. 6E shows NFKBIA and JUN expression on UMAP from FIG. 6B and reduced expression in NF-kB signaling in effector memory CD8+T cell cluster 3. FIG. 6F shows box-and-whisker plots of scores for gene signatures for antigen-directed CD8+T cell types indicated in FIG. 6C. P-values, t-test and Bonferroni correction (p.adjFIG. 6G shows a heatmap of selected differentially expressed genes within effector memory CD8+T cell clusters 0, 1, 3, and 5 from FIG. 6B. FIG. 6H shows, at the left: same UMAP as in FIG. 6B but instead, T cells are marked on basis of TCR clone frequency defined through scTCR- Seq. FIG. 6H shows, at the right: proportional representation of clone sizes within each cluster and type of antigen-directed CD8+T cell. FIG. 61 shows a gene set enrichment analysis of differentially expressed genes in hyperexpanded TCR clonotypes in viral- antigen versus neoantigen-directed CD8+T cells from FIG. 6H.

[0137] FIGs 7A-7L show neoantigen-reactive T cells and TCRs pre and post HLA-matched allogeneic transplantation in splicing factor mutant leukemia patients, according to aspects of the disclosure. FIG. 7A shows UMAP of CD8+T cells pre- and post- transplant for Patient 2 (see Table S4). FIG. 7B shows results as in FIG. 7A but indicates a fraction of total cells within each cluster for pre- and post-transplant samples.FIG. 7C shows proportional representation of TCR clone sizes within pre- versus posttransplant antigen-directed CD8+T cells. FIG. 7D shows results as in FIG. 7A but indicating a CDR3P TCR clonotype specific to RH0T2 peptide #5. This TCR clonotype largely maps to post-transplant effector memory CD8+T cells. FIG. 7E shows proportion of dextramer barcodes amongst cells with the TCR clonotype indicated in FIG. 7D. FIG. 7F shows IFNy and TNFa ICS FACS plots of CD8+and CD4+T cells transduced with RH0T2 TCR from FIG. 7D and exposed to K562 cells pulsed with increasing concentrations of RH0T2 peptide. FIG. 7G shows percent maximum T cell activation across peptide concentrations from FIG. 7F. EC50concentrations below graph. FIG. 7H shows IFNy and TNFa ICS FACS plots of CD8+or CD4+T cells transduced with RH0T2 TCR and exposed to K562 cells electroporated with RH0T2 wild-type or mis-spliced mRNA. FIG. 71 shows percent lysis of HLA-A*02:01 K562 cells expressing RH0T2 mRNA isoforms and exposed to CD8+RH0T2 TCR-T cells across E:T ratios. Mean+SEM. P values, unpaired Student’s t test (p. adj *<0.05, **<0.01, ***<0.001, ****<0.0001). FIG. 7J shows 24-hour co-culture of RHOT2 TCR-T cells with KO52 cells with or without HLA-A*02:01 at 1 : 1 E:T ratio. TCR-T cell activation assessed by CD69 and 4-1BB expression in CD8+mTCR+cells, and AML cell killing by activated caspase 3 / 7 and 7- AAD expression in CD8" cells. FIG. 7K shows time course detection of HLA-A*02:01+KO52 cell apoptosis upon co-culture with CD8+RHOT2 TCR-T cells at 1 : 1 E:T ratio, using caspase 3 / 7-based eGFP probe. Mean+SEM. P-values, one-way ANOVA (p.adj *<0.05). FIG. 7L shows normalized number of HLA-A*02:01+eGFP+KO52 cells in coculture with CD8+T cells expressing RHOT2 or CMV TCR over time. E:T ratio of 4: 1. Mean+SEM. P-values, ordinary one-way ANOVA (p.adj ****<0.0001).

[0138] FIGs 8A-8I show anti-tumor efficacy CLK3 neoantigen-reactive TCR-T cells against SRSF2 mutant AML cells, related to FIGs 5A-5I, according to aspects of the disclosure. FIG. 8A shows FACS histograms of HLA-A2 surface expression in SRSF2 mutant KO52 and SRSF2 wild-type THP-1 (endogenously expresses HLA-A*02:01) and MV4;11 (transduced with HLA-A*02:01 cDNA). FIG. 8B shows time course detection of KO52 cell apoptosis using the caspase 3 / 7-based eGFP probe via live cell imaging. KO52 parental cells were co-cultured with CD8+T cells transduced with CLK3 neoantigen- reactive TCR clonotypes 2, 3, 9, or 11 (or CMV-reactive TCR) at 1 : 1 E:T ratio over time. Mean+SEM. P-values, ordinary one-way ANOVA (p.adj *<0.05, **<0.01, ****<0.0001). FIG. 8C shows percent lysis of HLA-A2+SRSF2 wild-type leukemia cells loaded with orwithout 1 pg / ml CLK3 peptide and co-cultured with CD8+CLK3 TCR9-T cells across E:T ratios. Mean+SEM. P-values, unpaired Student’s t test (p.adj *<0.05, ***<0.001, ****<0.0001). FIG. 8D shows FACS plots of CD69 and 4-1BB surface expression in CLK3 or CMV TCR-T cells, upon co-culture with BM MNCs or PBMCs from HLA- A*02:01+SRSF2 wild-type or mutant AML patients or a normal donor. FACS plots are gated on CellTrace+, live, mouse TCR+T cells. FIG. 8E shows identification of peptide recognition motif for CLK3 TCR3 (top) and TCR2 (bottom) using alanine scanning. Frequency of TNFa+and / or IFNy+TCR-T cells upon 1 : 1 E:T co-culture with peptide- loaded K562 cells (1 pg / ml). % maximum response relative to unsubstituted CLK3 peptide (y-axis). n = 3 technical replicates. FIG. 8F shows bar graph enumerating the frequency of TNFa+and / or IFNy+CLK3 TCR2-T cells upon co-culture with HLA-A*02:01+K562 cells loaded with potential off-target peptides derived from the proteins indicated on the x-axis. DMSO and native CLK3 peptide used as controls. FIG. 8G shows representative BLI images of animals treated with CLK3 TCR9-T cells or either of the controls as described in FIG. 5G. FIG. 8H shows weight of tumor-bearing mice after two doses of CD8+CLK3 TCR9-T cells or either of the controls. FIG. 81 shows Kaplan-Meier curves of the mice treated with CLK3 TCR11-T cells or either PBS (left) or CMV TCR-T cells (right). P- values, log-rank test (p.adj *<0.05).DETAILED DESCRIPTION

[0139] The present disclosure provides novel neoantigens that are specific to cancer cells with recurrent mutations in SRSF2 or ZRSR2 and shared across patients with those mutational genotypes. The present disclosure also provides novel cognate T cell receptors (TCRs) that specifically recognize the neoantigens. The identification of the neoantigens, and their cognate TCRs, enables the preparation of new and useful cancer vaccines based on the neoantigens, as well as the preparation of TCR-based therapeutic compositions that can be used to treat cancer.

[0140] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings and example below. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure can be practiced without these specific details and in other instances, well-known methods, procedures, and elements have not been described in detail so as to not unnecessarily obscure aspects of the embodiments.

[0141] Unless otherwise defined in the present disclosure, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the disclosure belongs. The following references provide one of ordinary skill in the art with a general definition of many terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rded. 2006); Cambridge Dictionary of Science and Technology (Walker ed. 1990); The Glossary of Genetics: Classical and Molecular, 5thed., Rieger et al. ed. 1991, Springer-Verlag, and Hale and Marham, The Harper Collins Dictionary of Biology (1991); Molecular Cloning: a Laboratory Manual 3rdedition, Sambrook and Russell, ed. Cold Spring Harbor Laboratory Press 2001; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “oligonucleotide Synthesis” (M.J. Gait, ed. 1984); “Animal Cell Culture” (RI Freshney, ed. 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (FM Ausubel et al. eds. 1987, and periodic updates); ““PCR: The Polymerase Chain Reaction”, (Mullis et al., eds. 1994); “A Practical Guide to Molecular Cloning” (P. Bernard., 1988); and “Phage Display: A Laboratory Manual” (Barbas et al. eds. 2001). The contents of these references and other references containing standard protocols, widely known to and relied upon by those skilled in the art, including manufacturers’ instructions are hereby incorporated by reference as part of the presently disclosed subject matter.

[0142] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is described, measured, or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold, or within 2-fold, of a value. In the case of a stated percentage, such as “85%”, the term “about” means within 3, 4, or 5 percentage points above and within 3, 4, or 5 percentage points below the stated percentage; for example, “about 85%” means “80-90%”, “81-89%”, or “82-88%”, as the case may be, unless the context clearly indicates otherwise.

[0143] As used herein, the term “neoantigen”, “neoepitope,” or “neopeptide” refers to a tumor-specific antigen that arises from one or more tumor-specific mutation(s),which alters the amino acid sequence of genome encoded proteins and makes it distinct from the corresponding wild-type, parental protein, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence.

[0144] As used herein, the term “mutation” refers to permanent change in the DNA sequence that makes up a gene. In certain embodiments, mutations range in size from a single DNA building block (DNA base) to a large segment of a chromosome. In certain embodiments, mutations can include missense mutations, frameshift mutations, duplications, insertions, nonsense mutations, deletions and repeat expansions. In certain embodiments, a missense mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene. In certain embodiments, a nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. In certain embodiments, an insertion changes the number of DNA bases in a gene by adding a piece of DNA. In certain embodiments, a deletion changes the number of DNA bases by removing a piece of DNA. In certain embodiments, small deletions can remove one or a few base pairs within a gene, while larger deletions can remove an entire gene or several neighboring genes. In certain embodiments, a duplication consists of a piece of DNA that is abnormally copied one or more times. In certain embodiments, frameshift mutations occur when the addition or loss of DNA bases changes a gene’s reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. In certain embodiments, a frameshift mutation shifts the grouping of these bases and changes the code for amino acids. In certain embodiments, insertions, deletions, and duplications can all be frameshift mutations. In certain embodiments, a repeat expansion is another type of mutation. In certain embodiments, nucleotide repeats are short DNA sequences that are repeated a number of times in a row. For example, a trinucleotide repeat is made up of 3-base-pair sequences, and a tetranucleotide repeat is made up of 4-base-pair sequences. In certain embodiments, a repeat expansion is a mutation that increases the number of times that the short DNA sequence is repeated. A mutation can also include a splice variant. Post-translational modifications specific to a tumor cell can also include a proteasome-generated spliced antigen.

[0145] As used herein, the term “tumor neoantigen” is a neoantigen present in a subject’s tumor cell or tissue but not in the subject’s corresponding normal cell or tissue.

[0146] As used herein, the terms “antibody” and “antibodies” refer to antigenbinding proteins of the immune system. As used herein, the term “antibody” includes whole, full-length antibodies having an antigen-binding region, and any fragment thereof in which the “antigen-binding portion” or “antigen-binding region” is retained, or single chains, for example, single chain variable fragment (scFv), thereof. The term “antibody” means not only intact antibody molecules but also fragments of antibody molecules that retain immunogen binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab’)2, and Fab. F(ab’)2, and Fab fragments that lack the Fe fragment of intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding of an intact antibody (Wahl et al., J. NucL Med. 24:316-325 (1983). In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further sub-divided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0147] The term “antigen-binding portion”, “antigen-binding fragment”, or “antigen binding region” of an antibody, as used herein, refers to that region or portion of an antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins. It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding portions encompassed within the term “antibody fragments” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et aL, Nature 341 :544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR).

[0148] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: :VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide- encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston etal. (Proc. Natl. Acad. Set. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513; 5,132,405; and 4,956,778; and U.S. Patent Publication No. 2005 / 0196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al. Hyrbidoma (Larchmt) 2008 27(6):455-451; Peter et al., J. Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J. ImunoL 2009 183(4):2277- 2285; Giomarelli et al., Thromb. Haemost. 2007 97(6):955-963; Fife et al., J. Clin. Invest. 2006 116(8):2252-2261; Brocks et al., Immunotechnology 1997 3(3): 173-184; Mooscaner etal., Ther. Immunol. 1995 2(10):31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J. Bio. Chem. 2003 25278(38):36740-36747; Xie et al., Nat. Biotech. 1997 15(8):768-771 ; Ledbetter et al., Crit. Rev. Immunol. 1997 17(5- 6):427-455; Ho et al., Bio. Chim. Biophys. Acta 2003 1638(3) :257-266).

[0149] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0150] As used herein, “F(ab’)2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab’) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab’)2” fragment can be split into two individual Fab’ fragments.

[0151] As used herein, the term “antigen-binding protein” refers to a protein or polypeptide that comprises an antigen-binding region or antigen-binding portion, that is, has a strong affinity to another molecule to which it binds. Antigen-binding proteins encompass antibodies, chimeric antigen receptors (CARs) and fusion proteins.

[0152] Suitable antibody fragments for practicing some embodiments of the present disclosure include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementaritydetermining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.

[0153] As used herein, the terms “complementarity-determining region” or “CDR” are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR Hl or Hl; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3). Examples of such CDR sequences are provided, including according to the Example below.

[0154] The identity of the amino acid residues in a particular antibody that make up a variable region CDR can be determined using one or more suitable methods, such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia etal., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular’s AbM antibody modeling software (now Accelrys®, see, Martin et al., Proc. Natl. Acad. Set. USA 86:9268, 1989, and the world wide web for various databases providing CDR sequence information, available complex crystal structures as defined bythe contact definition (see MacCallum et aL, J. Mol. Biol. 262:732-745, 1996), the “conformational definition” (see, e.g., Makabe et al. , J. Biol. Chem. 15 283: 1156-1166, 2008) and IMGT (Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and 1g superfamily V-like domains. Dev. Comp. Immunol. 27: 55-77, 2003).

[0155] As used herein, the “variable regions” and “CDRs” can refer to variable regions and CDRs defined by any one or more suitable approaches, including combinations of approaches. Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:

[0156] (I) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;

[0157] (ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule;

[0158] (iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond;

[0159] (iv) Fab, a fragment of an antibody molecule containing a monovalent antigen binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;

[0160] (v) Fab’, a fragment of an antibody molecule containing a monovalent antigen binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);

[0161] (vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (z.e., a dimer of Fab’ fragments held together by two disulfide bonds); and

[0162] (vii) Single domain antibodies or nanobodies are composed of single VH or VL domains which exhibit sufficient affinity to the antigen.

[0163] Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are envisioned (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0164] Antibody fragments according to some embodiments in the present disclosure can be prepared by proteolytic hydrolysis of the antibody or by expression in, for example. E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab’)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab’ monovalent fragments.

[0165] Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab’ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter (Biochem. J. 73: 119-126, 1959). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques can also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0166] Fv fragments comprise an association of VH and VL chains. This association can be noncovalent, as described in Inbar el al. (Proc. Natl Acad. Sci. USA 69:2659-2662, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond, cross-linked by chemicals such as glutaraldehyde, and the like. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These singlechain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow and Filpula (Methods2:97-105, 1991; Bird et aL, Science 242:423-426, 1988; Pack et al., Bio / Technology 11 : 1271-1277, 1993; and U.S. Pat. No. 4,946,778, which are hereby incorporated by reference in their entirety).

[0167] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry (Methods 2:106-110, 1991).

[0168] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of 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 which residues form a complementary determining region (CDR) of the recipient 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 can also comprise residues which 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 the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally will also 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, 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992).

[0169] Methods for humanizing non-human antibodies are envisioned. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which 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. Humanization can be essentially performed following suitable methods (See for example, 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.

[0170] Accordingly, such humanized antibodies are chimeric antibodies (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, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in, for example, rodent antibodies.

[0171] Human antibodies can also be produced using various suitable techniques, including, for example, phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 1991; Marks etal. J. Mol. Biol. 222:581, 1991). The techniques are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al. J. Immunol. 147(1): 86- 95, 1991). Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. Bio / Technology 10:779-783, 1992; Lonberg etal. Nature 368: 856-859 (1994); Morrison Nature 368 812-813, 1994; Fishwild et al. Nat. Biotechnol. 14:845-851, 1996; Neuberger Nat. Biotechnol. 14: 826, 1996; and Lonberg and Huszar Intern. Rev. Immunol. 13:65-93, 1995.

