Non-canonical peptide antigens as therapeutic targets in cancer

Abstract

Noncanonical peptides associated with cancers, including pancreatic cancer, that can be presented by human leukocyte antigens class I, T cell receptors that recognize such peptides, and uses for such peptides, T cell receptors and the nucleotide sequences that encode them in methods of activating immune cells and treating subjects with cancer are described herein.

Description

[0001] DFCI 3431.W01WO / / MRG 0680.003431WO01

[0002] NON-CANONICAL PEPTIDE ANTIGENS AS

[0003] THERAPEUTIC TARGETS IN CANCER

[0004] CONTINUING APPLICATION DATA

[0005] This application claims the benefit of U. S. Provisional Application No. 63 / 747,859, filed January 21, 2025, and U. S. Provisional Application No. 63 / 802,036, filed May 8, 2025, each of which is incorporated by reference herein.

[0006] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0007] This invention was made with government support under CA259621, CA274464, and CA268835 awarded by the National Institutes of Health. The government has certain rights in this invention

[0008] REFERENCE TO SEQUENCE LISTING

[0009] This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an XML file entitled “3431. W01 WO. xml” having a size of 551,280 bytes and created on January 20, 2026. The information contained in the Sequence Listing is incorporated by reference herein.

[0010] BACKGROUND

[0011] Cancer cells can translate ostensibly noncoding genetic elements to produce cryptic (noncanonical) peptides that can be presented by human leukocyte antigen class I (HLA-I). Cryptic peptides are poorly characterized in most solid tumors, where immunotherapies often yield limited efficacy. There is a need for a deeper understanding of cryptic peptide presentation and immunogenicity. Such an understanding will provide new therapeutic avenues in solid tumors, such as pancreatic cancer.

[0012] SUMMARY

[0013] There is provided a composition including two or more isolated non-canonical HLA-I bound peptides (ncHLAp), each isolated ncHLAp having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. In some aspects, the compositionincludes any two, any ten, any twenty-five, any fifty, or any hundred isolated ncHLAp selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. In some aspects, the composition further includes at least one more isolated ncHLAp selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. In some aspects, the composition further includes one or more adjuvants.

[0014] Als provided is an immunogenic composition including at least one isolated non-canonical HLA-I bound peptide (ncHLAp) and one or more adjuvants, said isolated ncHLAp having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

[0015] In some aspects for a composition provided herein, each said isolated ncHLAp is about 7 to about 12 amino acids in length. In some aspects, for a composition provided herein, each said isolated ncHLAp has only the amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. In some aspects for a composition provided herein, the composition includes about 2 to about 10, about 10 to about 20, about 21 to about 30, about 31 to about 40 or about 41 to about 50 isolated ncHLAp.

[0016] Also provided herein is an expression vector including a nucleic acid sequence coding for an isolated non-canonical HLA-I bound peptide (ncHLAp), said isolated ncHLAp having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. In some aspects of the expression vector, said isolated ncHLAp is about 7 to about 12 amino acids in length. In some aspects of the expression vector, said isolated ncHLAp has only an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

[0017] Also provided herein is a host cell including an expression vector as provided herein. In some aspects, the host cell is an antigen presenting cell. In some aspects, the antigen presenting cell is a dendritic cell.

[0018] Also provided herein is a T cell receptor (TCR) or functional fragment thereof having: a) a T cell receptor alpha chain (TCA) comprising a complementarity determining region (CDR) 1 comprising the sequence of SEQ ID NO:552, a CDR2 comprising the sequence of SEQ ID NO:575, and a CDR3 comprising the sequence of SEQ ID NO:600 and a T cell receptor beta chain (TCB) comprising a CDR1 comprising the sequence of SEQ ID NO:553, a CDR2 comprising the sequence of SEQ ID NO: 576, and CDR3 comprising the sequence of SEQ ID NO:601; orb) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:554, a CDR2 comprising the sequence of SEQ ID NO:577, and a CDR3 comprising the sequence of SEQ ID NO:602, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:555, a CDR2 comprising the sequence of SEQ ID NO:578, and a CDR3 comprising the sequence of SEQ ID NO:603; or

[0019] c) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:556, a CDR2 comprising the sequence of SEQ ID NO:579, and a CDR3 comprising the sequence of SEQ ID NO:604, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:557, a CDR2 comprising the sequence of SEQ ID NO:580, and a CDR3 comprising the sequence of SEQ ID NO:605;

[0020] d) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:572, a CDR2 comprising the sequence of SEQ ID NO:581, and a CDR3 comprising the sequence of SEQ ID NO:606, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:558, a CDR2 comprising the sequence of SEQ ID NO:582, and a CDR3 comprising the sequence of SEQ ID NO:607; or

[0021] e) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:559, a CDR2 comprising the sequence of SEQ ID NO:583, and a CDR3 comprising the sequence of SEQ ID NO:608, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:560, a CDR2 comprising the sequence of SEQ ID NO: 584, and a CDR3 comprising the sequence of SEQ ID NO:609; or

[0022] f) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:610, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:562, a CDR2 comprising the sequence of SEQ ID NO: 586, and a CDR3 comprising the sequence of SEQ ID NO:611; or

[0023] g) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:563, a CDR2 comprising the sequence of SEQ ID NO:587, and a CDR3 comprising the sequence of SEQ ID NO:612, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:557, a CDR2 comprising the sequence of SEQ ID NO: 580, and a CDR3 comprising the sequence of SEQ ID NO:613; orh) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:564, a CDR2 comprising the sequence of SEQ ID NO:588, and a CDR3 comprising the sequence of SEQ ID NO:614, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:565, a CDR2 comprising the sequence of SEQ ID NO:589, and a CDR3 comprising the sequence of SEQ ID NO:615; or

[0024] i) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:563, a CDR2 comprising the sequence of SEQ ID NO:587, and a CDR3 comprising the sequence of SEQ ID NO:616, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:555, a CDR2 comprising the sequence of SEQ ID NO:578, and a CDR3 comprising the sequence of SEQ ID NO:617; or

[0025] j) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:566, a CDR2 comprising the sequence of SEQ ID NO:590, and a CDR3 comprising the sequence of SEQ ID NO:618, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:567, a CDR2 comprising the sequence of SEQ ID NO:591, and a CDR3 comprising the sequence of SEQ ID NO:619; or

[0026] k) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:556, a CDR2 comprising the sequence of SEQ ID NO:579, and a CDR3 comprising the sequence of SEQ ID NO:620, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:568, a CDR2 comprising the sequence of SEQ ID NO:592, and a CDR3 comprising the sequence of SEQ ID NO:621; or

[0027] l) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:569, a CDR2 comprising the sequence of SEQ ID NO:593, and a CDR3 comprising the sequence of SEQ ID NO:622, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:565, a CDR2 comprising the sequence of SEQ ID NO: 589, and a CDR3 comprising the sequence of SEQ ID NO: 623; or

[0028] m) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO: 564, a CDR2 comprising the sequence of SEQ ID NO:594, and a CDR3 comprising the sequence of SEQ ID NO:624, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:570, a CDR2 comprising the sequence of SEQ ID NO:595, and a CDR3 comprising the sequence of SEQ ID NO:625; orn) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:626, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:571, a CDR2 comprising the sequence of SEQ ID NO:596, and a CDR3 comprising the sequence of SEQ ID NO:627; or

[0029] o) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:572, a CDR2 comprising the sequence of SEQ ID NO:597, and a CDR3 comprising the sequence of SEQ ID NO:628, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:573, a CDR2 comprising the sequence of SEQ ID NO:598, and a CDR3 comprising the sequence of SEQ ID NO:629; or

[0030] p) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:610, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:562, a CDR2 comprising the sequence of SEQ ID NO:586, and a CDR3 comprising the sequence of SEQ ID NO:630; or

[0031] q) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:574, a CDR2 comprising the sequence of SEQ ID NO:599, and a CDR3 comprising the sequence of SEQ ID NO:631, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:567, a CDR2 comprising the sequence of SEQ ID NO:591, and a CDR3 comprising the sequence of SEQ ID NO:632.

[0032] In some aspects of a TCR or functional fragment as provided herein:

[0033] a) the TCR of a) is specific for an isolated non-canonical HLA-I bound peptide (ncHLAp) having SEQ ID NO:9; or

[0034] b) the TCR of b) is specific for an isolated ncHLAp having SEQ ID NO:239; or c) the TCR of c) is specific for an isolated ncHLAp having SEQ ID NO:84; or d) the TCR of d) is specific for an isolated ncHLAp having SEQ ID NO:84; or e) the TCR of e) is specific for an isolated ncHLAp having SEQ ID NO:93; or f) the TCR of f) is specific for an isolated ncHLAp having SEQ ID NO:509; or g) the TCR of g) is specific for an isolated ncHLAp having SEQ ID NO:84; or h) the TCR of h) is specific for an isolated ncHLAp having SEQ ID NO:509; or i) the TCR of i) is specific for an isolated ncHLAp having SEQ ID NO:239; orj) the TCR of j) is specific for an isolated ncHLAp having SEQ ID NO:449; or k) the TCR of k) is specific for an isolated ncHLAp having SEQ ID NO:9; or

[0035] l) the TCR of l) is specific for an isolated ncHLAp having SEQ ID NO:9; or

[0036] m) the TCR of m) is specific for an isolated ncHLAp having SEQ ID NO:202; or n) the TCR of n) is specific for an isolated ncHLAp having SEQ ID NO:84; or o) the TCR of o) is specific for an isolated ncHLAp having SEQ ID NO:509; or p) the TCR of p) is specific for an isolated ncHLAp having SEQ ID NO:509; or q) the TCR of q) is specific for an isolated ncHLAp having SEQ ID NO:239.

[0037] In some aspects, a TCR or functional fragment as provided herein is a soluble T cell receptor (sTCR).

[0038] In some aspects, a TCR or functional fragment thereof as provided herein is a singlechain T cell receptor (scTCR).

[0039] Also provided herein is a bispecific T cell engager (BiTE) having a sTCR or a scTCR as provided herein and a second T cell-specific binding molecule. In some aspects, the T cellspecific binding molecule comprises an anti-CD3 immunoglobulin or scFv thereof.

[0040] Also provided herein is a nucleic acid sequence coding for the variable region of the alpha chain and / or the beta chain of a TCR as provided herein or a TCR or functional fragment thereof as provided herein. Also provided is a vector including such a nucleic acid sequence. Also provided is a cultured cell having a TCR or functional fragment thereof as provided herein, a nucleotide sequence provided herein, or a vector as provided herein.

[0041] Also provided is a T cell having a TCR or functional fragment thereof as provided herein, a nucleotide sequence provided herein, or a vector as provided herein of claim 22.

[0042] Also provided herein is an in vitro method for producing antigen-specific activated T lymphocytes, the method including contacting T cells in vitro with antigen presenting cells loaded with one or more isolated non-canonical HLA-I bound peptides (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517 for a period of time sufficient to activate said T cells in an antigen-specific manner to the one or more ncHLAp. Also provided is an antigen-specific activated T lymphocyte produced by the method. In some aspects, the antigen-specific activated T lymphocyte selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.Also provided herein is an in vitro method for producing antigen-specific activated T lymphocytes, the method including contacting T cells in vitro with antigen presenting cells loaded with a composition as provided herein for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. Also provided is an antigen-specific activated T lymphocyte produced by the method. In some aspects, the antigen-specific activated T lymphocyte selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

[0043] Also provided herein is an in vivo method for producing antigen specific activated T lymphocytes in a subject, the method including administering to the subject one or more isolated non-canonical HLA-I bound peptides (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517. Also provided is an antigen-specific activated T lymphocyte produced by the method. In some aspects, the antigen-specific activated T lymphocyte selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

[0044] Also provided herein is an in vivo method for producing antigen specific activated T lymphocytes in a subject, the method including administering to the subject a composition as provided herein. Also provided is an antigen-specific activated T lymphocyte produced by the method. In some aspects, the antigen-specific activated T lymphocyte selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

[0045] Also provided herein is a method of producing an antigen-specific T lymphocytes, the method including transforming a T lymphocyte with a nucleic acid provided herein coding for the variable region of the alpha chain and / or the beta chain of a TCR as provided herein or encoding a TCR or functional fragment thereof as provided herein. Also provided is a vector including such a nucleic acid sequence.

[0046] Also provided herein is a method of treating a subject having a cancer, the method comprising administering to the human subject:

[0047] a) one or more peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517;

[0048] b) a composition of ncHLAp as provided herein;c) a nucleic acid sequence coding for an isolated non-canonical HLA-I bound peptide (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517;

[0049] d) an expression vector as provided herein;

[0050] e) a host cell as provided herein;

[0051] f) a TCR or functional fragment thereof as provided herein;

[0052] g) a nucleic acid as provided herein;

[0053] h) a vector as provided herein;

[0054] i) a cultured cell as provided herein;

[0055] j) a T cell as provided herein; and / or

[0056] k) an antigen-specific activated T lymphocyte as provided herein.

[0057] In some aspects of a method of treating a subject having a cancer, the subject is human. In some aspects of a method of treating a subject having a cancer, the cancer is selected from glioblastoma, melanoma, ovarian cancer, meningioma, lung cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, B-acute lymphoblastic leukemia, lymphoma, T-acute lymphoblastic leukemia, chronic lymphocytic leukemia, colon carcinoma, breast cancer and neuroblastoma.

[0058] In some aspects of a method of treating a subject having a cancer, the cancer is pancreatic cancer.

[0059] In some aspects of a method of treating a subject having a cancer, the cancer expresses a peptide comprising an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID: 517 or a combination thereof.

[0060] In some aspects of a method of treating a subject having a cancer, the method further includes administering chemotherapy, immunotherapy, checkpoint inhibitor, radiation or surgery to the subject.

[0061] Definitions

[0062] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure. Accordingly, theterminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0063] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0064] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C”. The term “or” can be used conjunctively or disjunctively unless the context specifically refers to a disjunctive use.

[0065] Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting.

[0066] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0067] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0068] 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 measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of agiven value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, such as within 5-fold, and within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0069] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination.

[0070] Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there can be variations and modifications of the present disclosure, which can be encompassed within its scope.

[0071] The term "peptide" may include salts of a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The salts are pharmaceutical acceptable salts of the peptides, such as, for example, the chloride or acetate (trifluoroacetate) salts. It has to be noted that the salts of the peptides according to the present description differ from the peptides in their state(s) in vivo, as the peptides are not salts in vivo.

[0072] The term "isolated" means that the material is removed from its original environment (e g., the natural environment, if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. In an aspect, such polynucleotides are part of a vector and / or such polynucleotides or polypeptides are part of a composition and still are isolated in that such vector or composition is not part of its natural environment.

[0073] The polynucleotides, and recombinant or immunogenic polypeptides, disclosed in accordance with the present description may also be in “purified” form. The term “purified” doesnot require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity.

[0074] Purification of starting material or natural material to at least one order of magnitude, including two or three orders, including four or five orders of magnitude is expressly contemplated.

[0075] Furthermore, a claimed polypeptide which has a purity of 99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weight or greater is expressly encompassed.

[0076] The term “active fragment” means a fragment, usually of a peptide, polypeptide or nucleic acid sequence, that generates an immune response (i.e., has immunogenic activity) when administered, alone or optionally with a suitable adjuvant or in a vector, to an animal, such as a mammal, for example, a rabbit or a mouse, and also including a human, such immune response taking the form of stimulating a T-cell response within the recipient animal, such as a human. Alternatively, the "active fragment" may also be used to induce a T-cell response in vitro.

[0077] As used herein, the terms "portion", "segment" and "fragment", when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treatment of said polynucleotides with any of the endonucleases.

[0078] As used herein, "individual" (as in the subject of the treatment) means a mammal.

[0079] Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; and nonprimates, e.g., dogs, cats, rats, mice, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

[0080] All patent filings, websites, other publications, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of apriority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated.

[0081] Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise.

[0082] The following descriptions and examples illustrate embodiments of the present disclosure in detail. Although the present disclosure has been described in some details by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0083] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0084] BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0086] FIG. 1. Cancer-restriction of ncHLAp. translation-centric filtering pipeline was used to nominate PD AC-restricted ncHLAp. (A) the gating strategy to identify parental ORFs encoding ncHLAp that are not detected in any healthy tissue, including thymus, via HLA-I immunopeptidomics or Ribo-seq. Each vertical bar represents the percentage of samples (healthy tissue samples or PDAC organoids) in which expression of a ncHLAp-encoding ORF was identified. (B) the number of noncanonical HLA-I peptides retained after each filtration step in the translation-centric pipeline. (C) The percentage of noncanonical or canonical HLAp retained after each step of the translation-centric filtering pipeline. IP / MS-immunopeptidomics.

[0087] FIG. 2. (A) The number of CR ncHLAp detected in PDAC PDOs by category / biotype (only ncHLAp categories with >10 CR ncHLAp included). (B) The percent of detected ncHLAp that exhibit cancer-restriction within each category / biotype (only ncHLAp categories with >10CR ncHLAp included). (C) The frequency of shared and patient-specific HLA-A*02:01-restricted CR ncHLAp across PDOs.

[0088] FIG. 3. A histogram showing the number of CR ncHLAp and mutation-derived HLAp empirically detected in each PDO line.

[0089] FIG. 4. Immunogenic potential of empirically detected ncHLAp (CR ncHLAp = top; nonCR ncHLAp = bottom), following priming and two re-stimulations with autologous peptide-loaded APCs. Percent positivity over background in each CTL line indicated in heatmap.

[0090] Threshold for considering a positive response: >2% over the negative control, assessed by simultaneous staining of autologous pre-stimulated CD8 T cells (tetramer) or no peptide control (ICS). Table indicating #CTL lines evaluated, #CTL lines positive (red), HLA restriction tested, CR / nonCR, and recurrence (recurrent or private among PDOs) for each ncHLAp tested.

[0091] Private / recurrent designations are based on the pairing of each ncHLAp and its tested allele across all PDOs.

[0092] FIG. 5. PD AC-restricted ncHLAp exhibit immunogenic potential. (A) A Sankey plot depicting empirically identified CR and nonCR ncHLAp, detection in healthy tissues (including thymus), and immunogenicity. (B) The number of immunogenic or non-immunogenic CR ncHLAp from indicated biotypes. Here, 5’ uORF category is inclusive of 5’ uORFs and 5’ overlapping uORFs. ncRNA is inclusive of ncRNA processed transcript and ncRNA retained intron biotypes. 3’ dORF is inclusive of 3’ dORFs and 3’ overlap dORFs.

[0093] FIG. 6. PDAC-restricted ncHLAp exhibit immunogenic potential. (A) Shows candidate ncHLAp-reactive TCRs identified from BEAM-T with indicated HLA restriction, antigenspecificity score, and CDR3αβ sequences. (B) Functional avidity (as measured by cytotoxicity against peptide-loaded T2_eGFP_ffLuc cells) of HLA-A* 02: 01 -restricted, ncHLAp-directed TCR-T cells. Mean - / + SD (of n=2 biological replicates). Normalized to T2 cell viability alone. Note that some error bars are too small to be displayed.

[0094] FIG. 7. CR ncHLAp-specific T cells can recognize and kill pancreatic cancer organoids ex vivo and in vivo. (A) A schematic of the workfl ow / approach for organoid: TCR-T cell coculture. (B) IFN-y ELISA from co-culture of P0071_eGFP-ffLuc PDOs with the indicated ncHLAp-reactive TCR-T cells (10:1 E: T). Mean - / + SD (of n=3). (C) CD137 surface expression on indicated ncHLAp-reactive TCR-T cells following co-culture with indicated PDOs. P0151 (HLA-mismatched) PDOs were used as a negative control. Representative experiment of n=3biological replicates. Mean - / + SD of n=3 technical replicates displayed. An unpaired t-test was used to determine statistical significance in (B) and (C).

[0095] FIG. 8. CR ncHLAp-specific T cells can recognize and kill pancreatic cancer organoids ex vivo and in vivo. (A) Cytotoxicity after co-culture of P0071_eGFP-ffLuc PDOs with the indicated ncHLAp-reactive TCR-T cells at indicated E: T ratios. Mean - / + SD (of n=3). (B) presents alanine scanning of TCR001 TCR-T cells. Mean - / + SD (of n=2 biological replicates). An unpaired t-test was used to determine statistical significance.