[0172] In an embodiment in which the antibody is a full-length antibody, the heavy and light chains of an antibody of the disclosure can be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, or two, complete light chains) or can include an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (“scFv”)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGj, IgG2, IgG3, IgG4, IgM, IgAj, IgA2, IgD, and IgE. In some embodiments, the immunoglobulin isotype is selected from IgGj, IgG2, IgG3, and IgG4, more particularly, IgGj (e.g., human IgGj) or IgG4(e.g., human IgG4). The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.

[0173] Bispecific configurations of antibodies are also contemplated herein. A bispecific monoclonal antibody (MoAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen. According to a specific embodiment the bispecific MoAb is engineered to simultaneously bind to a cytotoxic cell e.g., using a receptor like CD3) and a target like a tumor cell to be destroyed (further described hereinbelow).

[0174] The methods of the present disclosure also include methods of determining a neoantigen peptide which is predicted to bind to an MHC molecule expressed by the subject. In particular, the methods can comprise the step of determining and selecting a neoantigen peptide which is predicted to bind strongly to an MHC molecule expressed by the subject. The exact definition of ‘binding strongly’ will depend on the method used to predict the MHC binding interaction; however, in all cases the neoantigen peptide selected will be predicted to be capable of binding to, and being presented in the context of, an MHC molecule expressed by the subject.

[0175] The binding affinity to a neoantigen peptide may be below 500 nM. “High affinity” can mean 0 to 50 nM binding affinity. In other embodiments the neoantigen peptide may bind the MHC molecule with an intermediate affinity of 50 to 150 nM binding affinity, or low affinity of 150 to 500 nM binding affinity.

[0176] In certain embodiments, the neoantigen peptide can be predicted to bind to the MHC molecule with a high affinity whilst a corresponding wild-type peptide (e.g., an equivalent peptide derived from the same region of the corresponding wild-type polypeptide) is predicted to bind to the same MHC molecule with low affinity.

[0177] As used herein, the phrase “major histocompatibility complex (MHC)” refers to a complex of antigens encoded by a group of loci, which are collectively termed H-2 in mice and HL A in humans. Two principal classes of MHC antigens, class I and class II, each comprise a set of cell surface glycoproteins which play a role in determining tissue type and transplant compatibility. In transplantation reactions, cytotoxic T cells (CTLs) respond mainly against foreign class I glycoproteins, while helper T cells respond mainly against foreign class II glycoproteins. According to certain embodiments disclosed herein, the MHC is a human MHC. According to certain embodiments disclosed herein the MHC is a class I MHC, and specifically is HLA-A*02:01.

[0178] As used herein, the term “HLA-restricted antigen” refers to a peptide capable of specifically binding an antigen-binding groove of an MHC. Such an antigen iscommonly referred to in the art as being “restricted” by such an MHC. A typical antigen, such as a pathogen-derived antigen, tumor antigen or autoantigen, is typically generated in a human cell by intracellular processing of a larger polypeptide such as derived from the pathogen or the tumor. The antigen generally has a characteristic dimension and / or chemical composition, for example, a characteristic amino acid length and set of anchor residues, respectively, in the case of a peptide antigen enabling it to specifically bind the antigen-binding groove of a particular MHC haplotype so as to form an MHC / antigen complex therewith having an antigen presenting portion capable of specifically binding a variable region of a cognate TCR.

[0179] For HLA-A2, ALA-A3, HLA-A*6801, HLA-B7 and HLA-B27, for example, the anchoring positions are P2 and P9. For HLA-B*08 the anchor positions are P5 and P9. For HLA-C*14:02 the anchor positions are P2 and P3.

[0180] HLA restricted peptide antigens can be from a tumor antigen (e.g., tumor specific antigen or a tumor associated antigen). In some instances, the peptide has an MHC- restricted tumor neoantigen structure.

[0181] HLA Restricted neoantigen peptides of the present disclosure include, for example, the following:Table 1. Neoantigen Peptides.Table 2, Summary of Reactivity of Neoantigen Peptides.

[0182] As used herein, the term “neoantigen-based vaccine” includes a vaccine construct based on one or more neoantigens, e.g., a plurality of neoantigens, and / or one or more polynucleic acids encoding one or more neoantigens. As a non-limiting example, a vaccine of the disclosure can comprise one or more proteins (e.g., neoantigen(s)) and / or one or more polynucleic acids (e.g., DNA and / or RNA encoding a neoantigen). A neoantigen-based vaccine can be formulated as a vaccine composition and can comprise one or more neoantigens and / or one or more polynucleic acids encoding one or more neoantigens, in combination with one or more formulation components such as a carrier or diluent, a salt, a buffer, a lipid, a detergent, or the like, or any combination thereof, for example, that serves to stabilize and / or increase or maintain bioactivity of the neoantigenbased vaccine. A neoantigen-based vaccine can be administered to a subject or patient for prevention, management, treatment, and / or curing of one or more cancers or cancer types including but not necessarily limited to those cancers or cancer types as set forth in the present disclosure. Non-limiting examples of routes of administration of a neoantigenbased vaccine to a subject or patient include intraperitoneal injection, intramuscular injection, intravenous injection, infusion, or the like, as would be recognized by a person skilled in the art upon review of the present disclosure.

[0183] The present disclosure also includes methods of providing an immune cell population which targets a neoantigen from a tumor. In some instances, the immune cell population is or comprises a T cell population. In some instances, the T cell population can comprise CD8+T cells, CD4+T cells or CD8+T cells and CD4+T cells.

[0184] The method can comprise providing at least a first and a second immune cell population, wherein the first immune cell population targets a first neoantigen generated by a first mutation and the second immune cell population targets a second neoantigen generated by a second mutation. The method can comprise providing two or more neoantigens made by two or more different splicing factor mutations. The method can comprise providing two or more neoantigens made by one splicing factor mutation. The method can comprise providing one neoantigen made by one or more splicing factor mutations.

[0185] The present disclosure also provides an immune cell composition which comprises a neoantigen specific immune cell or a population of immune cells as described herein. The neoantigen specific immune cell can express a chimeric antigen receptor (CAR). As above, in some instances the immune cell or immune cell population cancomprise a T cell or T cell population. In some instances, the neoantigen specific T cell population can express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) which specifically binds a neoantigen or neoantigen peptide, ie., a peptide derived from the neoantigen, as described herein.

[0186] Methods for generating TCRs are envisioned. Methods include, e.g., the isolation of TCR genes that encode TCRs from patient or normal donors. Methods of introducing such TCR genes into T cells are envisioned.

[0187] TCRs bind to neoantigen and HLA-restricted neoantigen peptides disclosed herein. In some instances, the TCRs specifically bind to an epitope comprised within at least one of the HLA-restricted neoantigen peptides of Table 1. In some instances, the TCR specifically binds the CLK3 mis-splicing derived peptide created by mutant SRSF2 and the mis-spliced neoantigen from the transcript encoding EZH2. As set forth herein, the TCR of the present disclosure preferably recognizes the neoantigen or neoantigen peptide when bound by MHC, and more specifically HLA-A*02.

[0188] The term “epitope” in general refers to a site on an antigen, typically a (poly-) peptide, which a binding domain recognizes. The term “binding domain” in its broadest sense refers to an “antigen binding site”, z.e., characterizes a domain of a molecule which binds / interacts with a specific epitope on an antigenic target. An antigenic target can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes depending on the size, conformation, and type of antigen. The term “epitope” in general encompasses linear epitopes and conformational epitopes. Linear epitopes are contiguous epitopes comprised in the amino acid primary sequence and typically include at least 2 amino acids or more. Conformational epitopes are formed by non-contiguous amino acids juxtaposed by folding of the target antigen, and in particular target (poly-) peptide.

[0189] In the context of the present disclosure, the term “binding domain” in particular refers to the variable region of the TCR alpha and / or beta chain and specifically the CDR3 alpha and CDR3 beta of the TCR.

[0190] Specifically, the TCR described herein is envisaged to recognize at least one epitope within the aforementioned amino acid sequences. The terms “binding to” and “recognizing” in all grammatical forms are used interchangeably herein. The antigenic target is particularly envisaged to be recognized by the TCRs described herein when being bound by a MHC class I molecule, specifically a HLA-A molecule, and preferably a HLA-A*02 molecule, in particular a HLA-A*02:01 molecule. The MHC molecule, in particular HLA-A and HLA-A*02 molecule, can be present on the surface of a cell, for instance a tumor cell, or on a (solid) carrier.

[0191] The TCRs disclosed herein specifically bind to their antigenic target. The term “specific(ally) binding” generally indicates that a TCR binds via its antigen binding site more readily to its intended antigenic target than to a random, unrelated non-target antigen. The term “specifically binds” indicates that the binding specificity of the TCR will be at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-30 fold, and most preferably 100-fold or more, greater for its antigenic target than its binding specificity for a non-target antigen.

[0192] Effector host cells expressing a native TCR as described herein are envisaged to bind to their antigenic target (z.e., preferably a neoantigen peptide described herein presented on HLA-A*02 by antigen presenting cells) with a high functional avidity. The term “functional avidity” refers to the capability of TCR expressing cells (in particular T-cells expressing native TCRs as described herein) to respond in vitro to a given concentration of a ligand and is thought to correlate with the in vivo effector capacity of TCR expressing cells. By definition, TCR expressing cells with high functional avidity respond in in vitro tests to very low antigen doses, while such cells of lower functional avidity require higher amounts of antigen before they mount an immune response similar to that of high-avidity TCR expressing cells. The functional avidity can be therefore considered as a quantitative determinant of the activation threshold of a TCR expressing cell. It is determined by exposing such cells in vitro to different amounts of cognate antigen. TCR expressing cells with high functional avidity respond to low antigen doses. For example, a TCR expressing cell will typically be considered to bind with “high” functional avidity to its antigenic target if it secretes at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, or 20,000 pg / mL or more) of interferon gamma (IFNy) upon coculture with antigen-negative HLA-A*02 expressing target cells loaded with a low concentration of the neoantigen peptide ranging from about 10'5to about 10'11M (z.e., about 0.05 ng / mL to about 5 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / ml, or 5 ng / mL).

[0193] Other methods to determine specific binding of the inventive TCRs include the51Cr release assay, the CD107a / b mobilization assay, and the peptide:MHC multimer binding analyses. All of which are envisioned.

[0194] The term “TCR” also encompasses TCR variants, which include TCR sequence variants, fragments, and constructs. All TCR variants are expected to be functional variants of the inventive TCR. The term “functional variant” as used herein refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, its variable regions or its antigen-binding regions and shares its biological activity, z.e., its ability to specifically bind to the antigenic target for which the parent TCR of the present disclosure has antigenic specificity to a similar, the same or even a higher extent as the TCR disclosed herein.

[0195] The term “TCR variants” includes “sequence variants” of the TCRs disclosed herein, z.e., variants substantially comprising the amino acid sequence of the inventive TCR as described above (also referred to as the “parent” TCR) but containing at least one amino acid modification (z.e., a substitution, deletion, or insertion) as compared to the “parent” TCR amino acid sequence, provided that the variant preferably retains the antigenic specificity of the “parent” TCR. TCR sequence variants of the present disclosure are typically prepared by introducing appropriate nucleotide changes into the nucleic acids encoding the “parent” TCR, or by peptide synthesis. Generally, the aforementioned amino acid modifications can be introduced into, or present in, the variable region or the constant region of the TCR and can serve to modulate properties like binding strength and specificity, post-translational processing (e.g., glycosylation), thermodynamic stability, solubility, surface expression or TCR assembly.

[0196] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the parent TCR. Example insertional variants of a TCR of the present disclosure include fusion products of said TCR and an enzyme or another functional polypeptide. Exemplary substitutional variants of a TCR of the present disclosure are those including example acid substitutions in variable regions or CDRs of the alpha and / or beta chain, the framework region or the constant region. In particular, the substitutions are conservative amino acid substitutions.

[0197] Conservative amino acid substitutions are envisioned and include amino acid substitutions in which one amino acid having certain physical and / or chemicalproperties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be in an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, and the like), a basic amino acid substituted for another basic amino acid (Lys, Arg, and the like), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, and the like), and the like that can be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.Cysteine modification

[0198] The addition of a disulfide bond in the constant region has been reported to foster correct pairing of the TCR alpha and beta chains (Kuball J et al. Blood 109(6):2331-2338, 2007). Thus, the addition of one or more cysteine bonds in the constant region is also considered part of the disclosure.

[0199] In general, TCR sequence variants comprise at least one of the CDR1, CDR2, CDR3, alpha chain variable regions, beta chain variable regions, alpha chains and / or beta chains as disclosed herein, or comprising or consisting of an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% , about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequences disclosed herein, provided that said variants exhibit comparable, the same or improved binding characteristics as compared to TCR evaluated in the appended examples.

[0200] As used herein the term “sequence identity” indicates the extent to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment and is often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several algorithms are available for determining sequence identity using standard parameters, for example Blast (Altschul et al. NucL Acids Res. 25:3389-3402, 1997), Blast2 (Altschul et al. J. Mol. Biol. 215:403-410, 1990), Smith- Waterman (Smith et al. J. Mol. Biol. 147: 195-197, 1981) and ClustalW.

[0201] The term “TCR” as used herein further comprises TCR constructs. The term “construct” includes proteins or polypeptides comprising at least one antigen binding domain of the TCRs disclosed herein, but do not necessarily share the basic structure of a native TCR (z.e., variable domains incorporated into a TCR alpha chain and a TCR beta chain forming a heterodimer). TCR constructs and fragments are typically obtained by routine methods of genetic engineering and are often artificially constructed to comprise additional functional protein or polypeptide domains. In accordance with the foregoing, TCR constructs and fragments of the disclosure are envisaged to comprise at least one CDR3 alpha and / or at least one CDR3 beta as disclosed elsewhere herein. Further as disclosed herein are constructs and fragments comprising at least one CDR1 alpha, CDR2 alpha, CDR1 beta, CDR2 beta, alpha chain variable region, beta chain variable region, alpha chain and / or beta chain, or combinations thereof, optionally in combination with further protein domains or moieties. The TCR constructs and fragments provided herein are capable of specifically binding to the same antigenic target as the inventive TCRs described above.

[0202] As disclosed herein, TCRs (or polynucleotides encoding TCRs) binding the CLK3 neoantigen derived from SRSF2 can comprise a sequence as follows:

[0203] As disclosed herein, TCRs (or polynucleotides encoding TCRs) binding the RHOT2 neoantigen derived from SRSF2 can comprise a sequence as follows:Multimers

[0204] The term “TCR construct” encompasses heterodimers and multimers in which at least one TCR alpha chain variable region or TCR alpha-chain and at least one TCR beta-chain variable region are covalently linked to each other. In its simplest form a multivalent TCR construct according to the present disclosure comprises a multimer of two or three or four or more TCRs associated, coupled, or conjugated (e.g., covalently, or otherwise linked) with one another, e.g., in some instances, via a linker molecule.

[0205] Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCRs can be formed into multimers having a plurality of TCR binding sites. The number of TCRs in the multimer will depend upon the quantity of TCR in relation to the quantity of linker molecule used to make the multimers, and also on the presence or absence of any other biotinylated molecules. Example multimers are dimeric, trimeric, tetrameric or pentameric or higher-order multimer TCR constructs. Multimers of the present disclosure can also comprise further functional entities such as labels or drugs or (solid) carriers.

[0206] The term “TCR construct” also encompasses TCR molecules which are linked via a suitable linker to a spheric body, preferably a uniform bead, more preferably a polystyrene bead, most preferably a bio-compatible polystyrene bead. Such TCR constructs can also be comprised of an inventive TCR and a bead having a pre-defined fluorescence dye incorporated into the bead.Fusion proteins

[0207] The term “TCR construct” also relates to fusion proteins or polypeptides comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3 alpha and / or at least one TCR beta chain, TCR beta chain variable region or CDR3 beta; and further one or more fusion component(s). Useful components include Fc receptors; Fc domains (derived from IgA, IgD, IgG, IgE, and IgM); cytokines (such as IL-2 or IL-15); toxins; antibodies or antigen binding fragments thereof (such as anti-CD3, anti-CD28, anti- CD5, anti-CD16 or anti-CD56 antibodies or antigen-binding fragments thereof); CD247 (CD3-zeta), CD28, CD137, CD134 domains; or any combinations thereof.

[0208] Example antibody fragments that can be used as fusion components include fragments of full-length antibodies, such as (s)dAb, Fv, Fd, Fab, Fab’, F(ab’)2or “rlgG” (“half antibody”); modified antibody fragments such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab’s), tandem di-scFv, tandem tri-scFv, minibodies, multibodies such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising only one variable domain, which might be VHH, VH or VL.

[0209] TCR constructs of the present disclosure can be fused to one or more antibody or antibody fragments, yielding monovalent, bivalent and polyvalent / multivalent constructs and thus monospecific constructs, specifically binding to only one target antigen as well as bispecific and polyspecific / multispecific constructs, which specifically bind to more than one target antigens, e.g., two, three or more, through distinct antigen binding sites. Optionally, a linker can be introduced between the one or more of the domains or 25 regions of the TCR construct of the present disclosure, z.e., between the TCR alpha chain CDR3, TCR alpha chain variable region, and / or a TCR alpha chain, the TCR beta chain CDR3, TCR beta chain variable region, and / or a TCR beta chain, and / or the one or morefusion component(s) described herein. Linkers are envisioned and have been reviewed, inter alia, by Chen etal. Adv. Drug Deliv. Rev. 65(10): 1357-1369, 2013. In general, linkers include flexible, cleavable and rigid linkers and will be selected depending on the type of construct and intended use / application. For example, for therapeutic application, non- immunogenic, flexible linkers are often preferred in order to ensure a certain degree of flexibility or interaction between the domains while reducing the risk of adverse immunogenic reactions. Such linkers are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids and include “GS” linkers consisting of stretches of Gly and Ser residues. An example of the most widely used flexible linker which is also intended for use in the TCR construct of the present disclosure has the sequence of (Gly- Gly-Gly-Gly-Ser)nwherein n is an integer of appropriate value. Other suitable linkers include for instance KESGSVSSEQLAQFRSLD (SEQ ID NO:509), EGKSSGSGSESKST (SEQ ID NO:510), and GSAGSAAGSGEF (SEQ ID NO:511).

[0210] Particularly useful TCR constructs in accordance with the present disclosure are those comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3 alpha as defined herein, at least one TCR beta chain, TCR beta chain variable region or CDR3 beta as defined herein, optionally linked to each other and fused, optionally via a linker, to at least one antibody or an antibody fragment (such as a single chain antibody fragment (scFv)) directed against an antigen or epitope on the surface of lymphocytes. In some instances, the antigenic targets recognized by the antibody or antibody fragment (e.g., scFv) include CD3, CD28, CD5, CD16 and CD56. The construct can in general have any structure as long as the “TCR portion” (i.e., TCR alpha and beta chain or variable regions or CDR3s thereof) retains its ability to recognize the antigenic target defined herein, and the “antibody portion” binds to the desired surface antigen or epitope, thereby recruiting and targeting the respective lymphocyte to the target cell. Such constructs can advantageously serve as “adapters” joining an antigen presenting cell displaying the antigenic target (such as a tumor cell) and a lymphocyte (such as a cytotoxic T cell or NK cell) together. An example of such a fusion protein is a construct engineered according to the principle of a bi-specific T-cell engager (BiTE®) consisting of two singlechain variable fragments (scFvs) of different antibodies, on a single peptide chain of about 55 kilodaltons (kD). Accordingly, a TCR construct of the present disclosure can comprise at least one TCR antigen binding domain as described herein (for instance a TCR variable alpha and variable beta chain fused to each other) linked to a scFv (or other binding domain)of the desired binding specificity, e.g., CD3 or CD56. The scFv (or other binding domain) binds to T cells such as via the CD3 receptor or to CD56 for NK cell activation, and the other to a tumor cell via an antigenic target specifically expressed on the tumor cell. Also encompassed herein are tribodies comprising at least one TCR antigen binding domain as described herein, an scFv (or other binding domain) and a further domain, e.g., for targeting the construct to a site of action within the body (e.g., an Fc domain).Isolated Form

[0211] The TCRs of the present disclosure can be provided in “isolated” or “substantially pure” form. “Isolated” or “substantially pure” when used herein means that the TCRs have been identified, separated, and / or recovered from a component of its production environment, such that the “isolated” TCR is free or substantially free of other contaminant components from its production environment that might interfere with its therapeutic or diagnostic use. Contaminant components can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. “Isolated” TCRs will thus be prepared by at least one purification step removing or substantially removing these contaminant components. The aforementioned definition is equally applicable to “isolated” polynucleotides / nucleic acids, mutatis mutandis.Soluble forms

[0212] The TCRs of the present invention can be provided in soluble form. Soluble TCRs are useful as diagnostic tools, and carriers or “adapters” that specifically target therapeutic agents or effector cells to, for instance, a cancer cell expressing the antigenic target recognized by the soluble TCR. Soluble TCRs (sTCRs) will typically be fragments or constructs comprising TCR alpha and / or beta chains, or variable regions or CDRs thereof and optionally stabilized via disulfide bonds or covalently linked via a suitable linker molecule, e.g., as described above in the context of TCR constructs of the present disclosure. They will typically not comprise a transmembrane region. In some circumstances amino acid modifications in the polypeptide sequence may be introduced in order to enhance solubility of the molecules, and / or correct folding and pairing of the alpha and beta chains (if desired), in particular when produced in a recombinant host that does not provide for the aforementioned features. For instance, when using E. coli as production host cells, folding, and pairing of the TCR alpha and beta chains is typically accomplished in vitro. TCRs according to the present disclosure can therefore for instance comprise additional cysteine residues, as described elsewhere herein.Besides additional cysteine bridges, other useful modifications include, for instance, the addition of leucine zippers and / or ribosomal skipping sequences, e.g., sequence 2A from picoma virus as described in Walseng et al. (PLoS ONE 10(4):e0119559, 2015) to increase folding, expression, and / or pairing of the TCR alpha and / or beta chains.Modifications

[0213] In some aspects the TCRs as described herein can further comprise one or more modifications as described in the following. The modifications described below will typically be covalent modifications and can be accomplished using suitable techniques. In some circumstances, amino acid modifications in the TCRs can be implemented to facilitate the introduction of said modifications.Labels

[0214] In some aspects, the TCRs, in particular (soluble) TCRs, of the present disclosure can be labelled. In some instances, labels can be coupled to the TCR or TCR variant using routine methods, optionally via linkers of various lengths. The term “label” or “labelling group” refers to any detectable label. In general, labels fall into a variety of classes, depending on the assay in which they are to be detected - the following examples include, but are not limited to: isotopic labels, which may be radioactive or heavy isotopes, such as radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,89Zr,90Y, "Tc,i nIn,125I,133I); magnetic labels (e.g, magnetic particles); redox active moi eties; optical dyes (including, but not limited to, chromophores, phosphors and fluorophores) such as fluorescent groups (e.g, FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores which can be either “small molecule” fluorophores or proteinaceous fluorophores; enzymatic groups (e.g., horseradish peroxidase, P- galactosidase, luciferase, alkaline phosphatase; biotinylated groups; or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, and the like). Labelling is particularly envisaged when the TCRs, TCR variants or especially soluble TCR constructs (such as those comprising at least one TCR alpha and / or TCR beta chain) that can be used for diagnostic purposes.Functional moieties

[0215] The TCRs, in particular soluble TCRs, of the present disclosure can be modified by attaching further functional moieties, e.g, for reducing immunogenicity,increasing hydrodynamic size (size in solution) solubility and / or stability (e.g., by enhanced protection to proteolytic degradation) and / or extending serum half-life.

[0216] Example functional moieties for use in accordance with the present disclosure include peptides or protein domains binding to other proteins in the human body (such as serum albumin, the immunoglobulin Fc region or the neonatal Fc receptor (FcRn) polypeptide chains of varying length (e.g., XTEN™-technology or PASylation®), non- proteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol (PEGylation), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, or of carbohydrates, such as hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology).

[0217] Other useful functional moieties include “suicide” or “safety switches” that can be used to shut off effector host cells carrying an inventive TCR in a patient’s body. An example of “suicide” or “safety switches” includes the inducible Caspase 9 (iCasp9) “safety switch” described by Gargett and Brown (Front. Pharmacol. 5:235, 2014). Briefly, effector host cells are modified to express a Caspase 9 domain whose dimerization depends on a small molecule dimerizer drug such as AP1903 / CIP, and results in rapid induction of apoptosis in the modified effector cells. Examples for other “suicide” “safety switches” are envisioned, e.g., Herpes Simplex Virus thymidine kinase (HSV-5 TK), expression of CD20 and subsequent depletion using anti-CD20 antibody or myc tags (Kieback et al. Proc. Natl. Acad. Set. USA 105(2):623-628, 2008).Glycosylation

[0218] TCRs with an altered glycosylation pattern are also envisaged herein. As is envisioned, glycosylation patterns can depend on the amino acid sequence (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below) and / or the host cell or organism in which the protein is produced. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Addition of N-linked glycosylation sites to the binding molecule is conveniently accomplished by altering the amino acid sequence such that it contains one or more tri-peptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except praline). O-linked glycosylation sites can be introduced by the addition of or substitution by, one or more serine or threonine residues to the starting sequence.

[0219] Another means of glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to the protein. Depending on the coupling mode used, the sugar(s) can be attached to (A) arginine and histidine, (B) free carboxyl groups, (C) free sulfhydryl groups such as those of cysteine, (D) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (E) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (F) the amide group of glutamine.

[0220] Similarly, deglycosylation (z.e., removal of carbohydrate moi eties present on the binding molecule) can be accomplished chemically, e.g., by exposing the TCRs to trifluoromethanesulfonic acid, or enzymatically by employing endo- and exo-glycosidases. Tags

[0221] The TCRs, in particular soluble TCRs, of the present disclosure can be modified to introduce additional domains which aid in identification, tracking, purification and / or isolation of the respective molecules (tags). Non-limiting examples of such tags comprise peptide motives known as Myc-tag, HAT -tag, HA-tag, TAP -tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants thereof (e.g., Strep 11 -tag), CD20, Her2 / neu tags, FLAG-tag, T7-tag, HA(hemagglutinin}-tag, or green fluorescent protein-tags (GFP-tags).

[0222] In some instances, the epitope tag is incorporated into a TCR. Epitope tags are short stretches of amino acids that allow for binding of a specific antibody and therefore enable identification and tracking of the binding and movement of soluble TCRs or host cells within the patient’s body or cultivated (host) cells. Detection of the epitope tag, and hence, the tagged TCR, can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (“Western”), and affinity chromatography. The epitope tags can for instance have a length of 6 to 15 amino acids, in particular 9 to 11 amino acids. It is also possible to include more than one epitope tag in the TCR of the present disclosure.

[0223] Tags can further be employed for stimulation and expansion of host cells carrying a TCR of the present disclosure by cultivating the cells in the presence of binding molecules (antibodies) specific for said tag.Nucleic acid

[0224] As set forth above and herein, the present disclosure provides nucleic acids encoding the TCRs described herein, specifically, polynucleotides encoding TCRalpha or beta chains, TCR alpha or beta chain variable regions, and TCR CDR3 alpha and CDR3 beta, as well as TCR variants, constructs and fragments of the present disclosure. The term “polynucleotide” or “nucleic acid” as used herein comprises a sequence of polyribonucleotides and poly deoxribonucleotides, e.g., modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form linear or circular, or mixtures thereof, including hybrid molecules. The nucleic acids according to this disclosure thus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA ivtRNA), combinations thereof or derivatives (such as PNA) thereof.

[0225] A polynucleotide may comprise a conventional phosphodiester bond or a nonconventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The polynucleotides of the present disclosure can also contain one or more modified bases, such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications, are also conceivable, as long as a binding molecule of the present disclosure can be expressed from the polynucleotide. The polynucleotide can be provided in isolated form as defined elsewhere herein. A polynucleotide may include regulatory sequences such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding site, in trans, or the like.

[0226]

[0092] In particular, the present disclosure provides a polynucleotide comprising or consisting of a nucleic acid that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to a reference polynucleotide sequence selected from the polynucleotide sequences disclosed herein.

[0227] The polynucleotides described above can or may not comprise additional or altered nucleotide sequences encoding e.g., altered amino acid residues, a signal peptide to direct secretion of the encoded TCR, constant regions or other heterologous polypeptides as described herein. Such polynucleotides may thus encode fusion, polypeptides, fragments, variants and other derivatives of the binding molecules described herein.

[0228] Also, the present disclosure includes compositions comprising one or more of the polynucleotides described herein. Also provided herein are compositions, comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a TCR alpha chain variable region as described herein and wherein said second polynucleotide encodes a TCR beta chain variable region as described herein.

[0229] The nucleic acid sequences of the present disclosure may be codon- optimized for optimal expression in the desired host cell, e.g., a human lymphocyte (for example, a T cell); or for expression in bacterial, yeast or insect cells that are particularly envisaged for the expression of soluble TCRs of the disclosure. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons that are being exchanged. Selection of optimum codons thus depends on codon usage of the host genome and the presence of several desirable and undesirable sequence motifs. Examples of codon optimized TCR polynucleotide sequences are provided above. Vector

[0230] Further provided herein is a vector, comprising one or more of the polynucleotides as described herein. A “vector” is a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a host cell where it can for instance be replicated and / or expressed.

[0231] The term “vector” encompasses, without limitation plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is generally a nucleotide sequence, commonly a DNA sequence that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Engineered vectors typically comprise an origin for autonomous replication in the host cells (if stable expression of the polynucleotide is desired), selection markers, and restriction enzyme cleavage sites (e.g., a multiple cloning site, MCS). Vectors can additionally comprise promoters, genetic markers, reporter genes, targeting sequences, and / or protein purification tags. Large numbers of suitable vectors are envisioned and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012) and include:Targeting vectors

[0232] Targeting vectors can be used to integrate a polynucleotide into the host cell’s chromosome by suitable methods. Briefly, suitable means include homologous recombination or use of a hybrid recombinase that specifically targets sequences at theintegration sites. Targeting vectors are typically circular and linearized before being used for homologous recombination. As an alternative, the foreign polynucleotides can be DNA fragments joined by fusion PCR or synthetically constructed DNA fragments which are then recombined into the host cell. It is also possible to use heterologous recombination which results in random or non-targeted integration.

[0233] The present disclosure also provides a vector comprising the nucleic acid described herein.Expression vectors

[0234] The vector of the present disclosure can also be an expression vector. “Expression vectors” or “expression constructs” can be used for the transcription of heterologous polynucleotide sequences, for instance those encoding the TCRs of the present disclosure, and translation of their mRNA in a suitable host cell. This process is also referred to as “expression” of the TCRs of the present disclosure.

[0235] Besides an origin of replication, selection markers, and restriction enzyme cleavage sites, expression vectors typically include one or more regulatory sequences operably linked to the heterologous polynucleotide to be expressed.

[0236] The term “regulatory sequence” refers to a nucleic acid sequence necessary for the expression of an operably linked coding sequence of a (heterologous) polynucleotide in a particular host organism or host cell and thus include transcriptional and translational regulatory sequences. Typically, regulatory sequences for expression of heterologous polynucleotide sequences in prokaryotes include a promoter(s), optionally operator sequence(s), and ribosome binding site(s). In eukaryotes, promoters, polyadenylation signals, enhancers and optionally splice signals are typically implemented. Moreover, specific initiation and secretory signals also may be introduced into the vector in order to allow for secretion of the polypeptide of interest into the culture medium.