[0096] FIG. 9. Longitudinal tumor volume assessment of subcutaneously transplanted P0071_eGFP-ffLuc PDOX in NSG mice (n = 8 per arm), block randomized to receive two administrations of indicated TCR-T cells (Day 0 [2 x 107] and Day 10 [1.5 x 107]). Mean - / + SD. A linear mixed effects regression model was used to determine statistical significance.

[0097] FIG. 10. ncHLAp are translated and presented in situ in pancreatic cancer tumors. (A) The number of uniquely mapping ncHLAp detected in each PDAC bulk tumor. (B) The proportion of HLAp mapping specifically to either canonical genes or nuORFs per bulk tumor. (C) A length distribution of canonical or noncanonical HLAp detected in bulk tumors. (D) The number of ncHLAp detected in PDAC bulk tumors deriving from different nuORF biotypes / categories.

[0098] FIG. 11. The overlap (Jaccard similarity index) of nuORFs (detected via immunopeptidomics) between all pairings of PDOs and bulk tumors, ranked in a descending fashion by cumulative overlap. Bulk tumor samples indicated in red.

[0099] FIG. 12. Synthetic validation of ncHLAp and mutHLAp used for immunogenicity assays. FIG. 12 shows the best representative (see methods section of Example 1) MS / MS spectra for synthetic peptides with indicated precursor charge were compared to the corresponding immunopeptidome MS / MS spectra for each organoid sample. A passing dot product score > 0.7 validates a peptide’s identification in the immunopeptidome dataset. All tested peptides were validated except ALYTVLDPV (SEQ ID NO: 192) (spectrum similarity too low), VLLENSSIVKI (SEQ ID NO: 469) (spectrum similarity too low in 4 / 5 samples), and TEICPPKKKYF (SEQ ID NO:77) (synthetic was not detected in carbamidomethyl form).

[0100] FLQEEAALKAL (SEQ ID NO: 29) passed in 4 / 5 samples.

[0101] FIG. 13. LC-MS / MS spectra of select peptides in the organoid immunopeptidome (upper) mirrored with the MS / MS spectrum of the corresponding synthetic peptide (lower). Pass:KLFLWPYKV (SEQ ID NO: 9) (NU11), ALFSKLASA (SEQ ID NO: 93) (NU57), and IQIGWVTQK (SEQ ID NO: 102) (NU92). Fail: ALYTVLDPV (SEQ ID NO: 192).

[0102] FIG. 14. TCR reconstruction for TCR-T. (A) A schematic of the modular-based Gibson cloning strategy. (B) A schematic of the final assembled product of TCR-encoding lentiviruses, with elongation factor 1 alpha (EFla) driving a polycistronic transcript encoding both TCRP and TCRa, separated by a porcine teschovirus-1 ribosome skipping sequence (P2A) and furin cleavage site.

[0103] FIG. 15. Evaluation of ncHLAp-directed TCR-T reactivity and cytotoxicity. (A) IFN-y ELISA from co-culture of P0071_eGFP-ffLuc PDOs with the indicated low-avidity ncHLAp-reactive TCR-T cells (10: 1 E: T). Mean - / + SD (of n=3 biological replicates). Dotted line represents limit of detection for assay. (B) Cytotoxicity after co-culture of P0071_eGFP-ffLuc PDOs with the indicated low-avidity ncHLAp-reactive TCR-T cells at indicated E: T ratios. Mean - / + SD (of n=3 biological replicates). (C) CD 137 surface expression on indicated ncHLAp-reactive TCR-T cells following co-culture with indicated PDOs at 1:5 effectortarget ratio.

[0104] Representative experiment of n=3 biologic replicates. Mean - / + SD of n=3 technical replicates displayed. Statistics: (E) unpaired t-test.

[0105] FIG. 16. In vivo evaluation of ncHLAp-directed TCR-T. show longitudinal tumor volume assessment of subcutaneously transplanted P0071_eGFP-ffLuc PDOX in athymic mice, block randomized to receive a single administration (dashed line) of (1 x 107) TCR001- (n = 10), TCR005- (n = 7), TCR012- (n = 8), TCRirr- (n = 10) T cells. Mean - / + SEM.

[0106] FIG. 17. Individual tumor plots from the same evaluation of ncHLAp-directed TCR-T shown in FIG. 16.

[0107] FIG. 18. Individual tumor plots of longitudinal tumor volume assessment of subcutaneously transplanted P0071_eGFP-ffLuc PDOX in NSG mice (related to FIG. 9), block randomized (n = 8 per arm) to receive two administrations (dashed lines) of indicated TCR-T cells (Day 0 [2 x 107] and Day 10 (1.5 x 107). Gray stars in plots indicate two mice that died during study (unrelated to study treatment). Statistics: linear mixed effects regression model.

[0108] Schematic drawings are not necessarily to scale. Like numbers used in the figures may refer to like components. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is notintended to indicate that the different numbered components cannot be the same or similar to other numbered components.

[0109] DETAILED DESCRIPTION

[0110] Pancreatic cancer has been refractory’ to immunotherapies. Translation of the non-coding genome in cancer can generate cryptic (non-canonical) peptides capable of presentation by human leukocyte antigen class I (HLA-I). As described herein, high-depth immunopeptidomics on organoids from pancreatic cancer patients identified 517 cancer-restricted noncanonical HLA-I-bound peptides (ncHLAp). These 517 peptides are shown in Table 1. A substantial subset of these peptides is shared among cancer patients. And, as shown in Tables 2 and 3, the expression of many of these peptides is not limited to pancreatic cancer, but also noted in cancers such as acute myeloid leukemia, B-acute lymphoblastic leukemia, breast cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon carcinoma, glioblastoma, lung cancer, lymphoma, melanoma, meningioma, neuroblastoma, ovarian cancer, and T-acute lymphoblastic leukemia. These cancer-restricted ncHLAp (CR ncHLAp) displayed robust immunogenic potential in a sensitive ex vivo T cell priming platform. ncHLAp-reactive, T cell receptor-redirected T cells exhibited tumoricidal activity against patient-derived pancreatic cancer organoids. These findings demonstrate that cancer such as pancreatic cancer harbor cancer- restricted ncHLAp that can be recognized by cytotoxic T cells.

[0111] As disclosed herein, the present description provides peptides comprising a sequence that is selected from the 517 CR ncHLAp peptides of SEQ ID NO:1 to SEQ ID NO:517 or a variant thereof. Such peptides have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I. In humans there are three different genetic loci that encode classical MHC class I molecules (the MHC-molecules of the human are also designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C.

[0112] As used herein, the term "peptide" is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The disclosed herein are preferably less than about 30 amino acid residues in length, and at least about 8 amino acids in length. A peptide may include a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 517 and may be about 6 amino acids in length, about 7 amino acids in length, about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about llamino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, about 15 amino acids inlength, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids in length, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, about 30 amino acids in length, or any range thereof. For example, peptide including a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 517 may be about 5 to about 20 amino acids in length, about 7 to about 15 amino acids in length, or about 8 to about 11 amino acids in length.

[0113] By a "variant" of the given amino acid sequence it is meant that the side chains of, for example, one or two of the amino acid residues are altered (for example by replacing them with the side chain of another naturally occurring amino acid residue or some other side chain) such that the peptide is still able to bind to an HLA molecule in substantially the same way as a peptide consisting of the given amino acid sequence consisting of SEQ ID NO:1 to SEQ ID NO:517. For example, a peptide may be modified so that it at least maintains, if not improves, the ability to interact with and bind to the binding groove of a suitable MHC molecule and in that way it at least maintains, if not improves, the ability to bind to the TCR of activated T-cells.

[0114] The peptides disclosed herein can be modified by the substitution of one or more residues at different, possibly selective, sites within the peptide chain, if not otherwise stated. Those substitutions can be located at the end of the amino acid chain of said peptide. Such substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine. In studies of sequence variations in families of naturally occurring homologous proteins, certain amino acid substitutions are more often tolerated than others, and these often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, and such is the basis for defining "conservative substitutions."

[0115] Conservative substitutions are herein defined as exchanges within one of the following five groups: Group 1 -small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negatively charged residues and their amides (Asp, Asn, Glu, Gin); Group 3-polar, positively charged residues (His, Arg, Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu, He, Vai, Cys); and Group 5-large, aromatic residues (Phe, Tyr, Trp).

[0116] Less conservative substitutions might involve the replacement of one amino acid by another that has similar characteristics but is somewhat different in size, such as replacement of an alanine by an isoleucine residue. Highly non-conservative replacements might involve substituting an acidic amino acid for one that is polar, or even for one that is basic in character.

[0117] Of course, such substitutions may involve structures other than the common L-amino acids. Thus, D-amino acids might be substituted for the L-amino acids commonly found in the antigenic peptides of the description and yet still be encompassed by the disclosure herein. In addition, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present description.

[0118] If substitutions at more than one position are found to result in a peptide with substantially equivalent or greater antigenic activity, then combinations of those substitutions can be tested to determine if the combined substitutions result in additive or synergistic effects on the antigenicity of the peptide.

[0119] Of course, a peptide or variant according to the present description will have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class I. Binding of a peptide or a variant to a MHC complex may be tested by methods known in the art.

[0120] Table 1: CR ncHLAp (SEQ ID NOs:l-517)

[0121] QELHSSRGLW SSMTGERKW YSSEIWDLY HHARLVLYF AILVPQPPK SEQ ID NO:1 SEQ ID NO: 105 SEQ ID NO:209 SEQ ID NO:313 SEQ ID NO:417 EEFLGGKKTNW VPEAARSFSFL APRLLPATVV GLLETHPALLL MA VTTRHVLLW SEQ ID NO:2 SEQ ID NO: 106 SEQ ID NO:210 SEQ ID NO:2 14 SEQ ID NO:418 TLEEKRFCW APIDRYWTP TAPLTTRVL QSLLHKII STTKTAAFSL SEQ ID NO:2 SEQ ID NO: 107 SEQ ID NO:211 SEQ ID NO:2 15 SEQ ID NO:419 TLFRVVVAK FAIDQPELHL MKTGAYFTTW APGPRTYLA LPLPRLPVSA SEQ ID NO:4 SEQ ID NO: 108 SEQ ID NO:212 SEQ ID NO:216 SEQ ID NO:420 SVDLYKILTF TTRHVLLW SSMYTTIPR SPAPPDRAVL KLLDIGYRK SEQ ID NO:2 SEQ ID NO: 109 SEQ ID NO:213 SEQ ID NO:2 17 SEQ ID NO:421 KEQCGRSVLW EAMTRIKRW APGDRDMGFVL MTLLCVKTW VPILSRETL SEQ ID NO:2 SEQ ID NO: 110 SEQ ID NO:214 SEQ ID NO:2 18 SEQ ID NO:422 VTTRHVLLW AISPRRFLSW YLIQRLTSL RLFLIVSSGQK RLLLNLNQK SEQ ID NO:7 SEQ ID NO:111 SEQ ID NO:215 SEQ ID NO:2 19 SEQ ID NO:423 AVFVNWEQTK LIPKVFLKI AHAPPEQHL RSLSGSARRW VLWGHRFTF SEQ ID NO:8 SEQ ID NO: 112 SEQ ID NO:216 SEQ ID NO:220 SEQ ID NO:424 KLFLWPYKV GHSFPDPGL SYPLVLKEF MHFLENISL YMAGVLVGA

[0122]

[0123] SEQ ID NO:9 SEQ ID NO: 113 SEQ ID NO:217 SEQ ID NO:321 SEQ ID NO:425 STKDMALAWSR FRIPGTQSL EVARGA VPG ARLWFRPPL DLPSKSFHSF SEQ ID NO: 10 SEQ ID NO:114 SEQ ID NO:218 SEQ ID NO:322 SEQ ID NO:426 SSLRTGHLHHW NSRAKMGLFM AHASLSSVF KLFGYVQEF APVTPTPGP SEQ ID NO: 11 SEQ ID NO: 115 SEQ ID NO:219 SEQ ID NO:323 SEQ ID NO:427 EEIRALPLRKF VILLPGPAK SSILKRINM MHHHARLIF VPVAPCPGTAA SEQ ID NO: 12 SEQ ID NO: 116 SEQ ID NO:220 SEQ ID NO:324 SEQ ID NO:428 VVLYHLINK KTLWSPSTLW LQWLHSRL RPLKLLNKT EVVFLYTGL SEQ ID NO: 13 SEQ ID NO:117 SEQ ID NO:221 SEQ ID NO:325 SEQ ID NO:429 MLLSHTGKLAL STMHLITSW ALMTVGWLPK RTHPLTHSW DEFGDSRRRW SEQ ID NO: 14 SEQ ID NO: 118 SEQ ID NO:222 SEQ ID NO:326 SEQ ID NO:430 SVCDWTLTFPK GSVHDASDILW QPSSPASSIAA ITARVTVTGW VTSRGRALF SEQ ID NO: 15 SEQ ID NO: 119 SEQ ID NO:223 SEQ ID NO:327 SEQ ID NO:431 RVWEKRNPW STLEEKRFCW LWKQLLQM KLITMKILV MECLEGARRF SEQ ID NO: 16 SEQ ID NO: 120 SEQ ID NO:224 SEQ ID NO:328 SEQ ID NO:432 RSFPKASSSW GSAPVVQKV ERNIRIIAL FTQKINSTW LPRNGLLA SEQ ID NO: 17 SEQ ID NO: 121 SEQ ID NO:225 SEQ ID NO:329 SEQ ID NO:433 FTWKGTKTF ELRAGDTATW IFSVGTVLK IPWATPHQPA KMVFFLHMV SEQ ID NO: 18 SEQ ID NO: 122 SEQ ID NO:226 SEQ ID NO:330 SEQ ID NO:434 SPISAVRAPA RIYFQEQEK LTYHGDQVTL SSGPTRAIP TARSAETHW SEQ ID NO: 19 SEQ ID NO: 123 SEQ ID NO:227 SEQ ID NO:331 SEQ ID NO:435 EEGRIITKW YTKRWPNGW RPLHIQGWLLA SSWRWQSSW RHESLTLSFRL SEQ ID NO:20 SEQ ID NO: 124 SEQ ID NO:228 SEQ ID NO:332 SEQ ID NO:436 SPAIKHIL RPFYLLTKV ISHGILPNL LAQDRPLPL RFEELLLQL SEQ ID NO:21 SEQ ID NO: 125 SEQ ID NO:229 SEQ ID NO:333 SEQ ID NO:437 ASFHVVRMLK RIYLERRIY SSMQSFIEK VTRSSFLW KYPTIICGF SEQ ID NO:22 SEQ ID NO: 126 SEQ ID NO:230 SEQ ID NO:334 SEQ ID NO:438 AEMDRMLYF SRNISIWRF YLLDGQLQL LPPAPPPPG RRLETVLIK SEQ ID NO:23 SEQ ID NO: 127 SEQ ID NO:231 SEQ ID NO:335 SEQ ID NO:439 RTMIIGDLLQTK LVGPPGSSW SLLFMIRGV FSLKAARPW LPSQEDSIM SEQ ID NO:24 SEQ ID NO: 128 SEQ ID NO:232 SEQ ID NO:336 SEQ ID NO:440 LPAEVPEAA QLDNLKVEL EVTHKLLSV KVLVDFLLK MTDWANMFQHY SEQ ID NO:25 SEQ ID NO: 129 SEQ ID NO:233 SEQ ID NO:337 SEQ ID NO:441 RTLGKDMKW QLTHIHKI SADSVPHVL FILLWKSPK MLLLMLLYK SEQ ID NO:26 SEQ ID NO: 130 SEQ ID NO:234 SEQ ID NO:338 SEQ ID NO:442 VLYHLINK EAKNAKLRTW ITPTPGSSTFL QIVKQITSI LPRQPATSL SEQ ID NO:27 SEQ ID NO: 131 SEQ ID NO:235 SEQ ID NO:339 SEQ ID NO:443 ATESVLQKILK RVYWIGERK VPAPSRTAV APLYQVAAA LEAHLLPTP SEQ ID NO:28 SEQ ID NO: 132 SEQ ID NO:236 SEQ ID NO:340 SEQ ID NO:444 FLQEEAALKAL SPAMVFVTA SGAVAGLGVL RPFFNVRVA ATLSGVFSEG SEQ ID NO:29 SEQ ID NO: 133 SEQ ID NO:237 SEQ ID NO:341 SEQ ID NO:445 AVFVNWEQTKK HRHLLSVPQK RRIYLKLSLNQ REPL FIRS W VLPWQPLLL SEQ ID NO:30 SEQ ID NO: 134 SEQ ID NO:238 SEQ ID NO:342 SEQ ID NO:446 KVSSIFFINK LTAWALHLL RGNLPLPLK SVCGEVVLVK RPVWGGARAA SEQ ID NO:31 SEQ ID NO: 135 SEQ ID NO:239 SEQ ID NO:343 SEQ ID NO:447 RINYHLLELK SLNPLHWPK HHYYGPHIWI TSFDKPIFK MIASVLKAL SEQ ID NO:32 SEQ ID NO: 136 SEQ ID NO:240 SEQ ID NO:344 SEQ ID NO:448 FRPPQPQYL FQAAPDRGF IAAGNFRIY SLIPKVFLK ILQDRLWKL SEQ ID NO:33 SEQ ID NO: 137 SEQ ID NO:241 SEQ ID NO:345 SEQ ID NO:449 RLWTSVAVLK RAASWTSTW KARLLPRL FSLDHDSGRRW LRPELLNQL SEQ ID NO:34 SEQ ID NO: 138 SEQ ID NO:242 SEQ ID NO:346 SEQ ID NO:450 RTLEVNIMLKK RLWKHTLKY RVPDFSPEL ARISVTELIQK ASFSRRSLW SEQ ID NO:35 SEQ ID NO: 139 SEQ ID NO:243 SEQ ID NO:347 SEQ ID NO:451 HSVNQAGVH KTFHHPLSMNW TPSWPPPSV RLWGMSWPK LGLHFEKH

[0124]

[0125] SEQ ID NO:36 SEQ ID NO: 140 SEQ ID NO:244 SEQ ID NO: 348 SEQ ID NO:452 STLTLFLKK APLYQVAA QPFFGEWRP HLPSTFLLK KIWNANYFLTK SEQ ID NO:37 SEQ ID NO:141 SEQ ID NO:245 SEQ ID NO:349 SEQ ID NO:453 VPLPRIPTV EPGP AL VP A RRNWNGLLATM ILYQNPKTLK RTLQTVLPF SEQ ID NO:38 SEQ ID NO: 142 SEQ ID NO:246 SEQ ID NO: 350 SEQ ID NO:454 MPFWLTHWV APVSLIPAV REHWMGGFRW TRIDFFENR FLNNLEVNM SEQ ID NO:39 SEQ ID NO: 143 SEQ ID NO:247 SEQ ID NO:351 SEQ ID NO:455 LAARLAPGTW RLSQDGFMLK TVYRQEVSA GPSLPQPPA AVQSVAVSI SEQ ID NO:40 SEQ ID NO: 144 SEQ ID NO:248 SEQ ID NO: 352 SEQ ID NO:456 SEISPPPSF VPFAARLPA AEQPAPAHAY SPLPLLPVA AAAAPALDTL SEQ ID NO: 41 SEQ ID NO: 145 SEQ ID NO:249 SEQ ID NO:353 SEQ ID NO:457 ALWNVPTGRTW VSHPAVDAW PILSVRIL AERWQKKWW ITKELEGCVSW SEQ ID NO:42 SEQ ID NO: 146 SEQ ID NO:250 SEQ ID NO: 354 SEQ ID NO:458 RTNNILLPR APLLQPRAA KPFPGTSEF LQIPWKLLK VHTTTILYL SEQ ID NO:43 SEQ ID NO: 147 SEQ ID NO:251 SEQ ID NO:355 SEQ ID NO:459 ILAPPAILK KLLMTEIAL MPRWPGIWTAA RLYSLSTALR LGMCFLVYI SEQ ID NO:44 SEQ ID NO: 148 SEQ ID NO:252 SEQ ID NO: 356 SEQ ID NO:460 FLWEILERL YSNGLQHPL ITAQRAGPGW KEEGRIITKW ALWPKVPEV SEQ ID NO:45 SEQ ID NO: 149 SEQ ID NO:253 SEQ ID NO: 357 SEQ ID NO:461 VPWSLLPKA RPIWDVRSA RLAPLLLTH HLFSVLSAI MTNSWMMARH SEQ ID NO:46 SEQ ID NO: 150 SEQ ID NO:254 SEQ ID NO:358 W

[0126] SEQ ID NO:462 SLQKILHQL VEAPVVLEA VPMEIHFMEA RLKSYHVSF FTASGKYGMY SEQ ID NO:47 SEQ ID NO:151 SEQ ID NO:255 SEQ ID NO:359 SEQ ID NO:463 LFRVVVAK RLSLPLSSY VHSISGNFQF RAQPNGRHPW ILGTLVHAV SEQ ID NO:48 SEQ ID NO: 152 SEQ ID NO:256 SEQ ID NO:360 SEQ ID NO:464 RVSLPKLGYK KFVKSGLLL MVVLILVSRF RLFLNSNTL PEGLRLNL SEQ ID NO:49 SEQ ID NO: 153 SEQ ID NO:257 SEQ ID NO:361 SEQ ID NO:465 HPCPVLPIASA VLYLRQVGY RSLDFLLFR GLSDKTFLL SRITIHLNR SEQ ID NO:50 SEQ ID NO: 154 SEQ ID NO:258 SEQ ID NO:362 SEQ ID NO:466 SPIMPSASL LSTPLPEA HPEESLLLL LAPPAILK RVCVWGRDLQK SEQ ID NO:51 SEQ ID NO: 155 SEQ ID NO:259 SEQ ID NO:363 SEQ ID NO:467 ESLQVYRYW THSSWVSWL RVPFSHPPR LAGVRFSLEI TRHPFPGL SEQ ID NO:52 SEQ ID NO: 156 SEQ ID NO:260 SEQ ID NO:364 SEQ ID NO:468 LSNPRAVLW ATNPSTTRVIW KTSFIIRSL RPGWSTMAA VLLENSSIVKI SEQ ID NO:53 SEQ ID NO: 157 SEQ ID NO:261 SEQ ID NO:365 SEQ ID NO:469 RVNTMENLKLK DLGIKTSL FIKFHRTVTV AAYTKLFIK HLHRDSALI SEQ ID NO:54 SEQ ID NO: 158 SEQ ID NO:262 SEQ ID NO:366 SEQ ID NO:470 AADPGLGTL GEGGVPAAW AVGGLFEFCK RVFPEEIRI ALAVIPIPK SEQ ID NO:55 SEQ ID NO: 159 SEQ ID NO:263 SEQ ID NO:367 SEQ ID NO:471 LNLKRYLAL ISSSVFTSY TEALFTKEF SPGASPWAA TIKVRNIPL SEQ ID NO:56 SEQ ID NO: 160 SEQ ID NO:264 SEQ ID NO:368 SEQ ID NO:472 APSGLSPLPA PASSAFSK GLMKICISL GALLSMTFPLK MSRPPITTNYR SEQ ID NO:57 SEQ ID NO:161 SEQ ID NO:265 SEQ ID NO:369 SEQ ID NO:473 KILEGITLILK SLQSPPLRFK TLKIKSLQI RMWPNNLVHK SANSGIHLAL SEQ ID NO:58 SEQ ID NO: 162 SEQ ID NO:266 SEQ ID NO:370 SEQ ID NO:474 RDYHPRVGW RRKPEVIILNF LLAAGCPW ILLNFSTTTK HRMDIRTISL SEQ ID NO:59 SEQ ID NO: 163 SEQ ID NO:267 SEQ ID NO:371 SEQ ID NO:475 SPLPHLGPL RLNYRTTYR VVLGVSFSR SVISVLTTPK HHFYTLKNGTF SEQ ID NO:60 SEQ ID NO: 164 SEQ ID NO:268 SEQ ID NO: 372 SEQ ID NO:476 YLHPHRLPL MMPWRAASL RRIYLERRIY HLSSVKLLLF REPPQTPLP SEQ ID NO:61 SEQ ID NO: 165 SEQ ID NO:269 SEQ ID NO:373 SEQ ID NO:477 APHKYWAGP RLAPPCPPT LCMLLTVSL AVIQIYSHF LGELGLVDL

[0127]

[0128] SEQ ID NO:62 SEQ ID NO: 166 SEQ ID NO:270 SEQ ID NO: 374 SEQ ID NO:478HSVNQAGVHW GTIANILAH EVREGELDWW DVADVGRVSL MLARLVWNA SEQ ID NO:63 SEQ ID NO: 167 SEQ ID NO:271 SEQ ID NO:375 SEQ ID NO:479 KIFEKLVALK SSFTIIGRTW LPTLLGGANL ERLLRPIAL WMKEQTLEM SEQ ID NO:64 SEQ ID NO: 168 SEQ ID NO:272 SEQ ID NO:376 SEQ ID NO:480 HIMPHRVQLK ILWKNGILK TVIGTPMTK REGKVNVVW RLLLGLPQW SEQ ID NO:65 SEQ ID NO: 169 SEQ ID NO:273 SEQ ID NO: 377 SEQ ID NO:481 IPTTAFRFNVA HHEDLDFGARL REGAGNRWGW VSTKLKTIW IFHLSLARV SEQ ID NO:66 SEQ ID NO: 170 SEQ ID NO:274 SEQ ID NO:378 SEQ ID NO:482 RVLGKSGIPLK PEVTVFLK ELFAERFLHW LFRRLFSHL RVLPGRLSAF SEQ ID NO:67 SEQ ID NO: 171 SEQ ID NO:275 SEQ ID NO:379 SEQ ID NO:483 LCRWLVLLLK ATDAPISRLRK QQAFLIFSI DAPISRLRK GSVLERRCTW SEQ ID NO:68 SEQ ID NO: 172 SEQ ID NO:276 SEQ ID NO: 380 SEQ ID NO:484 MPSQEWKA KPAPFQGTST QEHTGCEEK YPVVETREE FPEAVTCPSV SEQ ID NO:69 SEQ ID NO:173 SEQ ID NO:277 SEQ ID NO:381 SEQ ID NO:485 LPIPYLISA SVLERRCTW WLHLLMIW MLPVFGNKL VPMWIPRA SEQ ID NO:70 SEQ ID NO: 174 SEQ ID NO:278 SEQ ID NO: 382 SEQ ID NO:486 RLWKGGEPLLK VARSSVVQW RLQWGIMRV ISSPGKRCW AEGFIISSWS SEQ ID NO:71 SEQ ID NO: 175 SEQ ID NO:279 SEQ ID NO: 383 SEQ ID NO:487 SHEEQSFLEPL SEEPVMEKP RLRQLDSADSF ARALLVRCF RQHQLKFLK SEQ ID NO:72 SEQ ID NO: 176 SEQ ID NO:280 SEQ ID NO: 384 SEQ ID NO:488 FARPVGPPA LPSAFSRDPA DVLADLPGGL ATDAPISRL LPAQSPAGL SEQ ID NO:73 SEQ ID NO: 177 SEQ ID NO:281 SEQ ID NO:385 SEQ ID NO:489 RLSALTFQSTK ALSPSHLASV IPLQVQVTA RRVHHVWLLQ GPAPLRPNL SEQ ID NO:74 SEQ ID NO: 178 SEQ ID NO:282 SEQ ID NO:386 SEQ ID NO:490 RAAPPPLRI SSTRLRPTW KIWHGTEPR LPLQATREA LSLPLSHVL SEQ ID NO:75 SEQ ID NO: 179 SEQ ID NO:283 SEQ ID NO: 387 SEQ ID NO:491 AISPVLQVK LSFGSWLVV TPPSHSLDFSW LPIQPAERSAA FIIHPLLL SEQ ID NO:76 SEQ ID NO: 180 SEQ ID NO:284 SEQ ID NO: 388 SEQ ID NO:492 TEICPPKKKYF REWSRLNNF VSAPVRSRR RPFFAVSGRA LPHGFRRPPA SEQ ID NO:77 SEQ ID NO: 181 SEQ ID NO:285 SEQ ID NO: 389 SEQ ID NO:493 CSVTRSSFLW ETFAASALLEK SIASAISMK LLLETVFHL SEAPSKITL SEQ ID NO:78 SEQ ID NO: 182 SEQ ID NO:286 SEQ ID NO: 390 SEQ ID NO:494 LLYEHIGTVSA TAPLTASSL HLLQEELLL RVAQLFLPR YLSHDVLLLI SEQ ID NO:79 SEQ ID NO: 183 SEQ ID NO:287 SEQ ID NO:391 SEQ ID NO:495 RPYDRIWAIIH TVPCTAKEL TTATEALWHY LAADPGLGTL LPRPGSGLDF SEQ ID NO:80 SEQ ID NO: 184 SEQ ID NO:288 SEQ ID NO: 392 SEQ ID NO:496 RIANIIQSWW QAKPPSGAPW AQVTLSWPK HTINSMSRF MSSSSSRTPTW SEQ ID NO:81 SEQ ID NO: 185 SEQ ID NO:289 SEQ ID NO: 393 SEQ ID NO:497 RRVSQLLDLFY RRIYLKLS TGERDAIRKY KRWSGSGIRI SLLAADLLS SEQ ID NO:82 SEQ ID NO: 186 SEQ ID NO:290 SEQ ID NO: 394 SEQ ID NO:498 SPSPAIFSLKA RSWPWWWTL MPTHSWPTA SRFMVQHLK FMKQLITTL SEQ ID NO:83 SEQ ID NO: 187 SEQ ID NO:291 SEQ ID NO: 395 SEQ ID NO:499 LLWRISSSV GSVPTVGEW AILGTLVHA RTHFNLLPTF RLYERSRATW SEQ ID NO:84 SEQ ID NO: 188 SEQ ID NO:292 SEQ ID NO: 396 SEQ ID NO:500 IAWQSIQRSW RSAESILKCF VPLPHLSLA KLQAVQNWW CLSPVFKLYK SEQ ID NO:85 SEQ ID NO: 189 SEQ ID NO:293 SEQ ID NO: 397 SEQ ID NO:501 MPAFLSRASAL ATAPSATPWR IQVLWSLQP QILKVFIVK VGSRVSQSW SEQ ID NO:86 SEQ ID NO: 190 SEQ ID NO:294 SEQ ID NO:398 SEQ ID NO:502 LSDVLVKQY LPLIIPLPGL PVPEFIDPAF ALEGIIISL ARDALMYIK SEQ ID NO:87 SEQ ID NO: 191 SEQ ID NO:295 SEQ ID NO: 399 SEQ ID NO:503 RTSTTTWLLY ALYTVLDPV HVYAAFLRK QILYFRQLW RTSSMTGERKW SEQ ID NO:88 SEQ ID NO: 192 SEQ ID NO:296 SEQ ID NO:400 SEQ ID NO:504 MTYQQPFSNR TSVRIRQLW TEWSTVRHQW LISAGVPGGA VSLRWENAATW

[0129]

[0130] SEQ ID NO:89 SEQ ID NO: 193 SEQ ID NO:297 SEQ ID NO:401 SEQ ID NO:505VILLPLSSK TLSTTPFTSL QIVGSLKSW LGDLPSKSF APVCWRTPL SEQ ID NO:90 SEQ ID NO: 194 SEQ ID NO:298 SEQ ID NO:402 SEQ ID NO:506 ARLRDSFLR ELHSSRGLW RLDPHRPLW YPTNLPTT APDLVLDRY SEQ ID NO:91 SEQ ID NO: 195 SEQ ID NO:299 SEQ ID NO:403 SEQ ID NO:507 MELSDVLVKQY QKAAALAPF QVMLLRISK THHGDAFHLL FLAPRIHQL SEQ ID NO:92 SEQ ID NO: 196 SEQ ID NO:2OO SEQ ID NO:404 SEQ ID NO:508 ALFSKLASA RLLEPRPRW TNQTIALTL RPWQGGLGL GAYFFRHLK SEQ ID NO:93 SEQ ID NO: 197 SEQ ID NO:201 SEQ ID NO:405 SEQ ID NO:509 MTWDGSYLGK FTHKVVLYL AVAEALISK TVINQVLVYK TSSPAHPTW SEQ ID NO:94 SEQ ID NO: 198 SEQ ID NO:202 SEQ ID NO:406 SEQ ID NO:2 10 MPAFLSRASA IVAKQAGRWW TPYIPAPA LPIQPAERSA LSSLEVAQEVW SEQ ID NO:95 SEQ ID NO: 199 SEQ ID NO:203 SEQ ID NO:407 SEQ ID NO:2 11 RASWWAWAR HLPTWHVPV FPLKLSVPP EVVDSHQMLQF MSNGPQRPW SEQ ID NO:96 SEQ ID NO:200 SEQ ID NO:204 SEQ ID NO:408 SEQ ID NO:512 KIFEIGPVFTL KLNMHILWR LPRPPGVPGVA VHDASDILW MPRWPGIWTA SEQ ID NO:97 SEQ ID NO:201 SEQ ID NO:305 SEQ ID NO:409 SEQ ID NO:313 SALTFQSTK HIFHLSLARV KVESVSVLV LLDLPGGKY RQAPPVGLSW SEQ ID NO:98 SEQ ID NO:202 SEQ ID NO:206 SEQ ID NO:410 SEQ ID NO:514 VTYQSLHPNIK RLPERALVILK FIFDKSCHL SVNQAGVHW LGVLSGISVP SEQ ID NO:99 SEQ ID NO:203 SEQ ID NO:207 SEQ ID NO:411 SEQ ID NO:2 15 RVSVAQAGVQW WARSRYQAF SEIETGWRM KSLAAELLVLK SLSSMSLQRTW SEQ ID NO: 100 SEQ ID NO:204 SEQ ID NO:208 SEQ ID NO:412 SEQ ID NO:2 16 MSFTQKINSTW YVGPTRLEL ATSATLTHLLK LSSKLAFGRI TPTLRVVSL SEQ ID NO: 101 SEQ ID NO:205 SEQ ID NO:209 SEQ ID NO:413 SEQ ID NO:2 17 IQIGWVTQK REAEARVRW LTWEKPLEW LVTQRALAF SEQ ID NO: 102 SEQ ID NO:206 SEQ ID NO:2 10 SEQ ID NO:414

[0131] RLQELLESL LQGEGLLGT APPWPGRAA VTEENLLY SEQ ID NO: 103 SEQ ID NO:207 SEQ ID NO:2 11 SEQ ID NO:415

[0132] AVLQITHSF YLHLQLQEA AILAPPAILK SPGPPLPTA

[0133]

[0134] SEQ ID NO: 104 SEQ ID NO:208 SEQ ID NO:2 12 SEQ ID NO:416

[0135] The present description provides compositions that are a combination of any two or more of the CR ncHLAp peptides of SEQ ID NO:1 to SEQ ID NO:517 or a variant thereof. Such a combination may include, for example, any 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, or all 517 peptides. Such a combination may include, for example, any 2-10, any 10-15, any 15-30, any 20-40, or any 2-100 different peptides.

[0136] Such a combination may be a combination of any 2, any 3, any 4, any 5, any 6, any 7, any 8, any 9, any 10, any 11, any 12, any 13, or all 14 of the NU10 (HIFHLSLARV, SEQ ID NO:202), NU11 (KLFLWPYKV, SEQ ID NO:9 ), NU42 (LLWRISSSV, SEQ ID NO:84), NU43 (SALTFQSTK, SEQ ID NO:98), NU44 (RLQELLESL, SEQ ID NO: 103 ), NU45 (TVINQVLVYK, SEQ ID NO: 406), NU46 (GAYFFRHLK, SEQ ID NO: 509), NU47 (ILQDRLWKL, SEQ ID NO:449), NU49 (RGNLPLPLK, SEQ ID NO: 239), NU50 (AILGTLVHA, SEQ ID NO:292), NU51 (QILKVFIVK, SEQ ID NO:398), NU53(YLLDGQLQL, SEQ ID NO:231), NU56 (SLQKILHQL, SEQ ID NO:47), and / or NU57 (ALFSKLASA, SEQ ID NO:93) peptides,

[0137] As shown in Tables 2 and 3, many of the 517 CR ncHLAp peptides of SEQ ID NO: 1 to SEQ ID NO:517 have been found to be associated with cancers other than pancreatic cancer. For this analysis immunopeptidomic data from 15 cancer types (1444 cancer samples), including non-canonical HLA-I bound peptides (ncHLAp), was abstracted from the IEAtlas database (Cai et al. Nucleic Acid Research. 2023; 51(D1): D409-D417). Empirically identified CR ncHLAp identified in pancreatic cancer (Example 1 and Ely at al., Science. 2025 May 8;388(6747)) were cross-referenced against these additional tumor types for immunopeptidomic evidence of ncHLAp detection in each additional cancer type using a custom R script.

[0138] Such a combination of peptides may include one or more peptides identified in Table 2 or Table 3 to be associated with a specific cancer, such as for example, acute myeloid leukemia, B-acute lymphoblastic leukemia, breast cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon carcinoma, glioblastoma, lung cancer, lymphoma, melanoma, meningioma, neuroblastoma, ovarian cancer, or T-acute lymphoblastic leukemia.Table 2: CR ncHLAp Found in Tumor Types in Addition to PDAC

[0139] SEQ Peptide Sequence Tumor types detected in (in addition to PDAC)*

[0140] ID Glio. Mel. Ovarian Menin. Lung AML CML B-ALL Lymph. T-ALL Colon Breast CLL Neuro.