[0237] A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence, in particular on the same polynucleotide molecule. For example, a promoter is operably linked with a coding sequence of a heterologous gene when it is capable of effecting the expression of that coding sequence. The promoter is typically placed upstream of the gene encoding the polypeptide of interest and regulates the expression of said gene.

[0238] Example regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells,such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. The expression vectors can also include origins of replication and selectable markers.

[0239] Vectors of the present disclosure can further comprise one or more selection markers. Suitable selection markers for use with eukaryotic host cells include, without limitation, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyl transferase (hgprt), and adenine phosphoribosyl transferase (aprt) genes. Other genes include dhfr (methotrexate resistance), gpt (mycophenolic acid resistance) neo (G-418 resistance) and hygro (hygromycin resistance). Vector amplification can be used to increase expression levels. In general, the selection marker gene can either be directly linked to the polynucleotide sequences to be expressed or introduced into the same host cell by co-transformation.

[0240] As such, the present disclosure further provides one or more of the nucleotide sequences described herein inserted into (z.e., comprised by) a vector. Specifically, the present disclosure provides (replicable) vectors comprising a nucleotide sequence encoding a TCR described herein, or an alpha or beta chain thereof, or an alpha or beta variable domain, or a CDR3 alpha or CDR3 beta operably linked to a promoter.

[0241] It is envisioned that it may be necessary to select a suitable expression vector based on, e.g., the host cell intended for TCR expression. Examples for suitable expression vectors are viral vectors, such as retroviral vectors, for example, MP71 vectors or retroviral SIN vectors; and lentiviral vectors or lentiviral SIN vectors. Viral vectors comprising polynucleotides encoding the TCRs disclosed herein are for instance capable of infecting T cells, which are envisaged to subsequently express the heterologous TCR. The nucleic acids and / or in particular expression constructs of the disclosure can also be transferred into cells by transient RNA transfection.

[0242] Currently used viral vectors for native TCR expression typically link the TCR-alpha and TCR-beta chain genes in one vector with either an internal ribosomal entry site (IRES) sequence or the 2A peptide sequence derived from a porcine teschovirus, resulting in the expression a single messenger RNA (mRNA) molecule under the control of the viral promoter within the transduced cell.Host Cell

[0243] The present disclosure further provides a host cell comprising the TCR, nucleic acid or the vector described herein.

[0244] A variety of host cells can be used in accordance with the present disclosure. As used herein, the term “host cell” encompasses cells which can be or has / have been recipients of polynucleotides or vectors described herein and / or express (and optionally secreting) the TCR of the present disclosure. The terms “cell” and “cell culture” are used interchangeably to denote the source of a TCR unless it is clearly specified otherwise. The term “host cell” also includes “host cell lines”.

[0245] In general, the term “host cell” includes prokaryotic or eukaryotic cells, and also includes without limitation bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, macaque or human cells.

[0246] As such, the present disclosure provides host cells comprising a polynucleotide or a vector, e.g., an expression vector comprising a nucleotide sequence encoding a TCR or TCR construct as described herein.

[0247] Polynucleotides and / or vectors of the present disclosure can be introduced into the host cells using suitable methods, e.g., by transfection, transformation, or the like.

[0248] “ Transfection” is the process of deliberately introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, i.e., the process of introducing RNA (such as in vitro transcribed RNA) into a host cell. The term is mostly used for non-viral methods in eukaryotic cells. The term “transduction” is often used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or “holes” in the cell membrane, to allow the uptake of material. Transfection can be carried out using calcium phosphate, by electroporation, by cell squeezing or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their nucleic acid molecules inside. Example techniques for transfecting eukaryotic host cells include lipid vesicle mediated uptake, heat shock mediated uptake, calcium phosphate mediated transfection (calcium phosphate / DNA co-precipitation), microinjection and electroporation.

[0249] The term “transformation” is used to describe non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, and also into nonanimal eukaryotic cells, including plant cells. Transformation is hence the genetic alteration of a bacterial or non-animal eukaryotic cell resulting from the direct uptake through the cellmembrane(s) from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be effected by artificial means. For transformation to happen, cells or bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. For prokaryotic transformation, techniques can include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection and electroporation. Techniques for plant transformation include Agrobacterium mediated transfer, such as by A. tumefaciens, rapidly propelled tungsten or gold microprojectiles, electroporation, microinjection and polyethylene glycol mediated uptake.

[0250] As such, the present disclosure further provides host cells comprising at least one polynucleotide sequence and / or vector as described herein.

[0251] For expression of the TCRs described herein, a host cell may be chosen that modulates the expression of the inserted polynucleotide sequences, and / or modifies and processes the gene product (z.e., RNA and / or protein) as desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of gene products may be important for the function of the TCR. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the product. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

[0252] The present disclosure further provides (I) host cell(s) for expressing and obtaining TCRs of the present disclosure, in particular in soluble form (“production host cells”) and II) host cell(s) expressing a TCR of the present disclosure and having effector function (“effector host cells”). Such “effector host cells” are particularly useful for therapeutic applications and are useful for administration to a subject in need thereof. Preferred “effector host cells” include lymphocytes such as cytotoxic T lymphocytes (CTLs), CD8+T cells, CD4+T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta-T -cell s.“Production host cell”Cells

[0253] “Production host cells” used in certain embodiments for the expression of soluble TCRs of the disclosure are preferably capable of expressing high amounts ofrecombinant protein. Example mammalian host cells that can be used as “production host cells” include Chinese Hamster Ovary (CHO cells) including DHFR minus CHO cells such as DG44 and DUXBI 1, NSO, COS (a derivative of CVI with SV40 T antigen), HEK293 (human kidney), and SP2 (mouse myeloma) cells. Other example host cell lines include, but are not limited to, HELA (human cervical carcinoma), CVI (monkey kidney line), VERY, BHK (baby hamster kidney), MOCK, 293, W138, R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), P3x63-Ag3.653 (mouse myeloma), BFAlclBPT (bovine endothelial cells), and RAJI (human lymphocyte). Host cell lines are typically available from commercial services, the American Type Culture Collection (ATCC) or from published literature.

[0254] Non-mammalian cells such as bacterial, yeast, insect or plant cells are also readily available and can also be used as “production host cells” as described above. Example bacterial host cells include enterobacteriaceae, such Escherichia coli, Salmonella, Bacillaceae, such as Bacillus suhlilis: Pneumococcus,' Streptococcus, and Haemophilus influenza. Other host cells include yeast cells, such as Saccharomyces cerevisiae, and Pichia pastoris. Insect cells include, without limitation, Spodoptera frugiperda cells.

[0255] In accordance with the foregoing, conceivable expressions systems (i.e., host cells comprising an expression vector as described above) include microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid). Mammalian expression systems harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter, the cytomegalovirus (CMV) major immediate-early promoter (MIEP) promoter) are often preferred. Suitable mammalian host cells can be selected from known cell lines (e.g., COS, CHO, BLK, 293, 3T3 cells); however, it is also conceivable to use immune cells, e.g., lymphocytes such as cytotoxic T lymphocytes (CTLs), CD8+T cells, CD4+T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta-T-cells.

[0256] In accordance with the foregoing, the present disclosure also provides a method for producing and obtaining a TCR as described herein comprising the steps of (I) incubating a host cell (z.e., a production host cell) under conditions causing expression of said TCR and (ii) purifying said TCR.Cultivation

[0257] The host cells harboring the expression vector are grown under conditions appropriate to the production of the TCRs provided herein, in particular alpha chains and / or beta chains as described elsewhere herein and assayed for alpha and / or beta chain protein synthesis. For the expression of double-chained TCRs, vectors encoding both the alpha and beta chains may be co- expressed in the host cell for expression of the entire molecule. Purification

[0258] Once a TCR of the present disclosure has been expressed, it can be purified by any purification method known in the art, for example, by chromatography (e.g., ion exchange chromatography (e.g., hydroxylapatite chromatography), affinity chromatography, particularly Protein A, Protein G or lectin affinity chromatography, sizing column chromatography), centrifugation, differential solubility, hydrophobic interaction chromatography, or by any other standard technique for the purification of proteins. The skilled person will readily be able to select a suitable purification method based on the individual characteristics of the TCR to be recovered.“Effector host cell”

[0259] As set forth above, the present disclosure also provides for “effector host cells” comprising a nucleotide sequence, vector or TCR disclosed herein. The effector host cells are modified using routine methods to comprise a nucleic acid sequence encoding the TCR disclosed herein, and are to express the TCR described herein, in particular on the cell surface. For the purposes of the present disclosure, “modified host cells expressing a TCR disclosed herein” generally refers to (effector or production) host cells treated or altered to express a TCR according to the present disclosure, for instance by RNA transfection. Other methods of modification or transfection or transduction, such as those described elsewhere herein, are also included. The term “modified host cell” thus includes “transfected”, “transduced” and “genetically engineered” host cells preferably expressing the TCR of the present disclosure.

[0260] In certain embodiments, such “(modified) effector host cells” (in particular “(modified) effector lymphocytes”) are capable of mediating effector functionsthrough intracellular signal transduction upon binding of the TCR to its specific antigenic target. Such effector functions include for instance the release of perforin (which creates holes in the target cell membrane), granzymes (which are proteases that act intracellularly to trigger apoptosis), the expression of Fas ligand (which activates apoptosis in a Fas- bearing target cell) and the release of cytokines, preferably Thl / Tcl cytokines such as IFNy, IL-2 and TNFa. Thus, an effector host cell engineered to express the TCR of the disclosure that is capable of recognizing and binding to its antigenic target in the subject to be treated can carry out the abovementioned effector functions, thereby killing the target (e.g., cancer) cells. Cytolysis of target cells can be assessed, e.g., with the CTL fluorescent killing assay (CTL, USA) detecting the disappearance of fluorescently labeled target cells during co-culture with TCR-transfected recipient T cells.

[0261] In certain embodiments, the effector host cells express a functional TCR, z.e., that typically comprises a TCR alpha and beta chain described herein; and also the signal transducing subunits CD3 gamma, delta, epsilon and zeta (CD3 complex). Moreover, expression of co-receptors CD4 or CD8 may also be desired. Generally, lymphocytes harboring genes involved in antigen binding, receptor activation and downstream signaling (e.g., Lek, FYN, CD45, and / or Zap70), T cells are suitable as effector host cells. However, effector host cells expressing the TCR of the disclosure as a “binding domain” without the CD3 signal transducing subunit and / or aforementioned downstream signaling molecules (i.e., being capable of recognizing the antigenic target described herein, but without effecting functions mediated by CD3 and / or the aforementioned downstream signaling molecules) are also considered an embodiment herein. Such effector cells are capable of recognizing the antigenic target described herein, and optionally of effecting other functions not associated with CD3 signaling and / or signaling of the aforementioned downstream signaling molecules. Examples include NK or NKT cells expressing a disclosed TCR and being capable of, e.g., releasing cytotoxic granules upon recognition of their antigenic target.

[0262] Thus, cytotoxic T lymphocytes (CTLs), CD8+T cells, CD4+T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta-T-cells are considered useful lymphocyte effector host cells. Such lymphocytes expressing the recombinant TCR of the present disclosure are also referred to as “modified effector lymphocytes” herein. Any component of the TCR signaling pathway leading to the desired effector function canbe introduced into a suitable host cell by recombinant genetic engineering methods that are also considered encompassed by the embodiments described herein.

[0263] An effector host cell, in particular lymphocytes such as T cells, can be autologous host cells that are obtained from the subject to be treated and transformed or transduced to express the TCR of the present disclosure. Typically, recombinant expression of the TCR will be accomplished by using a viral vector. Techniques for obtaining and isolating the cells from the patient are envisioned.

[0264] As set forth above, the effector host cells provided herein are particularly useful for therapeutic applications. Further genetic modifications of the host cells can be desirable to increase therapeutic efficacy. For example, when using autologous CD8+T cells as “effector host cells” suitable additional modifications include downregulation of the endogenous TCR, CTLA-4 and / or PD-1 expression; and / or amplification of costimulatory molecules such as CD28, CD134, CD137. Suitable means and methods for achieving the aforementioned genetic modifications are envisioned.

[0265] Methods for targeted genome engineering of host cells are envisioned and include, besides gene knockdown with siRNA, the use of so-called “programmable nucleases” such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR- associated) system. For instance, programmable nucleases such as TALENs can be employed to cut the DNA regions that code for “unwanted” proteins, such as PD-1, CTLA- 4 or an endogenous TCR, and thereby reducing their expression. When T cells are used as (effector) host cells, downregulation of the endogenous TCR has the benefit of reducing unwanted “mispairing” of endogenous and exogenous TCR alpha / beta chains.

[0266] As used herein a “T cell receptor-like antibody” or “TCRL” refers to an antibody which binds an MHC complexed with an HLA-restricted peptide antigen. Binding of the TCRL to its target is with an MHC -restricted specificity. The TCRL antibody does not bind the MHC in the absence of the complexed peptide, and the antibody does not bind the peptide in the absence of the MHC. Generally, to generate TCR-like antibodies a purified MHC-peptide complex folded in a native configuration that is recognized by a T cell is generated. Such complexes are typically formed using recombinant DNA technology. Suitable methods for producing such complexes are envisioned. TCR-like antibodies can be produced against the complex using standard hybridoma approaches orselected by employing immunized or non-immunized (e.g., phage) antibody libraries of full-length antibodies or antibody fragments, such as Fabs. Methods for screening any antibodies discovered for appropriate binding can be implemented. See, for example, WO 2016 / 199140, incorporated herein by reference.Pharmaceutical compositions

[0267] The present disclosure also provides pharmaceutical compositions comprising a neoantigen or neoantigen peptide as described herein. The pharmaceutical composition can also comprise a T cell or genetically modified effector host cells as described herein. Further, the pharmaceutical composition can comprise a TCR-like antibody, CAR, and any other active agent described herein.

[0268] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active agents described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term “active ingredient” refers to any of the agents described herein including, but not limited to a neoantigen, a neoantigen peptide, CAR, T cell, effector T cell, TCR-like antibody, and the like described herein accountable for a biological effect.

[0269] As the terms are used herein, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0270] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in, for example, “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0271] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, directintraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0272] In various implementations, one or more cancers are treatable with a TCR or TCR-like protein of the disclosure. In addition, in various implementations, one or more cancers are preventable with a TCR or TCR-like protein of the disclosure. Examples of cancers treatable and / or preventable include, but are not limited to, a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), a myelomonocytic leukemia (CMML), a clonal hematopoiesis, a clonal cytopenia of undetermined significance (CCUS), or any combination thereof.

[0273] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent, such as a neoantigen peptide that is itself incapable of crossing the Blood Brain Barrier (BBB)) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).

[0274] Alternately, one can administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

[0275] Pharmaceutical compositions of some embodiments of the present disclosure can be manufactured by suitable processes, e.g., by means of mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0276] Pharmaceutical compositions for use in accordance with some embodiments of the disclosure thus can be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen and is well known to the skilled artisan.

[0277] Pharmaceutical compositions suitable for use in context of some embodiments of the present disclosure include compositions wherein the active agents are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of an active agent effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0278] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0279] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition.

[0280] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0281] While each of the elements of the present disclosure is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present disclosure is capableof being used with each of the embodiments of the other elements of the present disclosure and each such use is intended to form a distinct embodiment of the present disclosure.

[0282] The referenced patents, patent applications, and scientific literature referred to herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[0283] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the present disclosure.

[0284] As can be appreciated from the disclosure above, the present disclosure has a wide variety of applications. The embodiments of the disclosure are further illustrated by the following examples, which are only illustrative and are not intended to limit the definition and scope of the disclosure in any way.Examples

[0285] Example 1. Mis-splicing-derived neoantigens and cognate TCRs in splicing factor mutant leukemias.

[0286] Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here a series of bona fide neoantigens, translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia- associated somatic splicing machinery mutations, are disclosed. Feature-barcoded peptide- MHC dextramers were utilized to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant- induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide an effective strategy for genetically redirecting T cells to recognize these targets.

[0287] Following five decades with few approved therapies for acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS), the last five years havebrought remarkable progress with multiple new FDA-approved therapies. Despite these advances, the 5-year survival rate for most AML patients is less than 20% and there are few effective therapies for high-risk MDS.

[0288] One major challenge in treatment of MDS and AML has been the lack of effective immunotherapies. Still, the promise for T cell-based immunotherapeutic approaches for treating myeloid leukemias is clear given the curative potential of allogeneic hematopoietic cell transplantation (allo-HCT) for AML and MDS patients, arising from the graft-versus-leukemia (GVL) activity of donor T cells.

[0289] To date, chimeric antigen receptor (CAR) T cell approaches for AML have nearly entirely relied on antigens which are shared across myeloid neoplasms and normal hematopoietic stem cells, risking unacceptable on-target but off-tumor toxicities. Given these challenges, identifying additional sources of leukemia-specific neoantigens shared across patients and applicable to multiple HLA alleles could represent a major therapeutic advance. In this example, it is tested whether cancer-associated mutations in genes encoding RNA splicing factors, which create stereotypical neomorphic changes in splicing consistently across patients, can yield novel HLA class-I (HLA-I) displayed peptides.

[0290] Mutations in the RNA splicing factor genes SF3B1. SRSF2, U2AF1, and ZRSR2 are seen in 50-70% of MDS and are also prevalent in AML and the related aggressive myeloid blood cancer chronic myelomonocytic leukemia (CMML). These mutations skew usage of alternative RNA splicing events observed in healthy cells but also create novel RNA isoforms which are reproducibly observed across patients with the same mutations. As such, aberrant mRNA splicing events have the potential to generate missplicing-derived neoantigens that are “public” (z.e., shared across patients with the same splicing factor mutation).

[0291] Putative aberrant RNA splicing-derived neoantigens have previously been identified in A7G7> / -mutant uveal melanoma; however, validation of such antigens as immunotherapeutic targets through TCR gene transfer was not performed. Moreover, no prior studies have evaluated mis-splicing-derived neoantigens in myeloid malignancies, where mutations affecting splicing factors occur at the highest frequency.

[0292] In this example, it was sought to identify mis-splicing-derived neoantigens in myeloid leukemias with mutations in the RNA splicing factor genes SRSF2 and ZRSR2. These two factors were selected for experiments in this example in view oftheir high mutation frequencies, associations with adverse outcomes, and unique mechanistic roles in RNA splicing. SRSF2 mutations in particular are especially common across myeloid neoplasms, as they occur in 30-50% of patients with CMML, 17-25% of patients with MDS, 18-20% of patients with AML over the age of 60, and 6-8% of younger patients with AML (<60yr).

[0293] Here a series of neoantigens derived from shared mis-splicing events observed across myeloid leukemia patients with SRSF2 or ZRSR2 mutations was identified. These empirically validated neoantigens were then used to construct HLA-I dextramers, which enabled discovery of rare, circulating neoantigen-reactive CD8+T cells in patients, evaluate their transcriptional characteristics, and isolate their TCRs. Finally, a panel of TCRs reactive against SRSF2 mutation-induced mis-splicing-derived neoantigens redirected primary CD8+T cells for specific recognition and cytotoxicity of 67%7’2-mutant leukemia cells. The present disclosure thereby describes endogenously produced neoantigens and their cognate TCRs in patients with splicing factor mutant leukemia and proposes neoantigen-based TCR-T cell therapies as novel immunotherapeutic strategies in at least this population.ResultsIdentification of putative RNA mis-splicing-derived neoantigens in SRSF2- or ZRSR2-mutant myeloid leukemias

[0294] Mutations in the splicing factors SF3B1. SRSF2, U2AF1, and ZRSR2 cause highly sequence-specific changes to splicing consistent across cancers and cell types which are uniformly observed across patients with mutations in the same RNA splicing factor. This example tested whether a subset of RNA mis-splicing events created by mutations in RNA splicing factors could give rise to novel HLA-I presented peptides that are immunogenic.

[0295] To test this, large-scale RNA-seq analyses were performed to identify recurrently mis-spliced isoforms in myeloid neoplasm patient samples from five patient cohorts with SRSF2 mutations (n=107), ZRSR2 mutations (n=33), or no mutations in SF3B1. SRSF2, U2AF1, or ZRSR2 (referred to as wild-type or WT; n=837). Mis-spliced isoforms that consistently produced across multiple patient cohorts and minimally expressed in healthy bone marrow, peripheral blood mononuclear cells (PBMCs), and 14 normal tissues were identified. Mis-spliced isoforms produced in 57?5F2-mutant MDS and AML affected diverse alternative splicing events (differentially spliced cassette exons,alternative 5’ or 3’ splice sites, mutually exclusive exons, and retention of constitutive or alternative introns), which could be broadly categorized into two groups (FIG. 1A). The first and larger group encompasses mis-splicing events that enhance production of isoforms that are normally produced in healthy tissues at a lower level. The second, smaller group is characterized by mis-splicing events that result in production of aberrant isoforms absent from healthy tissues and therefore represent candidates for neoepitope production. ZRSR2 mutations similarly gave rise to diverse mis-spliced isoforms, including strong representation of intron retention events and a large number of mis-spliced isoforms specific to Z / ,S7 2-mutant cells, and not observed in healthy tissues (FIG. IB). Cassette exons included in 57?5F2-mutant cells exhibited a strong enrichment for C-rich exonic splicing enhancer sequences (FIG. 1C), while G-rich exons were predominantly skipped (as described previously). ZRSR2 -mutant transcriptomes exhibited a striking retention of U12-type introns (FIG. ID), consistent with ZRSR2’s known role as a component of the minor (or U12-type) spliceosome.

[0296] Mis-spliced isoforms were next ranked based on the following criteria: (1) significant and consistent mis-splicing across patient cohorts, (2) minimal mis-splicing in healthy tissues, (3) high mis-spliced isoform expression (taking both mis-splicing rate and gene expression levels into account) in tumor and low expression in healthy tissues, (4) encode epitopes with predicted high-affinity binding to HLA-I which uniquely arise from the mis-spliced isoform. Each tumor-specific aberrant RNA isoform was translated in silico, and each split into 8- to 12-mer peptides, limited to those that arose uniquely from their parent mis-spliced isoforms, and predicted high-affinity binders to HLA-A*02:01 with NetMHCpan 4.0. Resulting candidates were further prioritized based on their parent isoforms’ mis-splicing patterns (Methods). In total, fifty-six candidate mis-splicing-derived neoantigens created by SRSF2 mutations and nineteen by ZRSR2 mutations were selected for further studies.Validation of immunogenicity

[0297] It was next tested whether candidate neoepitopes stably bind HLA- A*02:01 in vitro by performing a T2 HLA-A2 shift assay (FIG. IE). Fifty-six candidate 57?5F2-mutant and twelve Z / ,S7 2-mutant mis-splicing derived peptides were identified over a peptide concentration range of 0-100 pg / mL (FIG. IE). As positive controls, the well-studied HLA-A*02:01 -restricted Melan-A / MARTl (26-35) peptide (EAAGIGILTV; SEQ ID NO:508) and its high-affinity heteroclitic variant (ELAGIGILTV; SEQ IDNO:507) were used. As negative controls, three distinct 10-mer peptides predicted not to bind HLA-A*02:01 were used. Relative to controls, 42 out of 56 SRSF2 mutant-induced peptides (75%) and 9 out of 12 ZRSR2 mutant-induced peptides (75%) exhibited significant HLA-I binding (FIG. IE).

[0298] To evaluate the immunogenicity of the candidate peptides as well as to isolate antigen-reactive CD8+T cells with high-avidity cognate TCRs, in vitro sensitization of CD8+T cells from HLA-A*02:01+healthy donors was performed using the predicted neoantigens. Bulk PBMCs from 14 unique HLA-A*02:01 healthy donors were stimulated with candidate neopeptides to induce CD8+T cell priming. After one week, the expanded T cells were restimulated with the same peptide and IFNy / TNFa production measured using intracellular cytokine staining (ICS) and IFNy ELISpot. A number of SRSF2 mutant- induced peptides were immunogenic across multiple donors with statistical significance, which include those derived from mis-spliced transcripts encoding CLK3, RHOT2, and C16orf70 (FIG. 2A).

[0299] These immunogenic peptides and their related mis-splicing events were further interrogated. One example is RHOT2 peptide #5 (CLLPPALFL; SEQ ID NO:42), derived from an inclusion of intron 5 in RHOT2. This mis-splicing event is recurrently upregulated across SRSF2-mutant AML patients, compared to SRSF2 wild-type AML patients and normal human tissues across three cohorts of AML RNA-Seq datasets (FIG. 2B). Endogenous presentation of this peptide was validated by HLA-immunoprecipitation liquid chromatography-tandem mass spectrometry (HLA-IP LC-MS / MS) on HLA- A*02:01 -transduced K562 cells containing a knockin SRSF2P95H^WTmutation (FIG. 2C). As noted above, this RHOT2 neoantigen was immunogenic across multiple donors (FIG. 2A and FIG. 2D-E). To unequivocally prove the in vitro priming of antigen-reactive CD8+T cells using the immunogenicity assay, dual fluorescence-labeled dextramers were generated for RHOT2 peptide #5, which are composed of ten peptide / HLA-I (p / HLA-I) complexes assembled on a dextran scaffold. The in vitro primed T cells were stained with dextramers and dextramer double-positive CD8+T cells reactive to RHOT2 peptide #5 were identified (FIG. 2F).

[0300] Another example of immunogenic peptides is cl6orf70 peptide (RLLAAVLEA; SEQ ID NO:6). SRSF2 mutations promote inclusion of a poison exon in cl6orf70, and the resulting truncated protein product gives rise to the cl6orf70 neoantigen. The endogenous production and presentation of the peptide in SRSF2P95H / WTmutant cellswas confirmed. The immunogenicity of this peptide was also validated across multiple donors and in the presence of neoantigen-reactive CD8+T cells in in vitro primed T cells (FIG. 2G-2J).

[0301] For 12 ZRSR2 mutant-induced peptides, two peptides were immunogenic across multiple donors with statistical significance. These neoantigens are translated from minor introns in ATG3 and MYO IF. Dual-color p / HLA-I dextramer staining validated the successful in vitro priming of CD8+T cells reactive to ATG3 and MYO1F peptides.

[0302] To understand how broadly applicable the disclosed neoantigen-based therapies are for patients with MDS, CMML, and AML, RNA-seq data from cohorts of patients with each of these diseases was analyzed to quantify expression of SRSF2 or ZRSR2 mutant-induced mis-spliced mRNA encoding each neoantigen. These data demonstrate that expression of most of the neoantigens are consistent across patients with MDS, CMML, and AML bearing mutations in SRSF2 or ZRSR2.SRSF2 mutations induce CLK3 exon 4 skipping and give rise to a novel antigen

[0303] One peptide (RLWGTWVKA; SEQ ID NO:31) derived from SRSF2 mutation-induced aberrant splicing of CLK3 transcript was the most frequently antigenic peptide across donors (FIGs 2A, 3 A-3C). Given the robust capacity of this peptide to elicit T cell responses, the related mis-splicing event was investigated in more detail.

[0304] SRSF2 mutations promote skipping of exon 4 in CLK3 and the resulting transcript is a predicted substrate for nonsense-mediated mRNA decay (NMD) (FIG. 3D). This aberrant CLK3 isoform is significantly upregulated in SRSF2 mutant leukemia patients in RNA-Seq data across three AML cohorts and patients with MDS (FIGs 3D-3E). The splicing alteration was validated by RT-PCR in isogenic K562 cells with heterozygous knockin of the SRSF2P95H / WTmutation as well as in KO52 cells with a naturally occurring SRSF2P95H / WTmutation (FIG. 3F). Consistent with prediction that the CLK3 mis-spliced transcript is an NMD substrate, NMD inhibition (using an inhibitor of SMG1), led to a dose-dependent increase in the mis-spliced isoform in SRSF2-mutant cells (FIG. 3F). SMG1 inhibition also subtly increased CLK3 mis-spliced transcript in SRSF2 wild-type K562 cells but to a much lesser degree compared to SRSF2 mutant cells.

[0305] NMD transcripts can have a higher rate of RNA turnover accompanied by rapid decay of nascent peptides by the proteosome, that may drive frequent production of HLA-I peptides. To confirm that the CLK3 mis-spliced isoform gives rise to the predicted CLK3 neoantigen endogenously, HLA-IP LC-MS / MS was performed on HLA-A*02:01+SRSF2P95H / WTmutant K562 cells as well as KO52 cells. Eluted peptides exhibited the expected sequence preferences at anchor residues. In both cell lines, the eluted peptide matching MS / MS spectra was identified with that of synthetic CLK3 peptide (FIG. 3G).Characterization of TCRs recognizing CLK3 neoantigen

[0306] To isolate CLK3 neoantigen-reactive TCRs, dual-color CLK3 p / HLA-I dextramers were synthetized and CLK3 neoantigen-reactive CD8+T cells from two healthy donors were identified (FIG. 3H). Dextramer+CD8+populations were sorted and singlecell RNA- and TCR-sequencing was performed (FIG. 4A). Expression of cytotoxic markers (GZMB, CCL5, NKG7, and PRF1) indicated productive T cell activation and priming. TCR sequencing revealed 11 distinct TCR clonotypes across two donors (FIG. 4B).

[0307] It was next sought to functionally characterize the CLK3 neoantigen- reactive TCRs. Polyclonal human primary CD8+T cells were transduced with individual TCR panel members. T cells expressing each of the candidate TCRs bound the CLK3 p / HLA-I dextramers, establishing their specificity for the CLK3 neoantigen (FIG. 4C). As a negative control, the absence of dextramer staining on CD8+T cells transduced with an irrelevant CMV TCR was confirmed. The reactivity of each TCR to CLK3 neoantigen was next evaluated by co-culturing TCR-T cells with AML cells (1) loaded with increasing concentrations of CLK3 peptide or (2) electroporated with CLK3 full-length or exon 4 skipped mRNA (FIG. 4D). HLA-A*02:01 -expressing K562 cells co-transduced with CD80 and CD83 were utilized to mimic an artificial antigen presenting cell (aAPC) system. All CLK3 neoantigen-reactive TCR clonotypes elicited robust, dose-dependent production of IFNy and TNFa by TCR-T cells when co-cultured with peptide-loaded K562 cells (FIG. 4E). By contrast, CD8+T cells transduced with control CMV TCR did not react to the CLK3 peptide. The EC50 peptide concentration of each TCR clonotype was calculated, and two TCR clonotypes (TCR2 and TCR3) were found to be especially potent with EC50 values in the picomolar range (FIG. 4F). CD8 co-receptor independence of a TCR, which can be measured by TCR activity in CD4+cells, often correlates with TCR affinity and anti-tumor efficacy. Robust IFNy and TNFa production was measured in both CD8+and CD4+T cells transduced with candidate CLK3 TCRs following co-culture with K562 cells electroporated with CLK3 mis-spliced mRNA (FIG. 4G). These data indicate that the CLK3 neoantigen-reactive TCRs identified are potently reactive to their cognate neoantigen.

[0308] Finally, it was evaluated whether CLK3 neoantigen-reactive TCRs can redirect primary CD8+T cells for specific cytotoxicity of leukemic cells overexpressing CLK3 mis-spliced transcript. CD8+CLK3 TCR-T cells specifically lysed HLA-A*02:01- expressing K562 cells electroporated with CLK3 mis-spliced mRNA and did not lyse cells electroporated with CLK3 wild-type mRNA (FIG. 4H). In contrast, the same TCR-T cells did not lyse HLA-A*02: 01 -negative MV4;11 AML cells electroporated with either CLK3 mRNA isoform, confirming HLA restriction of these TCRs.CLK3 neoantigen-reactive TCR-T cells specifically lyse SRSF2 mutant AML cells

[0309] It was next asked if endogenous SRSF2 mutations can lead to production and HLA presentation of the CLK3 neoantigen at a sufficient level for recognition by CLK3 neoantigen-reactive TCR-T cells. KO52 cells, established from an HLA-A*02:01- AML patient with an endogenous SRSF2P95H / WTmutation, were engineered to express HLA- A*02:01 (FIG. 8A). These cells were co-cultured with CD8+T cells transduced with each of the most potent CLK3 neoantigen-reactive TCRs. All TCRs conferred cytolytic activity to CD8+T cells (FIGs 5A and 8B). No cytolytic activity was seen against KO52 cells without HLA-A*02:01 (FIG. 5B). CLK3 TCR-T cells upregulated canonical T cell activation markers CD69 and 4-1BB only when co-cultured with HLA-A*02:01+KO52 target cells, with a concomitant increase in frequencies of activated caspase 3 / 7+and / or 7- AAD+populations in target cells (FIG. 5B).