[0141] NO

[0142] 76 AISPVLQVK X X X X X

[0143] 391 RVAQLFLPR X

[0144] 167 GTIANILAH X X X

[0145] 172 ATDAPISRLRK X

[0146] 49 RVSLPKLGYK X X X X X X X X

[0147] 102 IQIGWVTQK X X X

[0148] 67 RVLGKSGIPLK X X X X X X X X X

[0149] 469 VLLENSSIVKI X X X

[0150] 71 RLWKGGEPLLK X X X X X X

[0151] 372 SVISVLTTPK X X

[0152] 47 SLQKILHQL X X X X X X X

[0153] 398 QILKVFIVK X X X X

[0154] 239 RGNLPLPLK X X X

[0155] 479 MLARLVWNA X X X

[0156] 449 ILQDRLWKL X

[0157] 406 TVINQVLVYK X X X X X X

[0158] 103 RLQELLESL X

[0159] 98 SALTFQSTK X X

[0160] 84 LLWRISSSV X X

[0161] 434 KMVFFLHMV X X

[0162] 358 HLFSVLSAI X X X

[0163] 45 FLWEILERL X X X

[0164] 417 AILVPQPPK X X X X

[0165] 43 RTNNILLPR X X X X X X X X

[0166] 268 VVLGVSFSR X

[0167] 202 HIFHLSLARV X

[0168] 55 AADPGLGTL X X X X

[0169] 366 AAYTKLFIK X X

[0170] 249 AEQPAPAHAY X

[0171] 216 AHAPPEQHL X X X X

[0172] 312 AILAPPAILK X

[0173] 471 ALAVIPIPK X X X X X X

[0174] 399 ALEGIIISL X

[0175]

[0176] 42 ALWNVPTGRTW X X461 ALWPKVPEV X

[0177] 107 APIDRYWTP X X X

[0178] 147 APLLQPRAA X X X X X X X 340 APLYQVAAA X

[0179] 210 APRLLPATW X X

[0180] 289 AQVTLSWPK X

[0181] 384 ARALLVRCF X

[0182] 347 ARISVTELIQK X

[0183] 91 ARLRDSFLR X X

[0184] 322 ARLWFRPPL X X

[0185] 22 ASFHWRMLK X

[0186] 385 ATDAPISRL X X X

[0187] 28 ATESVLQKILK X X

[0188] 309 ATSATLTHLLK X X X X X

[0189] 302 AVAEALISK X

[0190] 8 AVFVNWEQTK X X X X X 30 AVFVNWEQTKK X X X X X X

[0191] 374 AVIQIYSHF X X X

[0192] 456 AVQSVAVSI X X X X 430 DEFGDSRRRW X X X X 2 EEFLGGKKTNW X X X

[0193] 20 EEGRIITKW X X X X

[0194] 122 ELRAGDTATW X X X

[0195] 182 ETFAASALLEK X X

[0196] 218 EVARGAVPG X X X

[0197] 271 EVREGELDWW X

[0198] 408 EWDSHQMLQF X X X X X X

[0199] 108 FAIDQPELHL X

[0200] 338 FILLWKSPK X X

[0201] 508 FLAPRIHQL X X X

[0202] 455 FLNNLEVNM X X X

[0203] 29 FLQEEAALKAL X X X

[0204] 137 FQAAPDRGF X X X

[0205] 114 FRIPGTQSL X

[0206] 33 FRPPQPQYL X X X

[0207] 463 FTASGKYGMY X

[0208] 329 FTQKINSTW X X

[0209]

[0210] 113 GHSFPDPGL X X X X314 GLLETHPALLL X X 362 GLSDKTFLL X X

[0211] 490 GPAPLRPNL X X

[0212] 188 GSVPTVGEW X

[0213] 65 HIMPHRVQLK X X X X X X X X

[0214] 470 HLHRDSALI X

[0215] 287 HLLQEELLL X X X

[0216] 259 HPEESLLLL X

[0217] 296 HVYAAFLRK X

[0218] 44 ILAPPAILK X X X

[0219] 371 ILLNFSTTTK X X X

[0220] 169 ILWKNGILK X X X 350 ILYQNPKTLK X X

[0221] 66 IPTTAFRFNVA X X 160 ISSSVFTSY X 64 KIFEKLVALK X X X X X X X X

[0222] 453 KIWNANYFLTK X X X X

[0223] 323 KLFGYVQEF X X X

[0224] 421 KLLDIGYRK X X X X X

[0225] 251 KPFPGTSEF X X

[0226] 394 KRWSGSGIRI X

[0227] 412 KSLAAELLVLK X X X X X X X X X 337 KVLVDFLLK X X X

[0228] 392 LAADPGLGTL X X X X

[0229] 363 LAPPAILK X

[0230] 379 LFRRLFSHL X X X X

[0231] 48 LFRVWAK X

[0232] 478 LGELGLVDL X

[0233] 410 LLDLPGGKY X X X X X X

[0234] 390 LLLETVFHL X X X X 388 LPIQPAERSAA X

[0235] 191 LPLHPLPGL X 420 LPLPRLPVSA X

[0236] 496 LPRPGSGLDF X X X X X X

[0237] 443 LPRQPATSL X X X X X X

[0238] 440 LPSQEDSIM X 355 LQIPWKLLK X X

[0239]

[0240] 87 LSDVLVKQY X491 LSLPLSHVL X

[0241] 413 LSSKLAFGRI X

[0242] 128 LVGPPGSSW X X

[0243] 92 MELSDVLVKQY X X X 321 MHFLENISL X X X X 442 MLLLMLLYK X X

[0244] 86 MPAFLSRASAL X

[0245] 513 MPRWPGIWTA X

[0246] 473 MSRPPITTNYR X X X 441 MTDWANMFQHY X

[0247] 94 MTWDGSYLGK X X X X

[0248] 89 MTYQQPFSNR X X X X X X X X X 277 QLHTGCLLK X X 300 QVMLLRISK X

[0249] 138 RAASWTSTW X X

[0250] 360 RAQPNGRHPW X X X

[0251] 96 RASWWAWAR X

[0252] 32 RINYHLLELK X X

[0253] 123 RIYFQEQEK X

[0254] 254 RLAPLLLTH X X X X X X X

[0255] 319 RLFLIVSSGQK X X X

[0256] 361 RLFLNSNTL X X

[0257] 197 RLLEPRPRW X

[0258] 423 RLLLNLNQK X X X X

[0259] 164 RLNYRTTYR X X X X X 280 RLRQLDSADSF X

[0260] 74 RLSALTFQSTK X X

[0261] 152 RLSLPLSSY X X X X X 144 RLSQDGFMLK X

[0262] 348 RLWGMSWPK X X X X X

[0263] 139 RLWKHTLKY X X X X X X

[0264] 34 RLWTSVAVLK X X X X X X

[0265] 356 RLYSLSTALR X X X X X X

[0266] 370 RMWPNNLVHK X X X X X

[0267] 341 RPFFNVRVA X X X 125 RPFYLLTKV X

[0268] 150 RPIWDVRSA X

[0269]

[0270] 405 RPWQGGLGL X488 RQHQLKFLK X X X X X X 439 RRLETVLIK X

[0271] 246 RRNWNGLLATM

[0272] 17 RSFPKASSSW X

[0273] 258 RSLDFLLFR X

[0274] 326 RTHPLTHSW X

[0275] 24 RTMHGDLLQTK X X X X X

[0276] 504 RTSSMTGERKW X

[0277] 483 RVLPGRLSAF X

[0278] 54 RVNTMENLKLK X X

[0279] 100 RVSVAQAGVQW X

[0280] 132 RVYWIGERK X X X X

[0281] 234 SADSVPHVL X

[0282] 494 SEAPSKITL X X X X X X 176 SEEPVMEKP X X 286 SIASAISMK X X X X X X

[0283] 345 SLIPKVFLK X X X

[0284] 317 SPAPPDRAVL X X

[0285] 51 SPIMPSASL X X

[0286] 60 SPLPHLGPL X X 466 SRITIHLNR X X X X

[0287] 127 SRNISIWRF X

[0288] 331 SSGPTRAIP X

[0289] 213 SSMYTTIPR X X X

[0290] 10 STKDMALAWSR X X X X X X 5 SVDLYKILTF X X

[0291] 411 SVNQAGVHW X

[0292] 290 TGERDAIRKY X

[0293] 404 THHGDAFHLL X X

[0294] 156 THSSWVSWL X

[0295] 472 TIKVRNLPL X X X 4 TLFRVWAK X X X X X X X X X X 266 TLKIKSLQI X X

[0296] 351 TRIDFFENR X X X

[0297] 510 TSSPAHPTW X

[0298] 288 TTATEALWHY X

[0299] 248 TVYRQEVSA X X

[0300]

[0301] 502 VGSRVSQSW X409 VHDASDILW X X

[0302] 459 VHTTTILYL X X

[0303] 116 VILLPGPAK X

[0304] 90 VILLPLSSK X X X X

[0305] 424 VLWGHRFTF X

[0306] 154 VLYLRQVGY X X X

[0307] 422 VPILSRETL X

[0308] 38 VPLPRIPTV X X

[0309] 99 VTYQSLHPNIK X X

[0310] 13 WLYHLINK X

[0311] 480 WMKEQTLEM X

[0312] 208 YLHLQLQEA X X X

[0313] 61 YLHPHRLPL X X X X X X X

[0314] 215 YLIQRLTSL X X

[0315] 381 YPVVEIREL X X X X

[0316] 149 YSNGLQHPL X X X X X X

[0317] 209 YSSEIWDLY X X

[0318]

[0319] 205 YVGPTRLEL X X X X X

[0320] *“Glio." = glioblastoma; “Mel."= melanoma; “Ovarian"= ovarian cancer; “Menm.”= meningioma; “Lung" = lung cancer; “AML" = Acute Myeloid Leukemia; “CML” = Chronic Myelogenous Leukemia; “B-ALL” = B-Acute Lymphoblastic Leukemia; “Lymph," = lymphoma; “T-ALL” = T-Acute Lymphoblastic Leukemia; “Colon" = Colon carcinoma; “Breast” = Breast Cancer; “CLL" = Chronic Lymphoblastic Leukemia; “Neuro. ”= Neuroblastoma.Table 3 - Peptides Associated with Tumor Type Other Than PDAC

[0321] Glioblastoma

[0322] 76 AISPVLQVK 42 ALWNVPTGRTW 44 ILAPPAILK 89 MTYQQPFSNR 234 SADSVPHVL 49 RVSLPKLGYK 147 APLLQPRAA 371 ILLNFSTTTK 277 QLHTGCLLK 286 SIASAISMK 67 RVLGKSGIPLK 30 AVFVNWEQTKK 64 KIFEKLVALK 138 RAASWTSTW 345 SLIPKVFLK 469 VLLENSSIVKI 374 AVIQIYSHF 453 KIWNANYFLTK 32 RINYHLLELK 466 SRITIHLNR 71 RLWKGGEPLLK 430 DEFGDSRRRW 323 KLFGYVQEF 254 RLAPLLLTH 10 STKDMALAWSR 47 SLQKILHQL 20 EEGRIITKW 421 KLLDIGYRK 319 RLFLIVSSGQK 5 SVDLYKILTF 239 RGNLPLPLK 122 ELRAGDTATW 412 KSLAAELLVLK 361 RLFLNSNTL 4 TLFRVWAK 406 TVINQVLVYK 408 EWDSHQMLQF 337 KVLVDFLLK 164 RLNYRTTYR 248 TVYRQEVSA 84 LLWRISSSV 338 FILLWKSPK 392 LAADPGLGTL 74 RLSALTFQSTK 154 VLYLRQVGY 434 KMVFFLHMV 29 FLQEEAALKAL 363 LAPPAILK 152 RLSLPLSSY 38 VPLPRIPTV 358 HLFSVLSAI 137 FQAAPDRGF 48 LFRWVAK 348 RLWGMSWPK 99 VTYQSLHPNIK 417 AILVPQPPK 33 FRPPQPQYL 410 LLDLPGGKY 139 RLWKHTLKY 61 YLHPHRLPL 43 RTNNILLPR 329 FTQKINSTW 390 LLLETVFHL 34 RLWTSVAVLK 215 YLIQRLTSL 202 HIFHLSLARV 113 GHSFPDPGL 443 LPRQPATSL 356 RLYSLSTALR 381 YPVVEIREL 55 AADPGLGTL 314 GLLETHPALLL 321 MHFLENISL 370 RMWPNNLVHK 209 YSSEIWDLY 312 AILAPPAILK 65 HIMPHRVQLK 473 MSRPPITTNYR 488 RQHQLKFLK 234 SADSVPHVL 471 ALAVIPIPK 287 HLLQEELLL 441 MTDWANMFQHY 24 RTMHGDLLQTK 286 SIASAISMK Melanoma

[0323] 76 AISPVLQVK 91 ARLRDSFLR 296 HVYAAFLRK 360 RAQPNGRHPW 286 SIASAISMK 167 GTIANILAH 322 ARLWFRPPL 44 ILAPPAILK 254 RLAPLLLTH 345 SLIPKVFLK 49 RVSLPKLGYK 22 ASFHWRMLK 371 ILLNFSTTTK 319 RLFLIVSSGQK 466 SRITIHLNR 102 IQIGWVTQK 28 ATESVLQKILK 169 ILWKNGILK 361 RLFLNSNTL 127 SRNISIWRF 67 RVLGKSGIPLK 309 ATSATLTHLLK 350 ILYQNPKTLK 197 RLLEPRPRW 331 SSGPTRAIP 469 VLLENSSIVKI 302 AVAEALISK 66 IPTTAFRFNVA 423 RLLLNLNQK 213 SSMYTTIPR 71 RLWKGGEPLLK 8 AVFVNWEQTK 64 KIFEKLVALK 164 RLNYRTTYR 10 STKDMALAWSR

[0324]

[0325] 47 SLQKILHQL 30 AVFVNWEQTKK 323 KLFGYVQEF 280 RLRQLDSADSF 290 TGERDAIRKY398 QILKVFIVK 374 AVIQIYSHF 421 KLLDIGYRK 74 RLSALTFQSTK 404 THHGDAFHLL 239 RGNLPLPLK 430 DEFGDSRRRW 394 KRWSGSGIRI 152 RLSLPLSSY 472 TIKVRNIPL 479 MLARLVWNA 122 ELRAGDTATW 412 KSLAAELLVLK 348 RLWGMSWPK 4 TLFRVWAK 406 TVINQVLVYK 182 ETFAASALLEK 337 KVLVDFLLK 139 RLWKHTLKY 266 TLKIKSLQI 98 SALTFQSTK 218 EVARGAVPG 379 LFRRLFSHL 34 RLWTSVAVLK 351 TRIDFFENR 84 LLWRISSSV 408 EWDSHQMLQF 478 LGELGLVDL 356 RLYSLSTALR 248 TVYRQEVSA 45 FLWEILERL 508 FLAPRIHQL 410 LLDLPGGKY 370 RMWPNNLVHK 409 VHDASDILW 417 AILVPQPPK 455 FLNNLEVNM 390 LLLETVFHL 341 RPFFNVRVA 459 VHTTTILYL 43 RTNNILLPR 29 FLQEEAALKAL 388 LPIQPAERSAA 125 RPFYLLTKV 116 VILLPGPAK 268 WLGVSFSR 137 FQAAPDRGF 420 LPLPRLPVSA 488 RQHQLKFLK 90 VILLPLSSK 55 AADPGLGTL 114 FRIPGTQSL 496 LPRPGSGLDF 439 RRLETVLIK 424 VLWGHRFTF 366 AAYTKLFIK 33 FRPPQPQYL 443 LPRQPATSL 17 RSFPKASSSW 154 VLYLRQVGY 216 AHAPPEQHL 463 FTASGKYGMY 355 LQIPWKLLK 258 RSLDFLLFR 38 VPLPRIPTV 471 ALAVIPIPK 329 FTQKINSTW 491 LSLPLSHVL 326 RTHPLTHSW 480 WMKEQTLEM 399 ALEGIIISL 113 GHSFPDPGL 321 MHFLENISL 24 RTMHGDLLQTK 208 YLHLQLQEA 42 ALWNVPTGRTW 362 GLSDKTFLL 442 MLLLMLLYK 504 RTSSMTGERKW 61 YLHPHRLPL 461 ALWPKVPEV 490 GPAPLRPNL 86 MPAFLSRASAL 483 RVLPGRLSAF 215 YLIQRLTSL 107 APIDRYWTP 188 GSVPTVGEW 513 MPRWPGIWTA 54 RVNTMENLKLK 381 YPVVEIREL 147 APLLQPRAA 65 HIMPHRVQLK 94 MTWDGSYLGK 100 RVSVAQAGVQW 149 YSNGLQHPL 340 APLYQVAAA 470 HLHRDSALI 89 MTYQQPFSNR 132 RVYWIGERK 209 YSSEIWDLY 347 ARISVTELIQK 287 HLLQEELLL 138 RAASWTSTW 494 SEAPSKITL 205 YVGPTRLEL Ovarian Cancer

[0326] 76 AISPVLQVK 216 AHAPPEQHL 412 KSLAAELLVLK 144 RLSQDGFMLK 213 SSMYTTIPR 172 ATDAPISRLRK 147 APLLQPRAA 392 LAADPGLGTL 139 RLWKHTLKY 10 STKDMALAWSR 49 RVSLPKLGYK 210 APRLLPATW 390 LLLETVFHL 34 RLWTSVAVLK 4 TLFRVWAK 67 RVLGKSGIPLK 30 AVFVNWEQTKK 496 LPRPGSGLDF 356 RLYSLSTALR 510 TSSPAHPTW 71 RLWKGGEPLLK 2 EEFLGGKKTNW 443 LPRQPATSL 370 RMWPNNLVHK 502 VGSRVSQSW 372 SVISVLTTPK 20 EEGRIITKW 94 MTWDGSYLGK 405 RPWQGGLGL 90 VILLPLSSK 406 TVINQVLVYK 65 HIMPHRVQLK 89 MTYQQPFSNR 24 RTMHGDLLQTK 13 WLYHLINK

[0327]

[0328] 434 KMVFFLHMV 287 HLLQEELLL 360 RAQPNGRHPW 286 SIASAISMK 149 YSNGLQHPL45 FLWEILERL 64 KIFEKLVALK 254 RLAPLLLTH 317 SPAPPDRAVL 205 YVGPTRLEL 43 RTNNILLPR 251 KPFPGTSEF 423 RLLLNLNQK 51 SPIMPSASL

[0329] Meningioma

[0330] 76 AISPVLQVK 28 ATESVLQKILK 392 LAADPGLGTL 423 RLLLNLNQK 10 STKDMALAWSR 49 RVSLPKLGYK 309 ATSATLTHLLK 379 LFRRLFSHL 164 RLNYRTTYR 404 THHGDAFHLL 67 RVLGKSGIPLK 8 AVFVNWEQTK 410 LLDLPGGKY 139 RLWKHTLKY 472 TIKVRNIPL 71 RLWKGGEPLLK 30 AVFVNWEQTKK 496 LPRPGSGLDF 34 RLWTSVAVLK 4 TLFRVWAK 398 QILKVFIVK 20 EEGRIITKW 443 LPRQPATSL 356 RLYSLSTALR 409 VHDASDILW 239 RGNLPLPLK 122 ELRAGDTATW 87 LSDVLVKQY 370 RMWPNNLVHK 459 VHTTTILYL 98 SALTFQSTK 182 ETFAASALLEK 413 LSSKLAFGRI 150 RPIWDVRSA 90 VILLPLSSK 43 RTNNILLPR 408 EWDSHQMLQF 128 LVGPPGSSW 488 RQHQLKFLK 61 YLHPHRLPL 366 AAYTKLFIK 508 FLAPRIHQL 92 MELSDVLVKQY 24 RTMHGDLLQTK 381 YPVVEIREL 216 AHAPPEQHL 137 FQAAPDRGF 473 MSRPPITTNYR 54 RVNTMENLKLK 149 YSNGLQHPL 471 ALAVIPIPK 113 GHSFPDPGL 89 MTYQQPFSNR 132 RVYWIGERK 205 YVGPTRLEL 107 APIDRYWTP 65 HIMPHRVQLK 300 QVMLLRISK 286 SIASAISMK

[0331] 147 APLLQPRAA 350 ILYQNPKTLK 360 RAQPNGRHPW 317 SPAPPDRAVL

[0332] 210 APRLLPATVV 64 KIFEKLVALK 96 RASWWAWAR 51 SPIMPSASL

[0333] 384 ARALLVRCF 412 KSLAAELLVLK 254 RLAPLLLTH 213 SSMYTTIPR

[0334] Lung Cancer

[0335] 76 AISPVLQVK 2 EEFLGGKKTNW 44 ILAPPAILK 92 MELSDVLVKQY 4 TLFRVWAK 49 RVSLPKLGYK 20 EEGRIITKW 64 KIFEKLVALK 254 RLAPLLLTH 266 TLKIKSLQI 67 RVLGKSGIPLK 218 EVARGAVPG 251 KPFPGTSEF 488 RQHQLKFLK 351 TRIDFFENR 372 SVISVLTTPK 271 EVREGELDWW 412 KSLAAELLVLK 24 RTMHGDLLQTK 90 VILLPLSSK 47 SLQKILHQL 408 EWDSHQMLQF 379 LFRRLFSHL 494 SEAPSKITL 61 YLHPHRLPL 249 AEQPAPAHAY 508 FLAPRIHQL 410 LLDLPGGKY 286 SIASAISMK

[0336] 8 AVFVNWEQTK 362 GLSDKTFLL 443 LPRQPATSL 5 SVDLYKILTF

[0337] 374 AVIQIYSHF 65 HIMPHRVQLK 128 LVGPPGSSW 411 SVNQAGVHW

[0338] Acute Myeloid Leukemia

[0339] 391 RVAQLFLPR 91 ARLRDSFLR 89 MTYQQPFSNR 466 SRITIHLNR 351 TRIDFFENR

[0340]

[0341] 107 APIDRYWTP 322 ARLWFRPPL 254 RLAPLLLTH 10 STKDMALAWSR 61 YLHPHRLPL147 APLLQPRAA 408 EWDSHQMLQF 488 RQHQLKFLK 4 TLFRVWAK 149 YSNGLQHPL Chronic Myeloid Leukemia

[0342] 167 GTIANILAH 398 QILKVFIVK 471 ALAVIPIPK 2 ATSATLTHLLK 94 MTWDGSYLGK 102 IQIGWVTQK 417 AILVPQPPK 289 AQVTLSWPK 421 KLLDIGYRK 348 RLWGMSWPK 67 RVLGKSGIPLK 43 RTNNILLPR