[0310] As an orthogonal experiment, firefly luciferase was introduced into KO52 cells and co-cultured with CLK3 neoantigen-reactive TCR-T cells across varying effector- to-target (E:T) ratios. Specific lysis of HL A-A* 02:01+ KO52 cells by CLK3 TCR-T cells was again observed (FIG. 5C). The same TCR-T cells failed to recognize SRSF2-wild-type AML cells (THP-1 and MV4;11) that endogenously or exogenously express HLA- A*02:01, respectively (FIGs 5C and 8A). To confirm that these tumor lines expressed sufficient HLA-A*02:01 for immune recognition, CLK3 peptide was pulsed onto THP1 and MV4;11 cells and this rendered them susceptible to potent killing by the TCR-T cells (FIG. 8C).

[0311] In addition to KO52 cells, it was tested if CLK3 neoantigen-reactive TCR- T cells are reactive to primary cells from patients with SRSF2 mutant AML. CLK3 TCR- T cells were co-cultured with bone marrow mononuclear cells (BM MNCs) or PBMCs from HLA-A*02:01+SRSF2 mutant AML patients, and a significantly increased frequencyof CD69+and 4-1BB+ population was observed in CLK3 TCR-T cells (compared to CMV TCR-T cells) (FIGs 5D-5E and 8D). As negative controls, the same experiment was performed on primary cells from HLA-A*02:01+SRSF2 wild-type AML patients or normal donors, and significant activation of CLK3 TCR-T cells was not observed. It was concluded that SRSF2 mutant AML cells endogenously process and present a sufficient amount of the CLK3 neoantigen to allow their recognition and lysis by CLK3 neoantigen- reactive TCR-T cells.

[0312] The specificity of the CLK3 neoantigen-reactive TCRs and potential off- target peptides was next assessed using alanine scanning. A set of 9-mer peptides was generated, where each of the amino acid residues of the CLK3 neoantigen (RLWGTWVKA; SEQ ID NO:31) was substituted with alanine (or glycine for the amino acid #9 as it is already an alanine). HLA-A*02:01+K562 cells were pulsed with these peptides individually, and co-cultured with CD8+T cells transduced with each of the four CLK3 TCRs, and TNFa and IFNy production measured in TCR-T cells. Peptide positions were defined, resulting in TNFa and / or fFNy release >25% relative to the native CLK3 peptide as being permissive. These data established a peptide recognition motif for each TCR (FIGs 5F and 8E). ScanProsite was utilized to survey the human proteome for these motifs. Importantly, for TCR3, TCR9, and TCR11, the CLK3 neoantigen is the only sequence recognized. For TCR2, potential off-target peptides derived from the proteins BOC (cLWraWsKq; SEQ ID NO:22), DOC-1 (kLWipWmKs; SEQ ID NO:513), GVINP1 (qLWhhWcKk; SEQ ID NO:514), CD360 (pLWrlWkKi; SEQ ID NO:515), and TEX50 (yLWkkWkKh; SEQ ID NO:516) were identified (amino acids in uppercase constitute the TCR2 recognition motif identified from alanine scanning). These peptides were synthesized, and it was confirmed none of them resulted in activation of CD8+T cells transduced with TCR2 (FIG. 8F).

[0313] Finally, it was tested whether the anti-tumor activity of CLK3 neoantigen- reactive TCR-T cells translates into meaningful therapeutic efficacy in vivo. A xenograft model of SRSF2 mutant AML was generated by intravenously injecting firefly luciferase- labeled HLA-A*02:01+KO52 cells into sublethally irradiated NSG mice (FIG. 5G). In the first experiment, the therapeutic efficacy of CLK3 TCR9 was tested by randomizing tumorbearing mice to one of the following treatments: (1) PBS (n=5), (2) CMV-reactive TCR-T cells as a specificity control (n=5), and (3) CLK3 TCR9-T cells (n=8) (FIG. 5G). All mice received an assigned treatment on days 3 and 10 post tumor engraftment in addition to anextended half-life variant of IL-15.31 Mice treated with CLK3 TCR9-T cells had a significantly lower tumor burden compared to those that received PBS or CMV TCR-T controls (FIGs 51, 8G). None of the mice experienced significant weight loss (FIG. 8H).

[0314] In a second experiment, the therapeutic efficacy of another CLK3 neoantigen-reactive TCR (TCR11) was tested in a larger tumor burden model. Here, more time was allowed for the tumor to engraft in mice, and tumor engraftment was confirmed prior to treatment. Mice were randomized to one of the following treatments on days 13 and 20 post-tumor injections: (1) PBS (n=5), (2) CMV-reactive TCR-T cells (n=4), and (3) CLK3 neoantigen-reactive TCR11-T cells (n=9) (FIG. 5G). Mice treated with CLK3 TCR11-T cells had a significantly lower tumor burden and lived significantly longer compared to the controls (FIGs 5H-5I, 81).Isolation of neoantigen-reactive CD8+T cells in myeloid leukemia patients

[0315] The discovery of immunogenic neoantigens in splicing factor mutant leukemias raised the question of how such malignancies persist despite potential neoantigen-reactive immune responses. To establish whether mis-splicing-derived neoantigens can elicit productive CD8+T cell responses in AML patients, a panel of dextramers was generated for the predicted neoantigens. Each dextramer was labeled with a unique DNA barcode to detect, quantify, and characterize neoantigen-reactive CD8+T cells. By performing single-cell RNA" and TCR-sequencing on dextramer-positive patient CD8+T cells without in vitro manipulation, gene expression profiles of neoantigen-reactive CD8+T cells were interrogated in patients in vivo and captured TCR sequence of the individual clones (FIG. 6A). Separate pools of dextramers were generated for HLA- A*02:01+SRSF2 mutant or ZRSR2 mutant patients. The SRSF2 mutant dextramer pool consisted of one positive control dextramer against an HLA-A*02:01 CMV epitope, three negative control dextramers (1 HLA-matched and 2 HLA-mismatched), and 46 dextramers against SRSF2 mutation-induced neopeptides (FIG. 6A). The ZRSR2 mutant dextramer pool consisted of two positive control dextramers against HLA-A*02:01 CMV and EBV epitopes, three negative controls, and 12 dextramers against ZRSR2 mutation-induced neopeptides.

[0316] Bulk T cells from patient PBMCs were stained with a dextramer pool. Live CD3+CD8+dextramer-1-cells were sorted and followed by single-cell RNA-, TCR", and dextramer feature barcode sequencing. For samples where dextramer-1-cell numbers were limiting, live CD3+CD8+dextramer cells were stained with cell hashing antibodiesand spiked into dextramer+populations. Using this approach, single-cell profiling of CD8+T cells from nine samples was performed across five HLA-A*02:01+SRSF2 mutant leukemia patients (FIG. 6B) yielding 75,343 T cells, of which 46,030 were dextramer+. Neoantigen-reactive TCRs accounted for 56% of sequenced TCRs while viral-reactive were 4.5%. Neoantigen-reactive TCRs were present across naive and memory T cell clusters while viral-specific clones exclusively had a memory profile (FIG. 6B; lower right data groups). Based on differentially expressed genes, canonical immune markers, and curated gene signatures, cells were categorized into naive and memory subsets with unsupervised clustering revealing 12 distinct CD8+T cell clusters (FIG. 6B).

[0317] Prior studies in patients with solid tumors have identified differences in cell states between viral-reactive and tumor antigen-reactive CD8+T cells. Neoantigen- reactive T cells were most highly concentrated within cluster 3 marked by pronounced reduction in expression of NF-KB signaling components (FIGs 6B-6E). Scoring against reference gene signatures from datasets of human solid tumor TILs showed that neoantigen-reactive T cells consistently exhibited increased signatures of senescence, exhaustion, and apoptosis with reduced TCR and NF-KB signaling (FIG. 6F). Focusing on genes differentially expressed within effector memory CD8 T cells (clusters 0, 1, 3 and 5) revealed marked downregulation of TNFa via NF-KB signaling genes (NFKBIA, JUN, JUNB, TNF, FOS) in neoantigen-reactive T cells (FIG. 6G). Gene set enrichment analysis (GSEA) further highlighted diminished activity in TNFa via NF-KB pathways for neoantigen-reactive cells against the other two groups.

[0318] Peripheral blood neoantigen-reactive CD8+T cells were analyzed in a separate cohort of four HLA-A*02:01+ZRSR2 mutant myeloid leukemia patients using the ZRSR2 dextramer pool. This analysis also revealed enrichment of neoantigen-reactive CD8+T cells with an effector memory profile and reduced expression of TNFa via NF-KB pathway genes compared to CD8+T cells of undefined specificity.

[0319] The relationship between T cell phenotype and TCR clonal abundance amongst 10,976 unique clonotypes was also evaluated (FIG. 6H). As expected, highly expanded clones resided within the memory compartment. Although CMV-reactive TCRs comprised predominantly large clones, large and hyperexpanded neoantigen-reactive TCRs clones were also highly abundant. GSEA demonstrated significant loss of TNFa versus NF-KB signaling in hyperexpanded clones in neoantigen-reactive compared to CMV-reactive TCRs (FIG. 61). Taken together, these results revealed that neoantigen-reactive CD8+T cells are present in the peripheral blood of AML patients, are clonally expanded, and yet have a distinct phenotype from virus-reactive T cells with transcriptional evidence of impaired cytotoxicity.Characterizing neoantigen-reactive CD8+ T cells post curative allogeneic transplant

[0320] For patients with high-risk myeloid leukemia, allo-HCT remains the most established curative therapy. In the allo-HCT setting, donor alloreactive T cells recognizing host minor histocompatibility antigens contribute to the GVL effect. It was tested whether CD8+T cells reactive to SRSF2 mutation-induced neoantigens can be identifiable in this therapeutic setting.

[0321] To investigate this, dextramer-based single-cell profiling of pre- and posttransplant PBMCs from a 54-year-old patient with SRSF2R94duP-mutant MDS with excessive blasts- 1 (Patient 2) was performed. Pre-transplant samples were collected in the setting of persistent disease following two cycles of decitabine while post-transplant samples were collected 15 months post curative allo-HCT from an HLA-matched donor. Post-transplant T cells primarily fell within the memory T cell cluster characterized by high expression of NKG7, PRF1, and CCL5 (cluster 0, FIGs 7A-7B). GSEA of differentially expressed genes pre- versus post-transplant within memory CD8+T cell clusters (0, 1 and 3) revealed enrichment of multiple inflammatory and proliferation pathways in posttransplant T cells, indicative of T cell activation. Post-transplant effector T cells showed increased expression of hallmark T cell activation and cytotoxicity genes (PRF1, KLRF1, GZMB, GNLY). There was no sharing of neoantigen-reactive TCRs pre- and posttransplant, suggesting the emergence of likely donor-derived tumor-reactive TCR clonotypes post-transplant. Interestingly, when clone size of TCR clonotypes was compared between two timepoints, hyperexpanded clonotypes (>500 cells) were only present post-transplant (FIG. 7C). Another patient (Patient 1) was profiled with SRSR2P95H / WT-mutant CMML prior to and 1-y ear-post curative allo-HCT. Once again, post-transplant T cells were most abundant within effector memory CD8+T cell clusters, and GSEA revealed upregulated inflammatory (TNFa via NF-KB) and proliferation (mitotic spindle, mTORCl) pathways compared to pre-transplant T cells or other neoantigen-reactive T cells in the same cluster. TCR clonotypes were completely nonoverlapping pre- versus post-transplant, and hyper-expanded TCR clones were only seen post-transplant.

[0322] It was tested whether abundant and phenotypically activated neoantigen- reactive TCRs post-transplant might be driven by mis-splicing-derived neoantigens presented by splicing mutant leukemic cells. Expansion of one TCR clonotype (CAIRGGDSFLFNQPQHF; SEQ ID NO:512) was found in the post-transplant sample of Patient 2, which was predicted to bind dextramers created with a peptide (CLLPPALFL; SEQ ID NO:42) derived from an SRSF2 mutant-induced mis-splicing event in RHOT2 (FIGs. 7D-7E). The immunogenicity of this peptide was previously validated (FIGs. 2A- 2F).

[0323] To validate the antigen reactivity of the RHOT2 neoantigen-reactive TCR, human primary T cells were transduced with this TCR and binding confirmed to the RHOT2 #5 peptide by staining with RHOT2 #5 p / HLA-I dextramers. The TCR-T cells were then co-cultured with RHOT2 peptide-pulsed K562 aAPCs and dose-dependent production of IFNy and TNFa measured in CD8+and CD4+T cells (EC50125 and 930 pM, respectively) (FIGs 7F-7G). In addition, RHOT2 TCR-T cells were co-cultured with K562 aAPCs electroporated with RHOT2 wild-type or mis-spliced mRNA and RHOT2 neoantigen-induced T cell activation and cytotoxicity was confirmed (FIGs 7H-7I). Moreover, RHOT2 TCR-T cells were co-cultured with SRSF2 mutant KO52 cells. RHOT2 TCR-T cells upregulated surface CD69 and 4-1BB only when co-cultured with KO52 cells expressing HLA-A*02:01, while simultaneously inducing apoptosis of KO52 cells (FIGs 7J-7K). Lastly, RHOT2 TCR-T cells potently suppressed growth of KO52 cells in vitro (FIG. 7L). Overall, these data provide evidence of an endogenous T cell response to mis- splicing-derived neoantigens in myeloid leukemia patients in the post-transplant setting, with donor-derived T cell clones capable of contributing to long-term anti-leukemic T cell immunity.Discussion

[0324] In this example are presented a novel series of splicing factor mutation- induced neoantigens and cognate TCRs which can selectively recognize and lyse spliceosomal mutant AML cells. Neoantigens arising from RNA mis-splicing events that involve non-protein coding regions such as long non-coding RNAs, introns, circular RNAs, and transposable elements have been documented. While some of these neoantigens have been described as recurrent, the molecular basis for why these neoantigens would be shared between patients is elusive. By contrast, the molecular mechanisms of how mutations in RNA splicing factors lead to highly recurrent mis-splicing events across patients are nowincreasingly understood. The recurrent nature of mis-splicing occurs due to the defined roles that each splicing factor plays in RNA splicing and the sequence-specific impact of these mutations on RNA recognition. Thus, many neoantigens translated from these mis- splicing events are shared across patients. Moreover, RNA splicing factor mutations lead to numerous mis-splicing events which can generate hundreds to even thousands of potential mis-splicing-derived neoepitopes. The sheer number of neoepitopes created by these mutations increases the likelihood that neoantigens can be presented on a wide diversity of HLA alleles.

[0325] This example provides neoantigen-reactive CD8+T cells and provides new insights into the immune dysregulation that occurs in MDS / AML patients. It was found that CD8+T cells reactive to mis-splicing-derived neoantigens exist in the peripheral blood of patients with spliceosomal mutant leukemias. However, these T cells have defective NF-KB proinflammatory pathways, which might explain why these cells are unable to mount effective antigen-reactive immune responses in patients. Single-cell profiling of matched pre- and post-transplant samples suggested that allogeneic donor T cells have gene expression profiles of activation and cytotoxicity compared to the patient’s endogenous neoantigen-reactive T cells. Moreover, donor-derived CD8+T cells against RHOT2 neoantigen were discovered in one patient. Future studies can also be warranted to test the therapeutic efficacy and safety of this TCR, along with CLK3 neoantigen- reactive TCRs, against leukemias with splicing factor mutations. Of note, two recent clinical trials of TCR-edited T cells in solid cancers have created and infused gene-edited T cells with TCRs against up to three distinct epitopes into the same patient. It is envisioned that an approach can be implemented for myeloid leukemia patients with splicing factor mutations, given the multitude of public mis-splicing derived neoantigens created by these mutations.