[0343] B-Acute Lymphoblastic Leukemia

[0344] 167 GTIANILAH 43 RTNNILLPR 64 KIFEKLVALK 89 MTYQQPFSNR 356 RLYSLSTALR 49 RVSLPKLGYK 55 AADPGLGTL 453 KIWNANYFLTK 123 RIYFQEQEK 494 SEAPSKITL 102 IQIGWVTQK 471 ALAVIPIPK 421 KLLDIGYRK 164 RLNYRTTYR 4 TLFRVWAK 67 RVLGKSGIPLK 385 ATDAPISRL 412 KSLAAELLVLK 152 RLSLPLSSY 61 YLHPHRLPL 47 SLQKILHQL 309 ATSATLTHLLK 496 LPRPGSGLDF 348 RLWGMSWPK 149 YSNGLQHPL 406 TVINQVLVYK 8 AVFVNWEQTK 355 LQIPWKLLK 139 RLWKHTLKY 205 YVGPTRLEL 417 AILVPQPPK 65 HIMPHRVQLK 94 MTWDGSYLGK 34 RLWTSVAVLK

[0345] Lymphoma

[0346] co

[0347] co 49 RVSLPKLGYK 30 AVFVNWEQTKK 113 GHSFPDPGL 321 MHFLENISL 341 RPFFNVRVA 67 RVLGKSGIPLK 456 AVQSVAVSI 65 HIMPHRVQLK 442 MLLLMLLYK 132 RVYWIGERK 71 RLWKGGEPLLK 2 EEFLGGKKTNW 64 KIFEKLVALK 89 MTYQQPFSNR 156 THSSWVSWL 406 TVINQVLVYK 218 EVARGAVPG 412 KSLAAELLVLK 348 RLWGMSWPK 4 TLFRVWAK 103 RLQELLESL 408 EWDSHQMLQF 379 LFRRLFSHL 34 RLWTSVAVLK

[0348] 216 AHAPPEQHL 108 FAIDQPELHL 410 LLDLPGGKY 356 RLYSLSTALR

[0349] T-Acute Lymphoblastic Leukemia

[0350] 49 RVSLPKLGYK 43 RTNNILLPR 371 ILLNFSTTTK 32 RINYHLLELK 286 SIASAISMK 67 RVLGKSGIPLK 471 ALAVIPIPK 169 ILWKNGILK 254 RLAPLLLTH 345 SLIPKVFLK 71 RLWKGGEPLLK 30 AVFVNWEQTKK 64 KIFEKLVALK 319 RLFLIVSSGQK 60 SPLPHLGPL 47 SLQKILHQL 430 DEFGDSRRRW 453 KIWNANYFLTK 423 RLLLNLNQK 4 TLFRVWAK 398 QILKVFIVK 338 FILLWKSPK 421 KLLDIGYRK 152 RLSLPLSSY 154 VLYLRQVGY 479 MLARLVWNA 455 FLNNLEVNM 412 KSLAAELLVLK 139 RLWKHTLKY 99 VTYQSLHPNIK 449 ILQDRLWKL 490 GPAPLRPNL 337 KVLVDFLLK 370 RMWPNNLVHK 208 YLHLQLQEA 406 TVINQVLVYK 65 HIMPHRVQLK 496 LPRPGSGLDF 132 RVYWIGERK 381 YPVVEIREL

[0351]

[0352] 358 HLFSVLSAI 259 HPEESLLLL 89 MTYQQPFSNR 494 SEAPSKITLColon Carcinoma

[0353] 469 VLLENSSIVKI 385 ATDAPISRL 29 FLQEEAALKAL 410 LLDLPGGKY 466 SRITIHLNR 47 SLQKILHQL 455 FLNNLEVNM 314 GLLETHPALLL 176 SEEPVMEKP 288 TTATEALWHY 55 AADPGLGTL

[0354] Breast Cancer

[0355] 47 SLQKILHQL 453 KIWNANYFLTK 89 MTYQQPFSNR 176 SEEPVMEKP 61 YLHPHRLPL 43 RTNNILLPR 323 KLFGYVQEF 277 QLHTGCLLK 60 SPLPHLGPL 149 YSNGLQHPL 385 ATDAPISRL 392 LAADPGLGTL 164 RLNYRTTYR 10 STKDMALAWSR 205 YVGPTRLEL 309 ATSATLTHLLK 496 LPRPGSGLDF 152 RLSLPLSSY 472 TIKVRNIPL

[0356] 456 AVQSVAVSI 443 LPRQPATSL 488 RQHQLKFLK 4 TLFRVWAK

[0357] 33 FRPPQPQYL 92 MELSDVLVKQY 494 SEAPSKITL 422 VPILSRETL

[0358] Chronic Lymphoblastic Leukemia

[0359] 479 MLARLVWNA 8 AVFVNWEQTK 66 IPTTAFRFNVA 440 LPSQEDSIM 494 SEAPSKITL 358 HLFSVLSAI 456 AVQSVAVSI 160 ISSSVFTSY 321 MHFLENISL 208 YLHLQLQEA 45 FLWEILERL 430 DEFGDSRRRW 390 LLLETVFHL 473 MSRPPITTNYR

[0360] 147 APLLQPRAA 169 ILWKNGILK 191 LPLHPLPGL 341 RPFFNVRVA

[0361] Neuroblastoma

[0362]

[0363] 147 APLLQPRAA 456 AVQSVAVSI 412 KSLAAELLVLK

[0364] *SEQ ID NOs are shown to the right of each sequenceSuch a composition of peptides may be used as a vaccine for cancer, including, but not limited to, pancreatic cancer, acute myeloid leukemia, B-acute lymphoblastic leukemia, breast cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon carcinoma, glioblastoma, lung cancer, lymphoma, melanoma, meningioma, neuroblastoma, ovarian cancer, and / or or T-acute lymphoblastic leukemia. Such a cancer may express at least one of SEQ ID NO: 1 to SEQ ID NO: 517. Embodiments provide for two or more peptides of SEQ ID NO:1 to SEQ ID NO: 517 to be included in such anticancer vaccine. In some embodiments, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 517 different peptides are pooled and / or coded for in the vaccine composition, such as 2-100, including 10-15, 10-20, 15-30, 20-40 different peptides. Not all individual tumors necessarily express the same pattern of antigens. Therefore, a combination of several tumor-associated peptides can be used to ensure that every single tumor bears at least some of the targets. Thus, the vaccine can easily be used "off-the-shelf' for a larger patient population. This means that a pre-selection of patients does not require any additional biomarker assessments for antigen expression, but it is still ensured that several targets are simultaneously attacked by the induced immune response.

[0365] Such a combination of peptides may further include a pharmaceutically acceptable carrier or one or more adjuvants. A pharmaceutical composition is a composition suitable for administration to a human being in a medical setting, such as a sterile pharmaceutical composition produced according to GMP guidelines.

[0366] The pharmaceutical compositions comprise the peptides either in the free form or in the form of a pharmaceutically acceptable salt. As used herein, “a pharmaceutically acceptable salt” refers to a derivative of the disclosed peptides wherein the peptide is modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral -NH2 group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p toluenesulfonic acid, salicylic acid, and the

[0367] like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties whichmay be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethyl amine or the like.

[0368] The present description provides compositions that are a single CR ncHLAp peptide selected from SEQ ID NO: 1 to SEQ ID NO:517 and one or more adjuvants. Such a composition of a peptide and an adjuvant may be used as a vaccine for cancer, including, but not limited to, pancreatic cancer, acute myeloid leukemia, B-acute lymphoblastic leukemia, breast cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon carcinoma, glioblastoma, lung cancer, lymphoma, melanoma, meningioma, neuroblastoma, ovarian cancer, and / or or T-acute lymphoblastic leukemia. Such a cancer may express at least one of SEQ ID NO: 1 to SEQ ID NO: 517.

[0369] Compositions described herein may include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate the immune response. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX@, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA, resiquimod, ImuFact IMP321, Interleukins as IL-2, IL- 13, IL 21, Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel vector system, poly(lactid co-glycolid) [PLG]-based and dextran microparticles, talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, betaglucan, Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are included.

[0370] The composition can be used for parenteral administration, such as subcutaneous, intra dermal, intramuscular or oral administration. For this, the peptides or nucleotides, including vectors or naked nucleic acid segments, and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable carrier, such as an aqueous carrier. In addition, the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. The peptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for a prevention, prophylaxis and / or therapy of cancerous diseases.

[0371] The peptides and compositions described herein may be used in in vitro methods to producing antigen-specific activated T lymphocytes. Such a method includes contacting T cells in vitro with antigen presenting cells loaded with one or more isolated non-canonical HLA-I bound peptides (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517 for a period of time sufficient to activate said T cells in an antigen-specific manner to the one or more ncHLAp. Such T cells and / or antigen presenting cells may be derived from a subject with a cancer. Such a cancer may be associated with one or more of the peptides of SEQ ID NOS: 1-517. Such a cancer may include, for example, pancreatic cancer or other solid or hematological such as glioblastoma, melanoma, ovarian cancer, meningioma, lung cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, B-acute lymphoblastic leukemia, lymphoma, T-acute lymphoblastic leukemia, chronic lymphocytic leukemia, colon carcinoma, breast cancer, or neuroblastoma. The present description further includes the antigen-specific activated T lymphocytes produced by such methods. The antigenspecific activated T lymphocytes may be administered to the subject for the treatment of the cancer.

[0372] The present description further relates to in vivo methods for producing antigen-specific activated T lymphocytes in a subject. Such a method includes the administration of one or more peptides of SEQ ID NOS: 1-517 or a composition thereof to the subject. The subject may be suffering from a cancer, including but not limited to, pancreatic cancer or other solid or hematological such as glioblastoma, melanoma, ovarian cancer, meningioma, lung cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, B-acute lymphoblastic leukemia, lymphoma, T-acute lymphoblastic leukemia, chronic lymphocytic leukemia, colon carcinoma, breast cancer, neuroblastoma, or other a cancer that shows expression of at least one of SEQ ID NO: 1 to SEQ ID NO: 517. Such administration may include administration of an amount effective for the treatment of the cancer.

[0373] The present description further relates to methods for the treatment of cancer, by the administration of one or more peptides of SEQ ID NOS: 1-517 or a composition thereof to asubject with a cancer for the treatment of said cancer. Such a cancer includes, but is not limited to, pancreatic cancer or other solid or hematological such as glioblastoma, melanoma, ovarian cancer, meningioma, lung cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, B-acute lymphoblastic leukemia, lymphoma, T-acute lymphoblastic leukemia, chronic lymphocytic leukemia, colon carcinoma, breast cancer, neuroblastoma, or other cancers that show expression of at least one of SEQ ID NO: 1 to SEQ ID NO: 517. Such a treatment for cancer will include the administration of an effective amount of a composition as described herein to a subject with the cancer.

[0374] The present description further relates to particular marker proteins and biomarkers based on the peptides according to the present description, herein called "targets" that can be used in the diagnosis and / or prognosis of cancer, such a pancreatic cancer. The present description also relates to the use of these novel targets for cancer treatment.

[0375] A further aspect of the description provides a nucleic acid (for example a polynucleotide) coding for a peptide of SEQ ID NOS: 1-517 or peptide variant of the description. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA or combinations thereof, either single- and / or double-stranded, or native or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone and it may or may not contain introns so long as it codes for the peptide. A still further aspect of the description provides an expression vector capable of expressing a polypeptide according to the description. Such an expression vector may include a promoter and / or other regulatory elements.

[0376] The nucleotide sequence coding for a particular peptide, oligopeptide, or polypeptide may be naturally occurring or may be synthetically constructed. Generally, DNA segments encoding the peptides, polypeptides, and proteins of this description are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene that is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

[0377] As used herein the term “a nucleotide coding for (or encoding) a peptide” refers to a nucleotide sequence coding for the peptide. Such a nucleotide sequence may include artificial (man-made) start and stop codons compatible for the biological system the sequence is to be expressed by, for example, a cell system useful for the production of TCRs or for expression in vivo as a vaccine.As used herein, reference to a nucleic acid sequence includes both single stranded and double stranded nucleic acid. Thus, for example for DNA, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.

[0378] The term "coding region" refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.

[0379] The coding region can be derived from a non-mutated ("normal"), mutated or altered gene, or can even be derived from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

[0380] The term "expression product" means the polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).

[0381] The term "fragment," when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region, whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.

[0382] The term " DNA segment" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, by using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes.

[0383] A “vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as a transgene, may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic or interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide orpeptide suitable for eliciting an immune response in a mammal), and / or a selectable or detectable marker.

[0384] The term "primer" means a short nucleic acid sequence that can be paired with one strand of DNA and provides a free 3'-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.

[0385] The term "promoter" means a region of DNA involved in binding of RNA polymerase to initiate transcription.

[0386] A variety of methods have been developed to link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0387] Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc. New Haven, CN, USA.

[0388] A desirable method of modifying the DNA encoding the polypeptide of the description employs the polymerase chain reaction (PCR). This method may be used for introducing the DNA into a suitable vector, for example by engineering suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art. If viral vectors are used, pox- or adenovirus vectors are examples.

[0389] The nucleic acid (DNA, or in the case of retroviral vectors, RNA) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the description. Thus, the nucleic acid encoding the peptide or variant of the description may be used in accordance with known techniques, appropriately modified in view of the disclosure herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the description. Such techniques include those disclosed, for example, in US Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

[0390] The nucleic acid (DNA, or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the description may be joined to a wide variety ofother nucleic acid sequences for introduction into an appropriate host. The companion nucleic acid will depend upon the nature of the host, the manner of the introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired.

[0391] Generally, the nucleic acid is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the nucleic acid may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

[0392] Host cells that have been transformed by the recombinant nucleic acid of the description are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

[0393] Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus spec.), plant cells, animal cells and insect cells. For example, the system can be mammalian cells such as CHO cells available from the ATCC Cell Biology Collection.

[0394] A typical mammalian cell vector plasmid for constitutive expression comprises, as an example, the CMV or SV40 promoter with a suitable poly A tail and a resistance marker, such as neomycin. One example is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps). CMV promoter-based vectors (for example from Sigma- Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in variouscombinations of FLAG, 3xFLAG, c-myc or MAT. These fusion proteins allow for detection, purification and analysis of recombinant protein. Dual-tagged fusions provide flexibility in detection.

[0395] The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg / L in host cells. For less potent cell lines, protein levels are typically about 0.1 mg / L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive host cells. CMV vectors, for example, can contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the fl origin. Vectors containing the pre-pro-trypsin leader (PPT) sequence can direct the secretion of FLAG fusion proteins into the culture medium for purification using anti-flag antibodies, resins, and plates. Other vectors and expression systems are well known in the art for use with a variety of host cells.

[0396] In another embodiment two or more peptides or peptide variants of the description are encoded and thus expressed in a successive order (similar to "beads on a string" constructs). In doing so, the peptides or peptide variants may be linked or fused together by stretches of linker amino acids, such as for example LLLLLL, or may be linked without any additional peptide(s) between them. These constructs can also be used for cancer therapy and can induce immune responses both involving MHC I.

[0397] The present description also relates to a host cell transformed with a polynucleotide vector construct of the present description. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Eukaryotic host cells include yeast, insect and mammalian cells, such as vertebrate cells such as those from a mouse, rat, monkey or human cell lines. Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61 NS NIH Swiss mouse embryo cells NIH / 3T3 available from the ATCC as CRL 1658. Insect-cells include Sf9 cells which can be transfected with baculovirus expression vectors. An overviewregarding the choice of suitable host cells for expression can be found in, for example, the textbook of Paulina Balbes and Argelia Lorence " Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9, and other literature known to the person of skill.

[0398] Transformation of appropriate cell hosts with a DNA construct of the present description is accomplished by well-known methods that typically depend on the type of vector used.

[0399] Transformation methods for transforming prokaryotic host cells, yeast cells, vertebrate cells are well known in the art. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

[0400] Electroporation is also useful for transforming and / or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect-cells and vertebrate cells.

[0401] Successfully transformed cells, i.e., cells that contain a DNA construct of the present description, can be identified by well-known techniques such as PCR. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0402] It will be appreciated that certain host cells of the description are useful in the preparation of the peptides of the description, for example bacterial, yeast and insect cells. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells, such as dendritic cells, may usefully be used to express the peptides of the description such that they may be loaded into appropriate MHC molecules. Thus, the current description provides a host cell comprising a nucleic acid or an expression vector according to the description.

[0403] A further aspect of the description provides a method of producing a peptide or its variant, the method comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

[0404] In another embodiment the peptide(s), the nucleic acid or the expression vector of the description are used for treatment. For example, the peptide or its variant may be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. For example, methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. Methods of DNA injection include i.d., i.m., s.c., i.p. and i.v. Doses of e.g., between 50 pg and 1.5 mg, including 125 pg to 500 pg, of peptide or DNA may be given and will depend on the respective peptide or DNA. Said treatment further includeschemotherapy, immunotherapy, including checkpoint inhibitors, radiation, surgery or a combination thereof.

[0405] The polynucleotide used for vaccination may be substantially pure or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, mRNA, or a combination thereof. In some embodiments, the polynucleotide includes the nucleic acids encoding the ncHLAp. In some embodiments, the polynucleotide, in addition to the nucleic acids encoding the ncHLAp, includes additional nucleic acids from the nuORF form which the ncHALp is derived. Methods for designing and introducing such a nucleic acid are well known in the art. Suitable vectors and delivery systems include viral DNA and / or RNA, such as systems based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus or hybrids containing elements of more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the art of DNA delivery. Physical delivery, such as via a "gene-gun" may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein.

[0406] Also provided herein are anticancer vaccines or other immunogenic compositions, which can comprise a nucleic acid, DNA or RNA coding for a CR ncHLAp peptide. Embodiments provide for one or nucleic acids encoding one or more peptides of SEQ ID NO: 1 to SEQ ID NO: 517 to be included in an anticancer vaccine. In some embodiments, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, or 517 different nucleic acids are pooled and / or coded for in the vaccine composition, such as 1-100, including, for example, 10-15, 10-20, 15-30, 20-40 different nucleic acids. In some embodiments, the nucleic acid includes, in addition to the nucleotides encoding the ncHLAp, includes additional nucleotides from the nuORF form which the ncHALp is derived. The anticancer vaccines can further include a carrier and / or an adjuvant.

[0407] Vaccines or immune modulators that are or include nucleic acids can be administered by any method suitable for administration of nucleic acid agents, such as a DNA or RNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U. S. Patent No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U. S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as described in, interalia., Hamajima et al, Clin. Immunol. Immunopathol, 88(2), 205-10 (1998). Liposomes (e.g., as described in U. S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U. S. Patent No.

[0408] 6,471,996).

[0409] In one embodiment, composition and vaccines as described herein can be prepared with carriers that will protect against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Patent No. 4,522,811. Such compositions and vaccines can be included in a container, pack, or dispenser together with instructions for administration.

[0410] Further provided herein are novel T cell receptors and functional fragments thereof specific for an isolated non-canonical HLA-I bound peptide (ncHLAp) selected from SEQ ID NOS: 1-517. T-cell receptors include a heterodimer of T-cell receptor α (TCRα) and β (TCRβ) polypeptide chains which are linked by a disulfide bond. Both TCRα and TCRβ chains include N-terminal variable (V) regions, constant (C) regions, and a hinge region that forms the interchain disulfide bond via a cysteine residue followed by a transmembrane region, and a short cytoplasmic tail. The V and C regions represent extracellular domains, with the V region binding to the peptide / MHC complex. The variable (V) regions in both TCRα and TCRβ chains each include three hypervariable or complementarity-determining regions (CDRs), which are referred to herein as TCRα CDR1, TCRα CDR2, TCRα CDR3, TCRβ CDR1, TCRβ CDR2, and TCRβ CDR3.

[0411] The CDRs are interspersed with regions that are more conserved, referred to as framework regions (FRs). In TCRα and TCRβ chains, the variable region includes three CDRs and three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). Accordingly, the FRs are those variable domain residues other than the CDR residues. The terms FRla, FR2a, FR3a, and FR4arefer to the framework regions of the TCRα chain variable region, and the terms FR1β, FR2β, FR3β, and FR4β refer to the framework regions of the TCRβ chain variable region.

[0412] CDR3 (i.e., TCRα CDR3 and TCRβ CDR3) is the main CDR responsible for recognizing processed antigen peptide in the HLA / antigen peptide complex presented by an antigen presenting cell (APC). Amino acids of the CDR3 loops are in direct contact with the antigenic epitopes on the peptide-major histocompatibility complex. The CDR3 loops the have the greatest amount of variability in the TCR providing a large diversity of sequences allowing recognition of a wide array of antigenic peptides. While not believed to be important as the CDR3, TCRα CDR1 has also been shown to interact with the N-terminal part of the antigenic peptide, and TCRβ CDR1 interacts with the C-terminal part of the peptide. CDR2 is implicated in MHC recognition.

[0413] TCR αβ heterodimers are responsible for antigen recognition by most T cells and share high structural similarity to the Fab fragment of an immunoglobulin molecule. A small fraction of T cells in the body have an alternative but structurally similar TCR, made of TCR chains referred to as designated TCR γ and TCR δ. The membrane-bound immunoglobulin that functions as the B cell receptor has two binding sites and can be secreted as pentameric IgM as the initial antibody released by the B cells, whereas the TCRs have only one antigen-binding site are not secreted.

[0414] Examples 1 and 2 characterize seventeen T cell receptors specific for various ncHLAp. The amino acid sequences of the variable region of both the TCRα and TCRβ chains of these seventeen TCRs are shown in Table 4. In Table 4 the amino acid sequences of the complementarity-determining regions TCRα CDR1, TCRα CDR2, TCRα CDR3, TCRβ CDR1, TCRβ CDR2, and TCRβ CDR3 for these seventeen T cell receptors are identified with bold text and underlining.Table 4: ncHLAp-Reactive TCR Clonotypes

[0415] CDR1 CDR2 CDR3 Antigen Antigen TCR SEQ SEQ SEQ ID SEQ ID Antigen Number SEQ ID Specificity

[0416] Full AA sequence ID NO NO ID NO NO (Peptide) NO Code {)VKVT,OSSRYl. VKRTGEKVFLFCVQ[)MOHENMFWYR<)DPG

[0417] LGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTN KLFLWPY QTSMYLCASSLPGSNOPOHFGDGTRLSIL 518 552 575 600 KV 9 NU11 TCR001

[0418] KOEVTOIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKG LTSLLLIQSSQREQTSGRLNASLDKSSGRSTLY1AASOPGDSAT KLFLWPY YLCAVLGDSNYOLIWGAGTKLI1KP 519 553 576 601 KV 9 Nil 11 ETGVTOTPRI-ILYTvIGMTNKKSLKCEQHLGHNAMYWYKOSAK

[0419] KPLELMFWSLEERVENNSVPSRFSPECPNSSHLFLI-ILHTLOPE RGNLPLP DSALYLCASSPSGGGYNEOFFGPGTRLTVL 520 554 577 602 LK 239 NU49 TCR002

[0420] AOKVTOAOTEISVVEKEDVTLDCVYETRDTTYYLFWYKOPPS GELVFLIRRNSFDEQNEISGRYSWNFOKSTSSFNFTITASOVVD RGNLPLP SAVYFCALSEAOTSGSRLTFGEGTQLTVNP 521 555 578 603 LK 239 NU49 GAWSOHPSWVICKSGTSVKIECRST-DFOATTMFWYROFPKO

[0421] SLMLMATSNEGSKATYEOGVEKDKFLINHASLTLSTLTVTSAH LLWRISSS PEDSSFYICSVGVDEQYFGPGTRLTVT 522 556 579 604 V 84 NU42 TCR003

[0422] RKEVEQDPGPFNVPEGATVAFNCTYSNSASOSFFWYRODCRK EPKLLMSVYSSGNEDGRFTAQLNRASOYISLLIRDSKLSDSAIT LLWRISSS LCVVGDYKLSFGAGI'TVTVRA 523 557 580 605 V 84 NL42 GAGVSOSPRYKVAKRGODVALRCDPISGHVSLFWWQQALGO GPEFl..rtTONEAOLDKSGLPSDRFFAERPEGSVSTLKIORTOQE LLWRISSS DSAVYLCASSPEAGGEKLFFGSGTQLSVL 524 572 581 606 V 84 NU42 TCR004

[0423] GES VGLHLPTLS VQEGDNSIINCAY SNSASDYF1 W YKQESGKG

[0424] POFIIDIRSNMDKROGORVrVLLNKT'VKHLSLOIAAIOPGDSA LLWRISSS VYFCAETPPSGNTPLVFGKGTRLSVIA 525 558 582 607 V 84 NU42 AAGVIOSPRHLIKEKRFTATLKCYTIPRHDTVYWYOOGPGODP OFLISFYEKMOSDKGSIPDRFSAOQFSDYHSELNMSSLELGDS ALFSKLA ALYFCASSLSGSDOPOHFGDGTRLSIL 526 559 583 608 SA 93 NU57 TCR005

[0425] ELKVEONPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGKS LESLFVLLSNGAVKOEGRLMASLDTKARLSTLH1TAAVHDLSA ALFSKLA TYFCAVDPISGGYNKLIFGAGTRLAVHP 527 560 584 609 SA 93 NIJ57 EAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOOILGQGP TCR006 KLLIOFONNGWDDSOLPKDRFSAERLKGVDSTLKIQPAKLED GAYFFRH

[0426]

[0427] SAVYLCASSLSGEQFFGPGTRLTV’L 528 561 585 610 LK 509 NIJ46AQSWQLGSHVSVSEGALYYJ. RCNYSSSVPPYI. EW’YVQYPNQ

[0428] GLQLLLKYTTGATLVKGINGFEAEFKKSETSFHI.. TKPSAHMSD GAYFFRH AAEYFCAVSEDDYKLSFGAGTTVTVRA 529 562 586 611 LK 509 NU46 DSGVrOTPKHLITATGORVTLRCSPRSGDLSVYWYOOSLDQG

[0429] LOFLIQYYNGEERAKGN1LERFSAOQFPDLHSELNLSSLELGDS LLWRISSS ALYFCASSADSNEOYFGPGTRLTVT 530 563 587 612 84 NL42 TCR007 V RKEVEQDPGPFNVPEGATVAFNCTYSNSASOSFFWY'RODCRK

[0430] EPKLLMSVYSSGNEDGRFTAQLNRASOYISLLJRDSKLSDSATY LLWRISSS LCVVDSSYKLIFGSGTRLLVRP 531 557 580 613 V 84 NU42 EAOVTONPRYLITVTGKKLTVrCSQNMNHEYMSWYRODPGL

[0431] GLROIYYSWVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPN GAYFFRH QTSLYFCASSSLWGNOPOHFGDGTRLS1L 532 564 588 614 LK 509 NU46 TCR008

[0432] AOTVTOSOPEMSVOEAETVTL / R^TYDTSF. SDY^'L. FXVYKOPPS

[0433] ROM1LVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLG GAYFFRH

[0434] D AAM YFCAYAN SGG SN YKLTFGKGTLLTVNP 533 565 589 615 LK 509 NU46 DSGVTOTPKI-ILITATGORYTLRCSPRSGDLSVYWYOQSLDOG

[0435] LOFLIOYYNGEERAKGNILERFSAOQFPDLI-ISELNLSSLELGDS RGNLPLP ALYFCASSPGLVGEQYFGPGTRLTVT 534 563 587 616 LK 239 NU49 TCR009

[0436] AOKVTOAOTEISVV’EKEDVTLDCVYETRDTTYYLFWYKQPPS

[0437] GELVFLIRRNSFDEQNEISGRYSWNFOKSTSSFNFTITASOVVD RGNLPLP SAVYFCALSEAITGNQFYFGTGTSLTVIP 535 555 578 617 LK 239 NU49 DAGVTOSPTHLIKTRGOOVTLRCSPKSGHDTVSWYOQALGO GPQFIFOYYEEEERORGNFPDRFSGHQFPNYSSELNVN / XLLLG ILQDRLW DSALYLCASSOGALDGYTFGSGTRLTVV 536 566 590 618 KL 449 NU47 TCR010

[0438] ILNV'EOSPOSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKS

[0439] PEALFVMTLNGBEKKKGRISATLNTKEGYSYLYIKGSOPEDSA ILQDRLW TYLCASSNQAGTAL1FGKGTTLSVSS 537 567 591 619 KL 449 NU47 GAVVSOHPSWVKYSGTSVKIECRSLDFQATTMFWYROFPKO SLMLMATSNEGSKATYEOGVEKDKFLINWvSLTYSTLTVTSAH KLFLWPY^ PEDSSFYTCSASSYEOYFGPGTRLTVT 538 556 579 620 KV 9 TCRO11 NUM DOOVKONSPSLSVQEGRISILNCDYTWSMFDYFLWYKKYPAE GPTFLISISSIKDKNEDGRF'rVFLNKSAKHLSLHIVPSQPGDSAV KLFLWPY YFCAASAGTYKYIFGTGTRLKVLA 539 568 592 621 KV 9 NL11 SOTIHOWPATTARJPVGSPLSLFGYWGTSNPNLYWYROAAGR GLOLLFYSVGIGdlSSEVPONLSASRPODROFILSSKKLLLSDS KLFLWPY

[0440] 540 569 593 622 9 TCR012 GFYLCAWSVTGDYGYTFGSGTRLTW KV NUU AQTVTOSOPEMSVOEAETVTLSCTYDTSESDYYLFWYKQPPS RQMlLVIRQEAYKQQNArENRFSVNFOKAAKSFSLKISDSQLG KLFLWPY

[0441]

[0442] DAAMYFCVENSGYALNFGKGTSLLVTP 541 565 589 623 KV 9 NU11NAGVTOTPKFRVLKTGOSMTLLCAODMNHEYMYWYRODPG

[0443] MGLRLIHYSVGEGTTAKGEATDGYNVSRLKKONFLLGLESAA HIFHI. SL

[0444] 542 564 594 624 202 NU10 TCR013 PSQTSVYFCASTVGAGGFDEOFFGPGTRLTXT ARV

[0445] AQK1TQTQPGMF VQEKEAVTLDCT YDTSDPSYGLFWYKQPS S

[0446] GEMIFLIYOGSYDOONArEGRYSLNFOKARKSANLVISASOLG H1FHLSL DSAMYFCAftlRGVNYGONFVTGPGTRLSVLP 543 570 595 625 ARV 202 NG 10 EAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOQILGOGP KLLIOFQNNGWDDSOLPKDRFSAERLKGVDSTI.. KIQPAKi> ED LLWR1SSS SAVYLCASSPVAGIAPASTOTOYFGPGTRLTVL 544 561 585 626 84 TCR014 V NU42

[0447] OQPVOSPOAVILREGEDAVINCSSSKALYSVHWYROKHGEAP VFLMILLKGGEOKGHEKISASFNEKKQOSSLYLTASOLSYSGT LLWR1SSS YFCGIYTRMDSNYOLIWGAGTKLIIKP 545 571 596 627 V 84 NU42 GAGVSOSPRYKVTKRGODVALRCDPISGHVSLYWYROALGQ GPEFLTYFNYEAOODKSGLPNDRFSAERPEGSISTLTIQRTEQR GAYFFRH

[0448] 546 572 597 628 LK 509 TCR015 DSAMYRCASSFGPGTYEOYFGPGTRLTVT NU46

[0449] AORVTOPEKLLSXTKGAPVELKCNYSYSGSPELFWYVOYSRO RLQLLLRHISRESIKGFTADLNTCGETSFHLKKPFAQEEDSAMY GAYFFRH YCALSGLTOGGSEKLWGKGTKLTVNP 547 573 598 629 LK 509 NU46 EAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOQILGOGP KLLIQFONNGWDDSQLPKDRFSAERLKGVDSTLKIQPAKLED GAYFFRH SAVYLCASSLSGEOFFGPGTRLTVL 528 561 585 610 LK 509 NIJ46 TCR016

[0450] AQSVTOLGSHVSVSEGALY^LRCNYSSSWPYLFWYA / QYPNO

[0451] GLQLLLKYTTGATLVKGINGFEAEFKKSETSFHLTKPSAHMSD GAYFFRH AAEYFCAVSEDDYNLSFGAGTTVTVRA 549 562 586 630 LK 509 NU46 KAGVTOTPRYLIKTRGOQVTLSCSPISGHRSVSWYOQTPGOGL QFLFEYFSETQRNKGNFPGRFSGROFSNSRSEMNVSTLELGDS RGNLPLP TCR017 ALYLCASSLWQGNEOYFGPGTRLTVT 550 574 599 631 LK 239 NU49

[0452] ILNVEOSPOSLHVOEGDSTNFTCSFPSSNFYALHWYRWETAKS PEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSOPEDSA RGNLPLP

[0453]

[0454] TYLCACYNYGONFVFGPGTRLSVLP 551 567 591 632 LK 239 NU49The present description provides a T cell receptor (TCR) or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:552, a TCRα CDR2 of SEQ ID NO:575, a TCRα CDR3 of SEQ ID NO:600, a TCRβ CDR1 of SEQ ID NO:553, a TCRβ CDR2 of SEQ ID NO:576, and / or a TCRβ CDR3 of SEQ ID NO:601. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0455] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:554, a TCRa CDR2 of SEQ ID NO:577, a TCRa CDR3 of SEQ ID NO:602, a TCRβ CDR1 of SEQ ID NO:555, a TCRβ CDR2 of SEQ ID NO:578, and / or a TCRβ CDR3 of SEQ ID NO:603. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0456] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:556, a TCRα CDR2 of SEQ ID NO:579, a TCRα CDR3 of SEQ ID NO:604, a TCRβ CDR1 of SEQ ID NO:557, a TCRβ CDR2 of SEQ ID NO:580, and / or a TCRβ CDR3 of SEQ ID NO:605. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0457] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:572, a TCRα CDR2 of SEQ ID NO:581, a TCRα CDR3 of SEQ ID NO:606, a TCRβ CDR1 of SEQ ID NO:558, a TCRβ CDR2 of SEQ ID NO:582, and / or a TCRβ CDR3 of SEQ ID NO:607. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0458] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:559, a TCRα CDR2 of SEQ ID NO:583, a TCRα CDR3 of SEQ ID NO:608, a TCRβ CDR1 of SEQ ID NO:560, a TCRβ CDR2 of SEQ ID NO:584, and / or a TCRβ CDR3 of SEQ ID NO:609. Such a TCR may be specific for the ncHLAp SEQ ID NO:93.

[0459] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:561, a TCRα CDR2 of SEQ ID NO:585, a TCRα CDR3 of SEQ ID NO:610, a TCRβ CDR1 of SEQ ID NO:562, a TCRβ CDR2 of SEQ ID NO:586, and / or a TCRβ CDR3 of SEQ ID NO:611. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0460] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:563, a TCRα CDR2 of SEQ ID NO:587, a TCRα CDR3 of SEQ ID NO:612, a TCRβ CDR1 of SEQ ID NO:557, a TCRβ CDR2 of SEQ ID NO:580, and / or a TCRβ CDR3 of SEQ ID NO:613. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0461] The present description provides a TCR or functional fragment thereof having a TCRα CDR1 of SEQ ID NO:564, a TCRα CDR2 of SEQ ID NO:588, a TCRα CDR3 of SEQ IDNO:614, a TCRp CDR1 of SEQ ID NO:565, a TCRp CDR2 of SEQ ID NO:589, and / or a TCRp CDR3 of SEQ ID NO:615. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0462] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:563, a TCRa CDR2 of SEQ ID NO:587, a TCRa CDR3 of SEQ ID NO:616, a TCRp CDR1 of SEQ ID NO:555, a TCRp CDR2 of SEQ ID NO:578, and / or a TCRp CDR3 of SEQ ID NO:617. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0463] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO: 566, a TCRa CDR2 of SEQ ID NO: 590, a TCRa CDR3 of SEQ ID NO:618, a TCRβ CDR1 of SEQ ID NO:567, a TCRβ CDR2 of SEQ ID NO:591, and / or a TCRβ CDR3 of SEQ ID NO:619. Such a TCR may be specific for the ncHLAp SEQ ID NO:449.

[0464] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO: 556, a TCRa CDR2 of SEQ ID NO: 579, a TCRa CDR3 of SEQ ID NO:620, a TCRp CDR1 of SEQ ID NO:568, a TCRp CDR2 of SEQ ID NO:592, and / or a TCRp CDR3 of SEQ ID NO:621. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0465] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO: 569, a TCRa CDR2 of SEQ ID NO: 593, a TCRa CDR3 of SEQ ID NO:622, a TCRβ CDR1 of SEQ ID NO:565, a TCRβ CDR2 of SEQ ID NO:589, and / or a TCRβ CDR3 of SEQ ID NO:623. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0466] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:564, a TCRa CDR2 of SEQ ID NO:594, a TCRa CDR3 of SEQ ID NO:624, a TCRp CDR1 of SEQ ID NO:570, a TCRp CDR2 of SEQ ID NO:595, and / or a TCRp CDR3 of SEQ ID NO:625. Such a TCR may be specific for the ncHLAp SEQ ID NO:202.

[0467] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:561, a TCRa CDR2 of SEQ ID NO:585, a TCRa CDR3 of SEQ ID NO: 626, a TCRβ CDR1 of SEQ ID NO: 571, a TCRβ CDR2 of SEQ ID NO: 596, and / or a TCRβ CDR3 of SEQ ID NO:627. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0468] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:572, a TCRa CDR2 of SEQ ID NO:597, a TCRa CDR3 of SEQ ID NO:628, a TCRp CDR1 of SEQ ID NO:573, a TCRp CDR2 of SEQ ID NO:598, and / or a TCRp CDR3 of SEQ ID NO:629. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO:561, a TCRa CDR2 of SEQ ID NO:585, a TCRa CDR3 of SEQ ID NO:610, a TCRβ CDR1 of SEQ ID NO:562, a TCRβ CDR2 of SEQ ID NO:586, and / or a TCRβ CDR3 of SEQ ID NO:630. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0469] The present description provides a TCR or functional fragment thereof having a TCRa CDR1 of SEQ ID NO: 574, a TCRa CDR2 of SEQ ID NO: 599, a TCRa CDR3 of SEQ ID NO: 631, a TCRp CDR1 of SEQ ID NO: 567, a TCRp CDR2 of SEQ ID NO: 591, and / or a TCRp CDR3 of SEQ ID NO:632. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0470] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:518 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:519. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:518 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:519. In some aspects, the TCR has an a variable region with SEQ ID NO:518 and / or a β variable region with SEQ ID NO:519. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0471] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:520 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:521. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:520 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:521. In some aspects, the TCR has an a variable region with SEQ ID NO:520 and / or a β variable region with SEQ ID NO:521. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0472] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:522 and / or a β variableregion with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:523. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:522 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:523. In some aspects, the TCR has an a variable region with SEQ ID NO:522 and / or a variable region with SEQ ID NO:523. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0473] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:524 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:525. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:524 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:525. In some aspects, the TCR has an a variable region with SEQ ID NO:524 and / or a β variable region with SEQ ID NO:525. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0474] The present description provides a T cell receptor or functional fragment thereof having an α variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:526 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:527. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:526 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:527. In some aspects, the TCR has an a variable region with SEQ ID NO:526 and / or a β variable region with SEQ ID NO:527. Such a TCR may be specific for the ncHLAp SEQ ID NO:93.

[0475] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:528 and / or a P variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 99% sequence identity to SEQ ID NO:529. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:528 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:529. In some aspects, the TCR has an a variable region with SEQ ID NO:528 and / or a β variable region with SEQ ID NO:529. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0476] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:530 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:531. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:530 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:531. In some aspects, the TCR has an a variable region with SEQ ID NO:530 and / or a β variable region with SEQ ID NO:531. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0477] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:532 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:533. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:532 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:533. In some aspects, the TCR has an a variable region with SEQ ID NO:532 and / or a β variable region with SEQ ID NO:533. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0478] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:534 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:535. In some aspects, the TCR has ana variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:534 and / or a P variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:535. In some aspects, the TCR has an a variable region with SEQ ID NO:534 and / or a β variable region with SEQ ID NO:535. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0479] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:536 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:537. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:536 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:537. In some aspects, the TCR has an a variable region with SEQ ID NO:536 and / or a β variable region with SEQ ID NO:537. Such a TCR may be specific for the ncHLAp SEQ ID NO:449.