[0326] While this example focused on patients with myeloid neoplasms, it is important to note that mutations in splicing factors are recurrent in the pre-disease settings of clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS). Importantly, mutations in RNA splicing factors when present in CH confer higher risk of transformation to overt myeloid leukemia. One can also utilize a similar dextramer-based approach to study the characteristics of such neoantigen-reactive T cells in the earliest stages of pre-malignant disease. Such studies may motivate neoantigen vaccinationapproaches in splicing factor mutant CH and CCUS using collections of mis-splicing- derived peptides or the mRNAs encoding them, in the future.

[0327] This example focused on the most common HLA class I allele in the patient cohorts (z.e., HLA-A*02:01), however, additional studies can be implemented to identify neoantigens presentable on other common HLA-I alleles as well as their cognate TCRs. On a similar note, the disclosed therapeutic approach is achievable through targeting HLA class Il-restricted neoantigens, which is relevant in neoantigen-based vaccination.

[0328] In the disclosed in vivo experiments using immune-deficient mice, early timepoints after T cell transfer were focused on to evaluate the therapeutic efficacy of neoantigen-reactive TCR-T cells due to the potential for graft-versus-host-disease, which could confound interpretation of overall survival. Future in vivo studies in HLA-matched humanized mice can be implemented, including as neoantigen-reactive TCR constructs are optimized to improve TCR-T cell activation and persistence. Finally, while neoantigenbased vaccination is proposed as a viable therapeutic strategy in patients with myeloid malignancies, it is noted that endogenous neoantigen-reactive CD8+T cells in patients with active disease are dysfunctional. It is therefore unclear whether patients with active MDS or AML could respond to neoantigen vaccination. As such, careful consideration of when to perform vaccination would be critical, and future studies to better understand the molecular mechanisms of immune dysfunction in patients with MDS or AML will provide critical insight on how to maximize the therapeutic potential of the disclosed therapies.

[0329] Next, the TCRs recognizing the CLK3 mis-splicing derived peptide created by mutant SRSF2 were identified. Dextramer-double-positive CD8+T cells were identified in 2 unique healthy donors which underwent in vitro stimulation with CLK3 misspliced peptide, sorted these T cells, and performed (1) dextramer feature barcode and (2) VDJ sequencing to extract their TCR sequences (FIG. 7A). This resulted in the discovery of 14 distinct TCRs recognizing the CLK3 mis-spliced peptide (FIGs 7B-7C). The specificity of these 14 TCRs was next tested by expressing them retrovirally in primary human CD8 T cells and then co-culturing them with K562 cells or K562 cells loaded with the cognate CLK3 peptide or K562 cells electroporated with in vitro transcribed mRNA encoding the CLK3 neoantigenic peptide. Intracellular cytokine staining of the transgenic TCR expressing T-cells for IFNy and TNFa revealed strong activation of T cells bearing the clonotype 2 or 3 TCRs against the CLK3 peptide (FIG. 7D).

[0330] As a next step, antigen-specific T cells and their TCRs were identified directly from HLA-A*02:01 myeloid leukemia patients with the SRSF2 P95 mutation. This was performed using a panel of fluorescently labeled DNA-barcoded dextramers including (I) 46 distinct dextramers each against a unique SRSF2 mutant-induced mis-spliced peptide, (ii) three negative control dextramers, and (iii) positive control dextramers against antigens derived from CMV expected to be presented on HLA-A*02:01 in all individuals. This panel of dextramers was used and stained peripheral blood mononuclear cells from several HLA-A*02:01 myeloid leukemia patients with the SRSF2 P95 mutation and at distinct points of disease in treatment. This was applied to a patient with MDS prior to allogeneic stem cell transplant and 15 months post-transplant (FIG. 8 A). Using these data, an expanded clone of T-cells shared across pre- and post-transplant timepoints against a mis-spliced derived neoantigen from the transcript encoding RHOT2 (FIGs 8A-8C) with the TCR sequence of CASSSRTGGPDTQYF (SEQ ID NO:484) was identified. Similarly, in another patient, an expanded clone of T-cells was identified post-transplant against a mis-spliced derived neoantigen from the transcript encoding EZH2 (FIGs 8D-8F).NON-LIMITING EMBODIMENTS

[0331] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting Embodiments contain elements that are modular and can be combined with each other in any number, order, or combination to form a new non-limiting Embodiment, which can itself be further combined with other non-limiting Embodiments.

[0332] Embodiment 1. A neoantigen comprising a polypeptide sequence associated with an oncogenic mutation in one or more RNA splicing factors.

[0333] Embodiment 2. The neoantigen of Embodiment 1 or any other Embodiment, wherein the one or more RNA splicing factors comprises SRSF2, SF3B1 , U2A L ZRSR2.jor any combination thereof.

[0334] Embodiment s. The neoantigen of Embodiment 1 or any other Embodiment, wherein the polypeptide sequence is selected from the group consisting of SEQ ID NOs: 1-76.

[0335] Embodiment 4. The neoantigen of Embodiment 2 or any other Embodiment, wherein the one or more RNA splicing factors comprises SRSF2.

[0336] Embodiment s. The neoantigen of Embodiment 4 or any other Embodiment, wherein the oncogenic mutation comprises a P95 mutation of SRSF2 as a change-of-function mutation.

[0337] Embodiment 6. The neoantigen of Embodiment 5 or any other Embodiment, wherein the P95 mutation of SRSF2 comprises a P95H mutation, a deletion of the contiguous amino acid sequence from P95 to R102, or a P95-R insertion of SRSF2.

[0338] Embodiment 7. The neoantigen of any one of Embodiments 4-6 or any other Embodiment, wherein the neoantigen comprises the polypeptide sequence of SEQ ID NO:6, SEQ ID NO:31, or SEQ ID NO:32.

[0339] Embodiment 8. The neoantigen of Embodiment 2 or any other Embodiment, wherein the one or more RNA splicing factors comprises ZRSR2.

[0340] Embodiment 9. The neoantigen of Embodiment 8 or any other Embodiment, wherein the oncogenic mutation comprises a loss-of-function mutation of ZRSR2.

[0341] Embodiment 10. The neoantigen of Embodiment 9 or any other Embodiment, wherein the neoantigen comprises the polypeptide sequence of SEQ ID NO:7, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, or any combination thereof.

[0342] Embodiment 11. The neoantigen of Embodiment 2 or any other Embodiment, wherein the neoantigen comprises a polypeptide sequence associated with a mis-spliced CLK3 RNA transcript.

[0343] Embodiment 12. A vaccine comprising a neoantigen of any one of Embodiments 1-11 or any other Embodiment.

[0344] Embodiment 13. The vaccine of Embodiment 12 or any other Embodiment, further comprising a pharmaceutically acceptable carrier.

[0345] Embodiment 14. A method for vaccinating a subject against one or more cancers, the method comprising administering an effective amount of the vaccine of any one of Embodiments 12-13 or any other Embodiment to the subject.

[0346] Embodiment 15. A method for treating a subject suspected to develop one or more cancers, the method comprising administering an effective amount of the vaccine of any one of Embodiments 12-13 or any other Embodiment to the subject.

[0347] Embodiment 16. Use of a neoantigen of any one of Embodiments 1- 11 or any other Embodiment in the preparation of a medicament or a vaccine, wherein the medicament or the vaccine is for administration to a subject for treatment or prevention of one or more cancers.

[0348] Embodiment 17. The method or use of any one of Embodiments 14- 16 or any other Embodiment, wherein the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.

[0349] Embodiment 18. A T cell receptor (TCR) or TCR-like protein comprising an antigen binding domain configured to bind to the neoantigen of any one of Embodiments 1-11 or any other Embodiment.

[0350] Embodiment 19. A T cell receptor (TCR) or TCR-like protein configured to bind an antigen that comprises a polypeptide sequence associated with a misspliced CLK3 RNA transcript.

[0351] Embodiment 20. The T cell receptor (TCR) or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:79; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:80; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:81; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:83; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:84; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:85.

[0352] Embodiment 21. The TCR or TCR-like protein of Embodiment 20 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence comprising SEQ ID NO:78.

[0353] Embodiment 22. The TCR or TCR-like protein of any one of Embodiments 20-21 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence comprising SEQ ID NO:82.

[0354] Embodiment 23. The TCR or TCR-like protein of any one of Embodiments 20-22 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:86.

[0355] Embodiment 24. The TCR or TCR-like protein of any one of Embodiments 20-23 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:87.

[0356] Embodiment 25. The TCR or TCR-like protein of any one of Embodiments 20-24 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:77.

[0357] Embodiment 26. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:90; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:91; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:92; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:94; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:95; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:96.

[0358] Embodiment 27. The TCR or TCR-like protein of Embodiment 26 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:89.

[0359] Embodiment 28. The TCR or TCR-like protein of any one of Embodiments 26-27 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 93.

[0360] Embodiment 29. The TCR or TCR-like protein of any one of Embodiments 26-28 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:97.

[0361] Embodiment 30. The TCR or TCR-like protein of any one of Embodiments 26-29 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:98.

[0362] Embodiment 31. The TCR or TCR-like protein of any one of Embodiments 26-30 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:88.

[0363] Embodiment 32. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; whereinthe beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 101; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 102; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 103; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 105; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 106; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 107.

[0364] Embodiment 33. The TCR or TCR-like protein of Embodiment 32 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 100.

[0365] Embodiment 34. The TCR or TCR-like protein of any one of Embodiments 32-33 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 104.

[0366] Embodiment 35. The TCR or TCR-like protein of any one of Embodiments 32-34 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 108.

[0367] Embodiment 36. The TCR or TCR-like protein of any one of Embodiments 32-35 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 109.

[0368] Embodiment 37. The TCR or TCR-like protein of any one of Embodiments 32-36 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:99.

[0369] Embodiment 38. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 112; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 113; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 114; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 116; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 117; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 118.

[0370] Embodiment 39. The TCR or TCR-like protein of Embodiment 38 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 111.

[0371] Embodiment 40. The TCR or TCR-like protein of any one of Embodiments 38-39 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 115.

[0372] Embodiment 41. The TCR or TCR-like protein of any one of Embodiments 38-40 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 119.

[0373] Embodiment 42. The TCR or TCR-like protein of any one of Embodiments 38-41 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 120.

[0374] Embodiment 43. The TCR or TCR-like protein of any one of Embodiments 38-42 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 110.

[0375] Embodiment 44. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 123; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 124; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 125; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 127; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 128; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 129.

[0376] Embodiment 45. The TCR or TCR-like protein of Embodiment 44 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 122.

[0377] Embodiment 46. The TCR or TCR-like protein of any one of Embodiments 44-45 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 126.

[0378] Embodiment 47. The TCR or TCR-like protein of any one of Embodiments 44-46 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 130.

[0379] Embodiment 48. The TCR or TCR-like protein of any one of Embodiments 44-47 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 131.

[0380] Embodiment 49. The TCR or TCR-like protein of any one of Embodiments 44-48 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 121.

[0381] Embodiment 50. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 134; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 135; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 136; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 138; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 139; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 140.

[0382] Embodiment 51. The TCR or TCR-like protein of Embodiment 50 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 133.

[0383] Embodiment 52. The TCR or TCR-like protein of any one of Embodiments 50-51 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 137.

[0384] Embodiment 53. The TCR or TCR-like protein of any one of Embodiments 50-52 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 141.

[0385] Embodiment 54. The TCR or TCR-like protein of any one of Embodiments 50-53 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 142.

[0386] Embodiment 55. The TCR or TCR-like protein of any one of Embodiments 50-54 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 132.

[0387] Embodiment 56. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 145; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 146; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 147; and wherein the alphachain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 149; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 150; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 151.

[0388] Embodiment 57. The TCR or TCR-like protein of Embodiment 56 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 144.

[0389] Embodiment 58. The TCR or TCR-like protein of any one of Embodiments 56-57 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 148.

[0390] Embodiment 59. The TCR or TCR-like protein of any one of Embodiments 56-58 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 152.

[0391] Embodiment 60. The TCR or TCR-like protein of any one of Embodiments 56-59 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 153.

[0392] Embodiment 61. The TCR or TCR-like protein of any one of Embodiments 56-60 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 143.

[0393] Embodiment 62. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 156; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 157; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 158; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 160; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 161; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 162.

[0394] Embodiment 63. The TCR or TCR-like protein of Embodiment 62 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 155.

[0395] Embodiment 64. The TCR or TCR-like protein of any one of Embodiments 62-63 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 159.

[0396] Embodiment 65. The TCR or TCR-like protein of any one of Embodiments 62-64 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 163.

[0397] Embodiment 66. The TCR or TCR-like protein of any one of Embodiments 62-65 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 164.

[0398] Embodiment 67. The TCR or TCR-like protein of any one of Embodiments 62-66 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 154.

[0399] Embodiment 68. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 167; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 168; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 169; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 171; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 172; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 173.

[0400] Embodiment 69. The TCR or TCR-like protein of Embodiment 68 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 166.

[0401] Embodiment 70. The TCR or TCR-like protein of any one of Embodiments 68-69 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 170.

[0402] Embodiment 71. The TCR or TCR-like protein of one of Embodiments 68-70 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 174.

[0403] Embodiment 72. The TCR or TCR-like protein of one of Embodiments 68-71 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 175.

[0404] Embodiment 73. The TCR or TCR-like protein of one ofEmbodiments 68-72 or any other Embodiment, comprising a polypeptide sequence havingat least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 165.

[0405] Embodiment 74. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 178; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 179; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 180; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 182; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 183; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 184.

[0406] Embodiment 75. The TCR or TCR-like protein of Embodiment 74 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 177.

[0407] Embodiment 76. The TCR or TCR-like protein of any one of Embodiments 74-75 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 181.

[0408] Embodiment 77. The TCR or TCR-like protein of any one of Embodiments 74-76 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 185.

[0409] Embodiment 78. The TCR or TCR-like protein of any one of Embodiments 74-77 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 186.

[0410] Embodiment 79. The TCR or TCR-like protein of any one of Embodiments 74-78 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 186.

[0411] Embodiment 80. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 189; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 190; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 191; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ IDNO: 193; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 194; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 195.

[0412] Embodiment 81. The TCR or TCR-like protein of Embodiment 80 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 188.

[0413] Embodiment 82. The TCR or TCR-like protein of any one of Embodiments 80-81 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 192.

[0414] Embodiment 83. The TCR or TCR-like protein of any one of Embodiments 80-82 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 196.

[0415] Embodiment 84. The TCR or TCR-like protein of any one of Embodiments 80-83 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 197.

[0416] Embodiment 85. The TCR or TCR-like protein of any one of Embodiments 80-84 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 187.

[0417] Embodiment 86. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:200; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:201; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:202; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:204; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:205; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:206.

[0418] Embodiment 87. The TCR or TCR-like protein of Embodiment 86 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 199.

[0419] Embodiment 88. The TCR or TCR-like protein of any one of Embodiments 86-87 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:203.

[0420] Embodiment 89. The TCR or TCR-like protein of any one of Embodiments 86-88 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:207.

[0421] Embodiment 90. The TCR or TCR-like protein of any one of Embodiments 86-89 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:208.

[0422] Embodiment 91. The TCR or TCR-like protein of any one of Embodiments 86-90 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 198.

[0423] Embodiment 92. The TCR or TCR-like protein of Embodiment 19 or any other Embodiment, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:211; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:212; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:213; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:215; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:216; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:217.

[0424] Embodiment 93. The TCR or TCR-like protein of Embodiment 92 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:210.

[0425] Embodiment 94. The TCR or TCR-like protein of any one of Embodiments 92-93 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:214.

[0426] Embodiment 95. The TCR or TCR-like protein of any one of Embodiments 92-94 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:218.

[0427] Embodiment 96. The TCR or TCR-like protein of any one of Embodiments 92-95 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:219.

[0428] Embodiment 97. The TCR or TCR-like protein of any one of Embodiments 92-96 or any other Embodiment, comprising a polypeptide sequence havingat least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:209.

[0429] Embodiment 98. A T cell receptor (TCR) or TCR-like protein configured to bind an antigen that comprises a polypeptide sequence associated with a misspliced RH0T2 RNA transcript, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:482; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:483; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:484; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:486; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:487; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:488.

[0430] Embodiment 99. The TCR or TCR-like protein of Embodiment 98 or any other Embodiment, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:481.

[0431] Embodiment 100. The TCR or TCR-like protein of any one of Embodiments 98-99 or any other Embodiment, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:485.

[0432] Embodiment 101. The TCR or TCR-like protein of any one of Embodiments 98-100 or any other Embodiment, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:489.

[0433] Embodiment 102. The TCR or TCR-like protein of any one of Embodiments 98-101 or any other Embodiment, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:490.

[0434] Embodiment 103. The TCR or TCR-like protein of any one of Embodiments 98-102 or any other Embodiment, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:480.

[0435] Embodiment 104. The TCR or TCR-like protein of any one of Embodiments 19-102 or any other Embodiment, wherein the TCR or TCR-like protein is a TCR-like antibody or a TCR-like bispecific antibody.

[0436] Embodiment 105. A nucleic acid comprising a polynucleotide sequence encoding the TCR or TCR-like protein of any one of Embodiments 19-104 or any other Embodiment.

[0437] Embodiment 106. A nucleic acid vector or nucleic acid vaccine comprising the polynucleotide sequence of Embodiment 105 or any other Embodiment.

[0438] Embodiment 107. A composition comprising the TCR or TCR-like protein of any one of Embodiments 19-104 or any other Embodiment, the nucleic acid of Embodiment 105 or any other Embodiment, the nucleic acid vector or nucleic acid vaccine of Embodiment 106 or any other Embodiment, or any combination thereof.

[0439] Embodiment 108. An immune cell comprising the nucleic acid of Embodiment 105 or any other Embodiment.

[0440] Embodiment 109. The immune cell of Embodiment 108 or any other Embodiment, wherein the immune cell is a T cell.

[0441] Embodiment 110. The immune cell of Embodiment 109 or any other Embodiment, wherein the T cell is transgenic and comprises a polynucleotide sequence encoding the TCR or TCR-like protein of any one of Embodiments 19-104 stably integrated within a genome of the T cell.

[0442] Embodiment 111. A composition for immune therapy by administration of the composition to a subject for treatment of one or more cancers in the subject, the composition comprising the immune cell of any one of Embodiments 108-110 or any other Embodiment.

[0443] Embodiment 112. The composition of Embodiment 111 or any other Embodiment, further comprising a pharmaceutically acceptable carrier.

[0444] Embodiment 113. A method for treatment of one or more cancers in a subject in need thereof, the method comprising administering, to the subject, an effective amount of: the TCR or TCR-like protein of any one of Embodiments 19-104 or any other Embodiment; the nucleic acid of Embodiment 105 or any other Embodiment; the nucleic acid vector or nucleic acid vaccine of Embodiment 106 or any other Embodiment; the immune cell of any one of Embodiments 108-110 or any other Embodiment; the composition of any one of Embodiments 107 and 111-112 or any other Embodiment; or any combination thereof.

[0445] Embodiment 114. Use of an agent in the preparation of a medicament, wherein the medicament is for administration to a subject for treatment or prevention of one or more cancers and the agent comprises: the TCR or TCR-like protein of any one of Embodiments 19-104 or any other Embodiment; the nucleic acid of Embodiment 105 or any other Embodiment; the nucleic acid vector or nucleic acid vaccine of Embodiment 106or any other Embodiment; the immune cell of any one of Embodiments 108-110 or any other Embodiment; the composition of any one of Embodiments 107 and 111-112 or any other Embodiment; or any combination thereof.

[0446] Embodiment 115. The method of Embodiment 113 or any other Embodiment or the use of Embodiment 114 or any other Embodiment, wherein the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.

[0447] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A neoantigen comprising a polypeptide sequence associated with an oncogenic mutation in one or more RNA splicing factors.

2. The neoantigen of claim 1, wherein the one or more RNA splicing factors comprises SRSF2, SF3B1, U2AF1, ZRSR2, or any combination thereof.

3. The neoantigen of claim 1, wherein the polypeptide sequence is selected from the group consisting of: SEQ ID NOs: l-76.

4. The neoantigen of claim 2, wherein the one or more RNA splicing factors comprises SRSF2.

5. The neoantigen of claim 4, wherein the oncogenic mutation comprises a P95 mutation of SRSF2 as a change-of-function mutation.

6. The neoantigen of claim 5, wherein the P95 mutation of SRSF2 comprises a P95H mutation, a deletion of the contiguous amino acid sequence from P95 to R102, or a P95-R insertion of SRSF2.

7. The neoantigen of any one of claims 4-6, wherein the neoantigen comprises the polypeptide sequence of SEQ ID NO:6, SEQ ID NO:31, or SEQ ID NO:32.

8. The neoantigen of claim 2, wherein the one or more RNA splicing factors comprises ZRSR2.

9. The neoantigen of claim 8, wherein the oncogenic mutation comprises a loss-of-function mutation of ZRSR2.

10. The neoantigen of claim 9, wherein the neoantigen comprises the polypeptide sequence of SEQ ID NO:7, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, or any combination thereof.

11. The neoantigen of claim 2, wherein the neoantigen comprises a polypeptide sequence associated with a mis-spliced CLK3 RNA transcript.

12. A vaccine comprising a neoantigen of any one of claims 1-11.

13. The vaccine of claim 12, further comprising a pharmaceutically acceptable carrier.

14. A method for vaccinating a subject against one or more cancers, the method comprising administering an effective amount of the vaccine of any one of claims 12-13 to the subject.

15. A method for treating a subject suspected to develop one or more cancers, the method comprising administering an effective amount of the vaccine of any one of claims 12-13 to the subject.

16. Use of a neoantigen of any one of claims 1-11 in the preparation of a medicament or a vaccine, wherein the medicament or the vaccine is for administration to a subject for treatment or prevention of one or more cancers.

17. The method or use of any one of claims 14-16, wherein the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.

18. A T cell receptor (TCR) or TCR-like protein comprising an antigen binding domain configured to bind to the neoantigen of any one of claims 1-11.

19. A T cell receptor (TCR) or TCR-like protein configured to bind an antigen that comprises a polypeptide sequence associated with a mis-spliced CLK3 RNA transcript.

20. The T cell receptor (TCR) or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:79; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:80; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:81; andwherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 83; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:84; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:85.

21. The TCR or TCR-like protein of claim 20, wherein the beta chain comprises: a beta variable polypeptide sequence comprising SEQ ID NO:78.

22. The TCR or TCR-like protein of any one of claims 20-21, wherein the alpha chain comprises: an alpha variable polypeptide sequence comprising SEQ ID NO:82.

23. The TCR or TCR-like protein of any one of claims 20-22, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:86.

24. The TCR or TCR-like protein of any one of claims 20-23, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:87.

25. The TCR or TCR-like protein of any one of claims 20-24, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:77.

26. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:90; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:91; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:92; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 94; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:95; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:96.

27. The TCR or TCR-like protein of claim 26, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:89.

28. The TCR or TCR-like protein of any one of claims 26-27, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:93.

29. The TCR or TCR-like protein of any one of claims 26-28, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:97.

30. The TCR or TCR-like protein of any one of claims 26-29, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 98.

31. The TCR or TCR-like protein of any one of claims 26-30, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:88.

32. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 101; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 102; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 103; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 105; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 106; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 107.

33. The TCR or TCR-like protein of claim 32, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 100.

34. The TCR or TCR-like protein of any one of claims 32-33, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 104.

35. The TCR or TCR-like protein of any one of claims 32-34, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 108.

36. The TCR or TCR-like protein of any one of claims 32-35, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 109.

37. The TCR or TCR-like protein of any one of claims 32-36, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:99.

38. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 112; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 113; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:114; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 116; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 117; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 118.

39. The TCR or TCR-like protein of claim 38, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 111.

40. The TCR or TCR-like protein of any one of claims 38-39, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 115.

41. The TCR or TCR-like protein of any one of claims 38-40, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 119.

42. The TCR or TCR-like protein of any one of claims 38-41, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 120.

43. The TCR or TCR-like protein of any one of claims 38-42, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 110.

44. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises:a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 123; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 124; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 125; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 127; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 128; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 129.

45. The TCR or TCR-like protein of claim 44, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 122.

46. The TCR or TCR-like protein of any one of claims 44-45, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 126.

47. The TCR or TCR-like protein of any one of claims 44-46, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 130.

48. The TCR or TCR-like protein of any one of claims 44-47, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 131.

49. The TCR or TCR-like protein of any one of claims 44-48, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 121.

50. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 134; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 135; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 136; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 138; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 139; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 140.

51. The TCR or TCR-like protein of claim 50, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 133.

52. The TCR or TCR-like protein of any one of claims 50-51, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 137.

53. The TCR or TCR-like protein of any one of claims 50-52, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 141.

54. The TCR or TCR-like protein of any one of claims 50-53, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 142.

55. The TCR or TCR-like protein of any one of claims 50-54, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 132.

56. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 145; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 146; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 147; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 149; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 150; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 151.

57. The TCR or TCR-like protein of claim 56, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 144.

58. The TCR or TCR-like protein of any one of claims 56-57, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 148.

59. The TCR or TCR-like protein of any one of claims 56-58, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 152.

60. The TCR or TCR-like protein of any one of claims 56-59, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 153.

61. The TCR or TCR-like protein of any one of claims 56-60, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 143.

62. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 156; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 157; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:158; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 160; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 161; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 162.

63. The TCR or TCR-like protein of claim 62, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 155.

64. The TCR or TCR-like protein of any one of claims 62-63, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 159.

65. The TCR or TCR-like protein of any one of claims 62-64, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 163.

66. The TCR or TCR-like protein of any one of claims 62-65, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 164.

67. The TCR or TCR-like protein of any one of claims 62-66, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 154.

68. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 167; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 168; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 169; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 171; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 172; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 173.

69. The TCR or TCR-like protein of claim 68, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 166.

70. The TCR or TCR-like protein of any one of claims 68-69, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 170.

71. The TCR or TCR-like protein of one of claims 68-70, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 174.

72. The TCR or TCR-like protein of one of claims 68-71, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 175.

73. The TCR or TCR-like protein of one of claims 68-72, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 165.

74. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 178; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 179; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 180; and wherein the alpha chain comprises:an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 182; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 183; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 184.

75. The TCR or TCR-like protein of claim 74, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 177.

76. The TCR or TCR-like protein of any one of claims 74-75, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 181.

77. The TCR or TCR-like protein of any one of claims 74-76 wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 185.

78. The TCR or TCR-like protein of any one of claims 74-77, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 186.

79. The TCR or TCR-like protein of any one of claims 74-78, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 186.

80. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO: 189; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO: 190; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO: 191; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO: 193; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO: 194; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO: 195.

81. The TCR or TCR-like protein of claim 80, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 188.

82. The TCR or TCR-like protein of any one of claims 80-81, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO: 192.

83. The TCR or TCR-like protein of any one of claims 80-82, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO: 196.

84. The TCR or TCR-like protein of any one of claims 80-83, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO: 197.

85. The TCR or TCR-like protein of any one of claims 80-84, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 187.

86. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:200; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:201; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:202; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:204; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:205; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:206.

87. The TCR or TCR-like protein of claim 86, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO: 199.

88. The TCR or TCR-like protein of any one of claims 86-87, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:203.

89. The TCR or TCR-like protein of any one of claims 86-88, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:207.

90. The TCR or TCR-like protein of any one of claims 86-89, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:208.

91. The TCR or TCR-like protein of any one of claims 86-90, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO: 198.

92. The TCR or TCR-like protein of claim 19, the protein comprising a beta chain and an alpha chain; wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:211; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:212; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:213; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:215; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:216; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:217.

93. The TCR or TCR-like protein of claim 92, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:210.

94. The TCR or TCR-like protein of any one of claims 92-93, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:214.

95. The TCR or TCR-like protein of any one of claims 92-94, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:218.

96. The TCR or TCR-like protein of any one of claims 92-95, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:219.

97. The TCR or TCR-like protein of any one of claims 92-96, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:209.

98. A T cell receptor (TCR) or TCR-like protein configured to bind an antigen that comprises a polypeptide sequence associated with a mis-spliced RH0T2 RNA transcript, the protein comprising a beta chain and an alpha chain;wherein the beta chain comprises: a beta variable CDR1 polypeptide sequence comprising SEQ ID NO:482; a beta variable CDR2 polypeptide sequence comprising SEQ ID NO:483; a beta variable CDR3 polypeptide sequence comprising SEQ ID NO:484; and wherein the alpha chain comprises: an alpha variable CDR1 polypeptide sequence comprising SEQ ID NO:486; an alpha variable CDR2 polypeptide sequence comprising SEQ ID NO:487; and an alpha variable CDR3 polypeptide sequence comprising SEQ ID NO:488.

99. The TCR or TCR-like protein of claim 98, wherein the beta chain comprises: a beta variable polypeptide sequence of SEQ ID NO:481.

100. The TCR or TCR-like protein of any one of claims 98-99, wherein the alpha chain comprises: an alpha variable polypeptide sequence of SEQ ID NO:485.

101. The TCR or TCR-like protein of any one of claims 98-100, wherein the beta chain comprises: a beta constant polypeptide sequence of SEQ ID NO:489.

102. The TCR or TCR-like protein of any one of claims 98-101, wherein the alpha chain comprises: an alpha constant polypeptide sequence of SEQ ID NO:490.

103. The TCR or TCR-like protein of any one of claims 98-102, comprising a polypeptide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the polypeptide sequence of SEQ ID NO:480.

104. The TCR or TCR-like protein of any one of claims 19-102, wherein the TCR or TCR-like protein is a TCR-like antibody or a TCR-like bispecific antibody.

105. A nucleic acid comprising a polynucleotide sequence encoding the TCR or TCR-like protein of any one of claims 19-104.

106. A nucleic acid vector or nucleic acid vaccine comprising the polynucleotide sequence of claim 105.

107. A composition comprising the TCR or TCR-like protein of any one of claims 19-104, the nucleic acid of claim 105, the nucleic acid vector or nucleic acid vaccine of claim 106, or any combination thereof.

108. An immune cell comprising the nucleic acid of claim 105.

109. The immune cell of claim 108, wherein the immune cell is a T cell.

110. The immune cell of claim 109, wherein the T cell is transgenic and comprises a polynucleotide sequence encoding the TCR or TCR-like protein of any one of claims 19-104 stably integrated within a genome of the T cell.

111. A composition for immune therapy by administration of the composition to a subject for treatment of one or more cancers in the subject, the composition comprising the immune cell of any one of claims 108-110.

112. The composition of claim 111, further comprising a pharmaceutically acceptable carrier.

113. A method for treatment of one or more cancers in a subj ect in need thereof, the method comprising administering, to the subject, an effective amount of: the TCR or TCR-like protein of any one of claims 19-104; the nucleic acid of claim 105; the nucleic acid vector or nucleic acid vaccine of claim 106; the immune cell of any one of claims 108-110; the composition of any one of claims 107 and 111-112; or any combination thereof.

114. Use of an agent in the preparation of a medicament, wherein the medicament is for administration to a subject for treatment or prevention of one or more cancers and the agent comprises: the TCR or TCR-like protein of any one of claims 19-104; the nucleic acid of claim 105; the nucleic acid vector or nucleic acid vaccine of claim 106; the immune cell of any one of claims 108-110; the composition of any one of claims 107 and 111-112; orany combination thereof.

115. The method of claim 113 or the use of claim 114, wherein the one or more cancers comprises a myelodysplastic syndrome (MDS), a chronic myelomonocytic leukemia (CMML), an acute myeloid leukemia (AML), or any combination thereof.

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