[0480] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:538 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:539. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:538 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:539. In some aspects, the TCR has an a variable region with SEQ ID NO:538 and / or a β variable region with SEQ ID NO:539. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0481] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:540 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:541. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ IDNO:540 and / or a P variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:541. In some aspects, the TCR has an a variable region with SEQ ID NO:540 and / or a β variable region with SEQ ID NO:541. Such a TCR may be specific for the ncHLAp SEQ ID NO:9.

[0482] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:542 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:543. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:542 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:543. In some aspects, the TCR has an a variable region with SEQ ID NO:542 and / or a β variable region with SEQ ID NO:543. Such a TCR may be specific for the ncHLAp SEQ ID NO:202.

[0483] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:544 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:545. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:544 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:545. In some aspects, the TCR has an a variable region with SEQ ID NO:544 and / or a β variable region with SEQ ID NO:545. Such a TCR may be specific for the ncHLAp SEQ ID NO:84.

[0484] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:546 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:547. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:546 and / or a P variable region with one, two, three, four, or more amino acid differencesfrom SEQ ID NO:547. In some aspects, the TCR has an a variable region with SEQ ID NO:546 and / or a β variable region with SEQ ID NO:547. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0485] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:528 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:549. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:528 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:549. In some aspects, the TCR has an a variable region with SEQ ID NO:528 and / or a β variable region with SEQ ID NO:549. Such a TCR may be specific for the ncHLAp SEQ ID NO:509.

[0486] The present description provides a T cell receptor or functional fragment thereof having an a variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:550 and / or a β variable region with at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:551. In some aspects, the TCR has an a variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:550 and / or a β variable region with one, two, three, four, or more amino acid differences from SEQ ID NO:551. In some aspects, the TCR has an a variable region with SEQ ID NO:550 and / or a β variable region with SEQ ID NO:551. Such a TCR may be specific for the ncHLAp SEQ ID NO:239.

[0487] The term “percent identity” in relation to a polypeptide or polynucleotide sequence refers to the percentage of amino acid or nucleotides that are the same when two sequences of the same molecular type (polypeptide or polynucleotide) are aligned. The higher the percentage identity, the more similar the two sequences are. Methods of aligning polypeptide or polynucleotide sequences for comparison are well known in the art, and various programs and alignment algorithms are described in, for example, Smith and Waterman, Adv Appl Math. 2:482, 1981; Needleman and Wunsch, J Mol Biol 48:443, 1970; Pearson and Lipman, Proc Natl Acad Sci USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS5:151, 1989; Corpet et al., NAR 16:10881, 1988; Pearson and Lipman, Proc Natl Acad Sci USA 85:2444, 1988; and Altschul et al., Nature Genet 6:119, 1994. These references present a detailed consideration of sequence alignment methods and homology calculations. Comparisons can be made using common platforms, such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) available from the National Center for Biotechnology Information (NCBI, Bethesda, MD), and sequence analysis programs blastp, blastn, can blastx can be used for sequence analysis.

[0488] T cell receptors or functional fragments thereof include soluble T cell receptors (sTCR) recognizing a specific peptide-MHC complex. Such soluble T cell receptors can be generated from specific T-cell clones, and their affinity can be increased by mutagenesis targeting the complementarity-determining regions. sTCR are described in more detail in, for example, Robinson et al., FEBS J. 2021; 288(21):6159-6173, and Dolgin, Nature Biotechnology. 2022; 40:441-444.

[0489] T cell receptors or functional fragments thereof include single-chain T-cell receptors. A single-chain T-cell receptor (scTCR) is a modified, soluble protein combining the variable (Va and VP) domains of a natural T-cell receptor (TCR) into one polypeptide, often linked by a flexible peptide, creating a stable, antigen-binding unit and may be used in therapies like CAR-T cells for cancer. scTCRs are described in more detail in, for example, Novotny et al., PNAS. 1991, 88(19):8646-8650; Oh etal., Scientific Reports. 2019; 9:17291; Stone etal., Cancer Immunol Immunother. 2014; 63(11): 1163-1176; and Zou et al., Journal of Medicinal Chemistry, 67(9): 7635 -7646

[0490] For the purpose of T-cell receptor selection, phage display can be used (US 2010 / 0113300, (Liddy et al., Nat Med. 2012; 18(6):980-7). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use as drug, alpha and beta chain can be linked e.g., by non-native disulfide bonds, other covalent bonds (single-chain T-cell receptor), or by dimerization domains (Boulter et al., Protein Eng. 2003; 16(9):707-l 1; Card et al., Cancer Immunol Immunother. 2004; 53(4):345-57; Willcox et al., Protein Sci. 1999; 8(11):2418-23). The T-cell receptor can be linked to toxins, drugs, cytokines (see, for example, US 2013 / 0115191), and domains recruiting effector cells such as an anti-CD3 domain, etc., in order to execute particular functions on target cells. In another aspect, it is expressed in T-cells used for adoptivetransfer. See, for example, WO 2004 / 033685 Al, WO 2004 / 074322A1, and WO 2013 / 057586A1, the contents of which are incorporated by reference in their entirety.

[0491] AT cell receptor or functional fragment thereof of the present description includes bispecific T cell engagers (BiTEs). BiTEs are a class of immunotherapeutic molecules for the treatment of cancer. BiTEs enhance the patient’s immune response to tumors by retargeting T cells to tumor cells. BiTEs are constructed of sTCR connected to a second T cell-specific binding molecule. The connection may be via a flexible linker. The T cell-specific binding molecule binds to a T cell while the sTCR binds to a tumor-associated antigen (e.g., CR ncHLAp), allowing the BiTE to physically link a T cell to a tumor cell, ultimately stimulating T cell activation, tumor killing and cytokine production (Huehls et al., Immunol Cell Biol. 2014;

[0492] 93(3):290-296). In some aspects, the T cell-specific binding molecule targets CD3, such as for example, an anti-CD3 antibody or scFv thereof. One example is tebentafusp, a T-cell receptor-bispecific molecule that targets glycoprotein 100 and CD3 (Hassel et al., N Engl J Med. 2023; 389:2256-2266). With January 2022, the US FDA approved tebentafusp for HLA-A*02:01-positive adult patients with unresectable or metastatic uveal melanoma.

[0493] The present description provides nucleic acids encoding a T cell receptor or functional fragment thereof as described herein. A nucleic acid encoding a T cell receptor or functional fragment thereof can be included in an expression vector for expression in engineered cells. Expressions vectors are as described above. Briefly, an expression vector can include a selectable marker gene or a reporter gene to select cells expressing the polypeptides of the disclosure. An expression vector can include appropriate regulatory sequences that allow expression in the engineered cell. The expression vector can include promoters, initiation sequences, and transcription and translation tenninators for regulation of the expression of the nucleic acid sequences. The present description provides host cells expressing a nucleic acid or expression vector as described herein. Again, such host cells are described in more detail above.

[0494] The present description also provides engineered T cells engineered to express a T cell receptor or functional fragment thereof as described herein. Such engineered T cells may be transformed to express a nucleic acid or expression vector encoding a T cell receptor or functional fragment thereof as described herein. The nucleic acids encoding a T cell receptor or functional fragment thereof, expression vector, host cell, and engineered T cells as described here can be provided in a therapeutic composition to a subject for the treatment of a cancer.

[0495] Adoptive cellular therapy (ACT) includes the transfer of cells into a patient. There are three forms of ACT that are used in cancer therapy: tumor infiltrating lymphocytes (TILs), Tcells that are engineered to express anti-tumor T cell receptors (TCRs) and T cells with chimeric antigen receptors (CARs).

[0496] The present description provides TCR-engineered T cells recognizing a CR ncHLAp of any one of peptides SEQ ID NOS 1-517. A " TCR-engineered T cell" (TCR-T) refers to a T cell that has been genetically modified to express a specific T cell receptor (TCR), allowing it to recognize and target a particular antigen, typically used in cancer immunotherapy to specifically attack tumor cells by recognizing unique peptides presented on their surface through the body's natural MHC complex; essentially, it's a patient's own T cell that has been engineered to better target cancer cells.

[0497] T cells recognize MHC -presented antigens through their T cell receptor (TCR). TCRs recognize enzymatically cleaved peptides that are presented at the cell surface by MHC molecules (pMHC). In humans, antigen-presenting MHC alleles are broadly classified as HLA class I (A, B, or C) or HLA class II (DR, DP, or DQ). The coreceptors CD8 and CD4 enhance TCR antigen sensitivity through interaction with MHC class I or II molecules, respectively. TCR binding to cognate pMHC results in T cell activation and initiation of effector functions including proliferation, cytokine secretion, and cytolysis via secretion of perforin and granzyme.

[0498] In TCR T therapy, T cells are edited to express TCR α and β chains as described herein that confer a desired specificity, such as specificity for the peptides of any one of SEQ ID NO:1 to SEQ ID NO:517.

[0499] To develop engineered TCR-T, the TCR on a TCR-T cell is specifically designed to bind to a known tumor-associated antigen, such as the CR ncHLAp described herein, allowing it to selectively target cancer cells. The process of generating engineered TCR-T is known in the art. Generally, T cells are collected from a patient’s blood, the cells are genetically engineered to express a specific TCR gene using viral transduction or CRISPR-Cas gene editing or other methods, wherein the TCR directed against a tumor antigen, such as the CR ncHLAp disclosed herein, the engineered T cells are then expanded in culture and then infused back into the patient for treatment. For example, peripheral blood mononuclear cells (PBMCs) from the patient can be collected and the T cell population can be isolated. Then, tumor-infiltrating lymphocytes (TILs) can be isolated from a patient's tumor and screened for T cells with reactivity against the chosen antigen (CR ncHLAp), healthy donor T cells can be stimulated with peptides derived from the tumor antigen (CR ncHLAp) to identify antigen-specific TCRs and scRNAseq can beused to analyze gene expression patterns of T cells exposed to tumor antigens (CR ncHLAp) to identify TCR sequences with high specificity. Once a TCR identified, TCR alpha and beta chain genes can be cloned. Viral vectors (like lentivirus or retrovirus) can carry the cloned TCR genes to introduce them into the patient's T cells or CRISPR, such as CRISPR-Cas9, technology can be used to precisely replace the endogenous TCR with the desired tumor-specific TCR. The engineered T cells can then be expanded in ex vivo for infusion into the patient. Alternatively, a patient’s T cells can be engineered directly in the body via delivery of genetic cargo.

[0500] The present description provides TCR-mimetic antibodies specifically recognizing a CR ncHLAp peptide of SEQ ID NOS:1 to 517 presented on a major histocompatibility complex (MHC). A " TCR-mimetic antibody" targeting an antigen for CAR-T therapy is a type of antibody designed to mimic the function of a T cell receptor (TCR), allowing it to specifically recognize a peptide antigen presented on a tumor cell's Major Histocompatibility Complex (MHC) molecule, enabling CAR-T cells to target intracellular tumor antigens that wouldn't normally be accessible to standard CARs; this provides for treating solid tumors by targeting unique cancer-associated peptides presented on MHC molecules.

[0501] Unlike traditional CARs that target cell surface proteins, TCR-mimetic antibodies bind to the complex formed between a tumor-specific peptide and an MHC molecule, allowing CAR-T cells to recognize intracellular tumor antigens. To make TCR-mimetic antibodies targeting an antigen for CAR-T therapy, phage display technology can be used to screen an antibody library against a complex of the desired tumor-associated peptide antigen, including a CR ncHLAp peptide of SEQ ID NOS: 1 to 517, presented on a major histocompatibility complex (MHC) molecule (HLA-I), thereby generating antibodies that specifically recognize the peptide-MHC complex, mimicking the natural recognition pattern of a T cell receptor (TCR); this antibody can then be engineered into a CAR construct (fuse the selected TCR-mimetic antibody's variable regions (scFv) to a transmembrane domain and intracellular signaling domain to create a chimeric antigen receptor) for CAR-T cell development. Patient derived T cells and be transduced with the engineered CAR construct using viral vectors (e.g., lentivirus, retrovirus). In some embodiments, the cellular immunotherapy comprises chimeric antigen receptor (CAR)-T cells (CAR-T cells) derived from the human’s T-cells and modified to bind one or more CR nHLAp antigens on a surface of cancer cells in the human.The peptides of the present description can be used to generate and develop specific antibodies against MHC / peptide complexes. These can be used for therapy, targeting toxins or radioactive substances to the diseased tissue. Another use of these antibodies can be targeting radionuclides to the diseased tissue for imaging purposes such as PET. This use can help to detect small metastases or to determine the size and precise localization of diseased tissues.

[0502] Therefore, it is a further aspect of the description to provide a method for producing a recombinant antibody specifically binding to a human major histocompatibility complex (MHC) class I being complexed with a HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility complex (MHC) class I with a soluble form of a MHC class I molecule being complexed with said HLA restricted antigen; isolating mRNA molecules from antibody producing cells of said non-human mammal; producing a phage display library displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, said at least one phage displaying said antibody specifically binding to said human major histocompatibility complex (MHC) class I being complexed with said HLA-restricted antigen.

[0503] It is a further aspect of the description to provide an antibody that specifically binds to a human major histocompatibility complex (MHC) class I being complexed with a HLA-restricted antigen, wherein the antibody is a polyclonal antibody, monoclonal antibody, bi-specific antibody and / or a chimeric antibody.

[0504] Respective methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other tools for the production of these antibodies are disclosed for example, in WO 03 / 068201, WO 2004 / 084798, WO 01 / 72768, WO 03 / 070752, which for the purposes of the present description are incorporated by reference in their entireties.

[0505] The term “antibody” or “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact or “full” immunoglobulin molecules, also included in the term “antibodies” are fragments (e.g., CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties (e.g., specific binding of a cancer marker (poly)peptide or delivery of a toxin to a cancer cell expressing a cancer marker gene).The antibodies of the description may also be generated using well known methods. The skilled artisan will understand that either full length marker polypeptides or fragments thereof may be used to generate the antibodies of the description. A polypeptide to be used for generating an antibody of the description may be partially or fully purified from a natural source or may be produced using recombinant DNA techniques.

[0506] EXAMPLES

[0507] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0508] Example 1

[0509] Pancreatic cancer-restricted cryptic antigens are targets for T cell recognition

[0510] Cancer-restricted cryptic antigens harbor robust immunogenicity

[0511] Cytotoxic T lymphocytes (CTLs) continuously surveil the peptidomes displayed on major histocompatibility complex class 1 (MHC-1; HLA-1 in humans) for “non-self ’ HLA-1 bound peptides (HLAp). CTL recognition of mutated or otherwise foreign HLAp is thought to underlie the success of many cancer immunotherapies (Leko and Rosenberg, Cancer Cell 38, 454-472 (2020)). While neoantigen-directed therapies have primarily focused on tumor-specific somatic mutations, emerging evidence suggests that cancer cells can translate regions of the genome outside of annotated open reading frames (ORFs), leading to expression and HLA-I presentation of cryptic peptides derived from genomic elements like 5’ and 3’ untranslated regions (UTRs), long noncoding RNAs (IncRNAs), and alternative reading frames (intORFs) of protein-coding genes (Chong et al., Nat Commun 11, 1293 (2020); Laumont et al., Nat Commun 7, 10238 (2016); Laumont et al, Sci Transl Med 10, eaau5516 (2018); Lozano-Rabella et al., Clinical Cancer Research 29, 2250-2265 (2023); and Ouspenskaia et al., Nat Biotechnol 40, 209-217 (2022)). These non-canonical HLA-I bound peptides have been observed in a small subset of cancers (Chong et al., Nat Commun 11, 1293 (2020); Ouspenskaia et al., Nat Biotechnol 40, 209-217 (2022); and Wang et al., Journal of Experimental Medicine 183, 1131-1140 (1996), but they remain largely unexplored in most solid tumor types. Furthermore, it is unclear whethertranslation of unannotated ORFs encoding ncHLAp is entirely tumor-specific or to what extent this also occurs in healthy tissues (Leko and Rosenberg, Cancer Cell 38, 454-472 (2020)).

[0512] Likewise, it is incompletely understood to what extent ncHLAp may be recognized by the adaptive immune system, as efforts to systematically evaluate ncHLAp immunogenicity with a sensitive approach have been lacking. It is therefore essential to elucidate the cancer restriction and immunogenic potential of ncHLAp, as they could enable novel immunotherapies for cancers, such as those unresponsive to immune checkpoint inhibition.

[0513] Pancreatic ductal adenocarcinoma (PDAC) represents an example of solid tumors recalcitrant to currently available immunotherapies. It is the third leading cause of cancer-related deaths in the United States (Siegel et al., CA Cancer J Clin 74, 12-49 (2024)), and it is typified by a complex tumor-immune microenvironment (TME) and a notoriously low neoplastic cellularity (Cancer Genome Atlas Research Network, Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell 32, 185-203.e13 (2017); and Cao et al., Cell 184, 5031 (2021)). Immunotherapies have been largely ineffective in this disease (Ho et al., Nat Rev Clin Oncol 17, 527-540 (2020)), owing in part to the low-to-intermediate mutational burden of this cancer (Cancer Genome Atlas Research Network, Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell 32, 185-203.e13 (2017)), as well as defects in T cell priming, function, and / or localization (Ho et al., Nat Rev Clin Oncol 17, 527-540 (2020)). The low neoplastic cellularity of PDAC has hampered efforts to characterize its antigenic landscape (Bradley et al., Nat Commun 11, 5332 (2020)), but a deeper understanding of the PDAC immunopeptidome could accelerate novel immunotherapies for this disease.

[0514] With this example, patient-derived organoids (PDOs) were generated and used to enrich for the malignant compartment and empirically identified PDAC HLAp via immunopeptidomics. It was hypothesized that ncHLAp might broaden the landscape of targetable antigens in this malignancy, characterized by a relatively low mutational burden (Cancer Genome Atlas Research Network, Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell 32, 185-203.e13 (2017); and Aguirre et al., Cancer Discov 8, 1096-1111 (2018)). With this approach, thousands of cryptic peptides within the PDAC immunopeptidome were identified. As many ncHLAp are thought to arise from aberrant translation events, a novel, translation-centric filtering approach was developed to probe healthy tissues for evidence of unannotated ORF translation and nominated ncHLAp exhibiting cancer restriction. Finally, a highly sensitive exvivo platform was employed to enable priming and expansion of T cells capable of recognizing cancer-restricted ncHLAp, isolated and characterized a suite of ncHLAp-reactive T cell receptors (TCRs) and investigated whether ncHLAp-reactive TCRs can recognize and kill pancreatic cancer cells using patient-derived preclinical models.

[0515] MATERIAL AND METHODS

[0516] Patient specimens

[0517] Eligible participants were recruited from outpatient clinics and inpatient units at Dana-Farber Cancer Institute and Brigham and Women’s Hospital. Investigators obtained written, informed consent from patients at least 18 years old with pancreatic cancer for Dana-Farber / Harvard Cancer Center Institutional Review Board (IRB)-approved protocols for tissue collection, molecular analysis, and organoid generation. Organoid samples for bulk genomic and transcriptomic analyses were collected. For resection specimens, tissue was initially submitted for pathologic analysis, and additional tissue was allocated for organoid generation if available. For metastatic lesions, core needle biopsy specimens were collected, and the first core was sent for pathologic analysis. One or more additional cores were then allocated for organoid generation. PDOs were additionally generated from malignant ascites. All human studies were conducted according to the principles expressed in the Declaration of Helsinki.

[0518] Patient-derived organoid (PDO) generation

[0519] PDOs were generated as previously described (Boj and Hwang, Cell 160, 324-338 (2015); and Raghavan, Cell 184, 6119-6137. e26 (2021)). Briefly, tissue samples were minced and digested at 37°C for 15 min using complete organoid medium, 1 mg / mL collagenase XI (Sigma Aldrich), 10 pg / mL DNase (STEMCELL Technologies), and 10 pM Y27632 (Selleck) (Tiriac et al., Cancer Discov 8, 1112-1129 (2018)). Digested cells were seeded in growth-factor reduced Matrigel (Corning) and fed with complete organoid medium as previously described (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021); and Raghavan, Cell 184, 6119-6137. e26 (2021)).Bulk RNA-sequencing and DNA-sequencing of organoids

[0520] RNA / DNA was isolated from PDOs and matched normal blood as previously described (Raghavan, Cell 184, 6119-6137. e26 (2021)). Briefly, snap frozen PDOs were homogenized using buffer RLT Plus (QIAGEN) and a Precellys homogenizer. Samples were then processed for both DNA extraction and RNA isolation using the QIAGEN AllPrep DNA / RNA / miRNA Universal kit per manufacturer instructions. Bulk RNA-sequencing was performed as previously described (Raghavan, Cell 184, 6119-6137. e26 (2021)) by the Broad Institute Genomics Platform using a NovaSeq 6000. Data were analyzed using the Broad Picard pipeline which includes de-multiplexing and data aggregation (available on the world-wide web at broadinstitute. github. i o / pi card / ).

[0521] Whole genome sequencing was performed as previously described (Raghavan, Cell 184, 6119-6137.e26 (2021)). Library preparation was performed using a KAPA HyperPrep without amplification kit (KAPA Biosystems) with palindromic forked adapters with unique 8-base index sequences embedded within the adaptor (Roche). Libraries were sequenced using a NovaSeq 6000 S4. Sequencing data was processed by the Broad Picard pipeline (available on the worldwide web at broadinstitute. github. io / picard / ) to yield BAM files containing demultiplexed, aggregated aligned reads. Sequence reads were aligned to the GRCh37 reference genome (b37 edition from the Human Genome Reference Consortium) using bwa, and further processed using Picard (version 1.90, available on the world-wide web at broadinstitute. github. io / picard / ) to remove duplicates and Genome Analysis Toolkit (GATK, version 1.6-5-g557da77) to perform localized realignment around indel sites. Single nucleotide variants were called using MuTect vl.1.45. Copy number variants (CNV) and structural variants were called using the internally developed algorithms RobustCNV and BreaKmer followed by manual review (Abo et al., Nucleic Acids Res, 19 (2014)).

[0522] Organoid propagation and preparation of organoids for immunopeptidomics

[0523] PDOs were cultured in Matrigel domes as previously described (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)) and expanded to 5 x 107– 1 x 108cells. Prior to HLA-I immunopeptidomics, organoids were treated with hlFNy (PeproTech; 100 ng / mL) for 36-40 hours to upregulate surface HLA-I (Sarkizova et al., Nature Biotechnology 201938:238, 199— 209 (2019)). Organoids were mechanically dissociated and then subjected to a gentle enzymaticdigestion (TrypLE for less than 10 min at 37°C) to digest Matrigel without cleavage of HLA-I. Cells were washed in lx PBS and cell pellets were snap frozen and stored at -80°C for batch processing.

[0524] Media for PDO expansion was formulated as previously described (F reed-Pastor et al., Cancer Cell 39, 1342-1360. el4 (2021)). Briefly, L-WRN CM was generated by collecting 10 days of supernatant from L-WRN cells (VanDussen et al., Stem Cell Res 37, 101430 (2019)) (ATCC; CRL-3276), grown in Advanced DMEM / F12 (Gibco) supplemented with 20% fetal bovine serum, 2 mM GlutaMAX, 0.5X pen / strep (Gibco), and 0.25 pg / mL amphotericin. L-WRN CM was diluted 1: 1 in Advanced DMEM / F 12 (Gibco) and supplemented with additional RSPO-1 conditioned media (10% v / v), generated using HA-R-Spondinl-Fc 293T Cells (Cultrex; #3710-001-K). The following molecules were also added to the growth media: B27 (Gibco), 1 pM N-acetyl cysteine (Sigma- Aldrich), 10 pM nicotinamide (Sigma- Aldrich), 50 ng / mL EGF (Thermo), 500 nM A83-01 (Cayman Chemical), 10 pM SB202190 (Cayman Chemical), and 500 nM PGE2 (Cayman Chemical).

[0525] Ex vivo priming and expansion of antigen-specific T cells

[0526] Ex vivo priming and expansion of antigen-specific T cells was carried out as previously described (Rollins et al., Curr Protoc Immunol 129, e97 (2020)). Briefly, monocytes were isolated from HLA-typed leukapheresis samples (STEMCELL Technologies) using the adherence method on Day 1. To generate immature dendritic cells, cells were cultured for 16-24 hours in hGM-CSF (800 U / mL) and hIL-4 (35 ng / mL) and subsequently differentiated using hTNFa (10 ng / mL), hIL-ip (10 ng / mL), hIL-6 (10 ng / mL), and PGE2 (1 pg / mL). For certain experiments, on Day 3, MoDCs were harvested for flow cytometric staining using the following antibodies: anti-CD14 (clone M5E2; BUV737), anti-CDllc (clone Bul5; PE), anti-CD83 (clone HB15e; FITC), anti-CD86 (clone 2331; BV650); anti-HLA-DR (clone G46-6; BUV661), anti-CD40 (clone 5C3; BV421) to validate maturation. All antibodies were used at recommended dilutions unless otherwise indicated.

[0527] Mature DCs were loaded with peptide pools (4-15 candidate peptides per pool; each at 1-2 pg / mL) and incubated for 2-4 hours at 37°C. Autologous CD8+T cells were isolated from PBMCs using the EasySep™ Human CD8+ T Cell Positive Selection Kit II (STEMCELL Technologies). Peptide-loaded DCs were then irradiated (4000 rad). Cytotoxic T lymphocytelines (CTL lines) were generated (3-20 per donor) by combining irradiated DCs with CD8+T cells (2.5 - 5 x 106CD8 T cells per CTL line) in a 2.5:1 ratio supplemented with IL-21 (30 ng / mL). CTL lines were maintained as previously described (Rollins et al., Curr Protoc Immunol 129, e97 (2020)), with media changes every 2-3 days with fresh IL-2 (12.5 U / mL), IL-7 (5 ng / mL), and IL- 15 (5 ng / mL) [IL-2, IL-7, IL- 15 were obtained through NCI Biological Resources Branch (BRB)]. CTL lines were restimulated every 12-14 days by combining candidate CTL lines with peptide-loaded autologous CD8negPBMCs and IL-21. Immunogenicity was evaluated after a total of three stimulations (prime + two restimulations). To analyze antigenspecificity of expanded CTL lines, we employed fluorescently conjugated multimer analysis and / or peptide-mediated stimulation of effector cytokine production. All peptides were synthesized by Gen Script.

[0528] Multimer analysis. Briefly, each CTL line was harvested independently, and 3 x 105cells (per CTL line) were transferred to 96-well U-bottom plates. Individual CTL lines were fluorescently barcoded (Ali et al., Nature Protocols 2019 14:6 14, 1926-1943 (2019)) using a 3x4 grid of serially diluted CellTrace CFSE (CFSE) and CellTrace Violet (CTV). Cells were barcoded on ice for 5 min in the dark and excess dye was quenched with heat-inactivated FBS (HLFBS). Following fluorescent-barcoding, experimental and control CTL lines were combined and then simultaneously stained for flow cytometric analysis. Prior to surface staining, cell pellets were resuspended with viability dye (Ghost Dye Red 780, Tonbo Biosciences) diluted 1: 1000 in PBS on ice for 20 min in the dark. Fluorescently-conjugated HLA-A*02:01 or HLA-A*ll:01 peptide-receptive tetramers (Saini et al., Sci Immunol 4, 9039 (2019)) (APC and BV605 dual staining for fluorescently-barcoded samples; APC, BV605, PE, or BV421 for individual stains) were freshly loaded with peptide prior to staining and cells were incubated with candidate tetramers at room temperature for 15 min, prior to surface staining using anti-CD8 (clone SKI; BUV737, BV711, or FITC) for 30 min on ice.

[0529] Intracellular cytokine staining (ICS) analysis. Briefly, each CTL line was harvested independently and 3 x 105CTLs were plated per well in 96-well U-bottom plates (one well for each experimental peptide and one well for a no peptide control). Peptide-loaded autologous CD8negPBMCs (1:1 ratio with CTLs) and GolgiPLUG / GolgiSTOP were added. Cells were allowed to incubate at 37°C for 5-6 hours prior to flow cytometric staining. Cells were washed, stained with viability dye (Ghost Dye Red 780, Tonbo Biosciences or Zombie Aqua, Biolegend)on ice for 20 min in the dark, followed by surface staining using anti-CD8 (clone SKI; FITC) for 30 min on ice. CTLs were then fixed and permeabilized using FoxP3 fix (eBioscience), followed by intracellular cytokine staining with anti-IFNy (clone B27; APC) and anti-TNFa (clone MAbll; PE), both used at 1:80 dilution. We preset a threshold for considering a positive immunogenic response as >2% above the maximum value within the negative control condition included for each assay (relevant negative controls being “no peptide control” performed in parallel for each CTL line [ICS] or simultaneous staining of autologous pre- stimulated CD8+T cells [peptide: HLA multimer]). We subtracted any background dual cytokine reactivity IFNy+TNFa+(ICS) (defined by simultaneous intracellular cytokine staining of “no peptide control” for each CTL line) to define the “percent positivity” for each peptide: CTL combination evaluated. For any assayed peptide that was predicted to bind to both HLA-A*02:01 or HLA-A*ll:01 (n = 3 for ncHLAp), we considered the allele with the strongest predicted binding affinity (best. MSi_allele; HLAthena [available on the world-wide web at hlathena.tools]) as the “assayed allele”. For all flow cytometry experiments, samples were acquired on BD LSR II, LSR Fortessa, or Symphony analyzers.

[0530] Single-cell TCR sequencing of ncHLAp-specific CTLs

[0531] Single-cell TCR sequencing (paired V(D)J, gene expression, and DNA-barcoded multimers) was performed using the Barcode-Enabled Antigen Mapping (BEAM-T, 10X Genomics) pipeline. Briefly, ncHLAp-reactive CTL lines were pooled in equi-cellular ratios and stained with a panel consisting of a viability dye (7-AAD), anti-CD8 (clone SKI, FITC), and the BEAM-T assembly, consisting of a PE-conjugated and molecularly barcoded peptide-loaded HLA multimers. Staining procedures were performed according to the manufacturer's instructions with minor modifications. Individual BEAM-T assemblies were prepared fresh for each peptide of interest, as well as a negative control assembly. Each candidate BEAM-T assembly was confirmed to have absent background staining prior to incorporation into the panel. Live, CD8+, Tetramer+singlets were sorted using low-pressure purity mode into an FBS-coated microcentrifuge tube containing 5% HI-FBS. Cells were washed, counted with a fluorescent cell counter, and concentrated to 1000 cells / pL in PBS for a targeted cell recovery of 10,000 cells. Gene expression, V(D)J and BEAM single-cell libraries were prepared following manufacturer instructions. Library concentrations were quantified with the Agilent BioanalyzerHigh Sensitivity assay throughout library construction steps, and final library concentration was determined with the Qubit dsDNA HS Assay kit. Paired-end, dual-index sequencing was performed with a NovaSeq SI (Illumina) at 10,000 read-pairs per cell, using the 10X recommended sequencing structure. FASTQs were processed with the Cellranger multi pipeline to acquire paired gene expression, TCR sequencing and antigen-specificity scoring. Antigen specificity scores were calculated directly with the cellranger pipeline. This measures the probability of a given antigen binding to a specific TCR compared to its negative control. The antigen specificity score was mathematically calculated using a beta distribution with a confidence level of 0.925 and above, using the expression: (1-beta.cdf(0.925, AntigenUMI+1, ControlUMI+3))*100 (available on the worldwide web at 10xgenomics.com / support / software / cell-ranger / latest / algorithms-overview / cr-5p-antigen-algorithm). TCR assemblies from cells with low-confidence gene expression data were removed from downstream analysis, and TCRs that were not full-length or productive TCRs were additionally removed. Data was analyzed using the 10X Loupe V(D)J browser (v5.0.1) and custom R scripts.

[0532] T cell receptor (TCR) cloning

[0533] We first generated a modular Gibson-compatible third-generation SIN lentiviral expression construct, harboring aBsmBI-flanked filler sequence downstream of the EFla promoter and immediately upstream of a gRNA-resistant version of human TRAC, for rapid TCR reconstruction (FIG. 14). We likewise generated a geneblock (IDT) encoding a gRNA-resistant version of human TRBC1 followed by a furin cleavage site (Yang et al., Gene Therapy 15, 1411— 1423 (2008), a flexible linker, and a porcine teschovirus-1 ribosome skipping sequence (P2A) (FIG. 14). Each candidate alpha and beta V(D)J with 30-bp Gibson-compatible 5’ and 3’ homology arms were then synthesized as EBlocks Gene Fragments (IDT), reconstituted in nuclease-free H2O, and used directly for Gibson assembly (Gibson et al., Nat Methods 6, 343-345 (2009)). We introduced T48C (TRAC) and S57C (TRBCl) to promote receptor pairing (Kuball et al., Blood 109, 2331-2338 (2007)), but no additional TCR enhancements were pursued.Lentiviral production and titering

[0534] Lentiviral constructs and packaging vectors were prepared using endotoxin-free midiprep kits (QIAGEN). Lentiviruses were produced by co-transfection of HEK293 cells with lentiviral constructs plus packaging vectors: PsPax2 (psPAX2 was a gift from Didier Trono - Addgene plasmid # 12260; available on the world-wide web at n2t.net / addgene: 12260; RRID: Addgene 12260) and Pmd2. G (pMD2. G was a gift from Didier Trono - Addgene plasmid # 12259; available on the world-wide web at n2t.net / addgene: 12259; RRID: Addgene_12259). Viral supernatant was harvested 48 and 72 hours post transfection, fdtered through a 0.45 pm low-protein binding PVDF fdter (EMD Millipore), and concentrated by ultracentrifugation (25,000 rpm for 2 hours at 4°C). Concentrated virus was resuspended in Opti-MEM (Gibco) and lentiviral aliquots were frozen and stored at -80°C. Functional lentiviral titers were determined by measuring CD3 surface stabilization after TCR reconstitution into TCR-knockout (KO) Jurkat cells.

[0535] TCR redirection (TCR-T)

[0536] CD8+T cells were isolated using negative selection (EasySep Human CD8+ T Cell Isolation Kit #17953) from healthy donor PBMCs. CD8+T cells were activated with Dynabeads Human T- Activator CD3 / CD28 (#11132D) and lentivirally transduced at 24 hrs post-activation to express the indicated TCR. Cells were maintained in RPMI + 5% heat-inactivated human AB male serum supplemented with IL-7 (5 ng / ml) and IL-15 (5 ng / ml), starting on Day 2. One day 5, magnetic beads were removed and cells were electroporated with Cas9:sgRNA ribonucleoprotein (RNP) complexes to simultaneously knockout endogenous TRAC, TRBC1, and TRBC2. RNP complexes were prepared by incubating Cas9 (40 pM) with each sgRNA (80 pM) for 15 min (separate RNPs were generated with sgTRAC and sgTRBCl / 2). T cells were resuspended in 20 pL P3 buffer (1 x 106cells per reaction) and immediately mixed with both RNP complexes. Cells were then electroporated using the Lonza 4D-Nucleofector X system (pulse code EO115) and recovered in T cell media (without antibiotics) for 15 minutes at 37°C. TCR-redirected T cells (TCR-T) were then maintained in media supplemented with IL-7 and IL-15 for 9 days prior to fluorescence activated cell sorting. On Day 14, TCR-redirected T cells were assessed phenotypically and FACS sorted to obtain live CD8+Tetramer+T cells. Sorted cells were immediately subjected to a rapid expansion protocol (REP) to expand sufficient redirected TCR-T cells for functional experiments. REP was performed as previously described (Anderson et al., J Immunother Cancer 10, e003959 (2022)) with slight modifications. Briefly, sorted TCR-T cells were expanded via co-culture with irradiated (4000 rad) pooled allogeneic PBMCs and irradiated (8000 rad) LCL cells in the presence of anti-CD3 antibody (OKT3; 30 ng / mL) and IL-2 (50 U / mL) for 11-14 days in culture flasks or G-rex plates (Wilson Wolf). Cultures were supplemented with IL-2 every 48 hours until culture termination. On day 11-14, TCR-T cells were assessed by flow cytometry to ensure maintained redirection efficiency and cryopreserved. For functional experiments (T2 functional avidity, alanine scanning, PDO recognition, PDO cytotoxicity), TCR-T cells were thawed and rested overnight in T cell media + IL-2 (50 U / mL) prior to indicated experiments.

[0537] Functional avidity evaluation and alanine scanning

[0538] T2 cells (ATCC; CRL-1992) were first transduced with pWF073 (Lenti-eGFP-ffLuc) and sorted to obtain a pure eGFP1population (T2_eGFP-ffLuc). T2_eGFP-ffLuc cells were peptide loaded for 18 hours at 37°C with peptides at concentrations indicated in figures.

[0539] Cryopreserved TCR-T cells were thawed and rested overnight prior to co-culture. Peptide-loaded T2_eGFP-ffLuc cells were then washed five times to remove unbound peptide and plated in 96-well black-wall flat-bottom plates at 2.5 x 104cells in co-culture with 1.25 x 105TCR-T cells (5:1 E: T) in 200 pL media containing IL-2 (50 U / mL) and anti-CD28 antibody (clone CD28.2; 100 ng / mL). Viability was analyzed after 24 hours using ONE-Glo Luciferase Assay System (PROMEGA) according to manufacturer instructions. Luciferase activity, a surrogate for viable target cells, was quantified with an EnVision plate reader (PerkinElmer). Luminescence values were normalized to T2_eGFP-ffLuc in the absence of T cells (used to define 100% viability). T2_eGFP-ffLuc (without peptide) in co-culture with each candidate TCR-T cells was also used as an additional control (for avidity plots, “no peptide” DMSO control was approximated as 1 x 10'4pg / mL). Alanine scanning was performed exactly as above, comparing theNUll peptide to NU11 peptides harboring one of nine possible alanine-substitutions. Sigmoidal curves were fit to the data using “log(agonist) vs. response - variable slope (four parameters)” in Prism to calculate EC50.Organoid + TCR-T co-culture

[0540] Briefly, PANFR0071 PDOs were first transduced with pWF073 (Lenti-eGFP-ffLuc) and sorted to obtain a pure eGFP+population. PANFR0071 eGFP fILuc cells were then dissociated using TrypLE and plated in 96-well black wall flat bottom plates (wells precoated with 10% Matrigel) at 2.5 x 104cells in organoid complete media and allowed to adhere and proliferate for 24 hours. Cryopreserved TCR-T cells were thawed and rested overnight prior to co-culture. For indicated wells, PANFR0071_eGFP_ffLuc cells were pre-blocked for 30 minutes using mouse anti-human HLA-I functional grade blocking antibody (clone W6 / 32; 50 pg / mL; InVivoMab BioXCell). TCR-T cells (resuspended in REP media) were added at E: T ratios indicated in figures in a total of 200 pL media containing IL -2 (50 U / mL) and anti-CD28 antibody (clone CD28.2; 100 ng / mL). Target cell (PDO) viability was analyzed after 48 hours of co-culture using ONE-Glo Luciferase Assay System (PROMEGA) according to manufacturer instructions.

[0541] Luciferase activity, a surrogate for viable target cells, was quantified with an EnVision plate reader (PerkinElmer). Luminescence values were normalized to PANFR0071_eGFP_ffLuc PDOs grown in the absence of T cells (used to define 100% viability for each experiment). % viability was transformed to %cytotoxicity by taking the inverse of %viability (%cytotoxicity = 100% - %viability). Prior to luciferase analysis 100 pL of conditioned media from each coculture was collected and IFN-y concentration was measured using the ELISA MAX Deluxe Set Human IFN-y (BioLegend, #430116) according to the manufacturer’s instructions.

[0542] Flow cytometry-based evaluation of TCR-T recognition

[0543] PANFR0071 or PANFR0151 (neg Ctrl; HLA-mismatched) PDOs were pre-treated with IFN-y (100 ng / mL) for 36 - 40 hours prior to co-culture. Cryopreserved TCR-T cells were thawed and rested overnight prior to co-culture. PDOs were dissociated with TrypLE (maximum 8-10 minutes to minimize cleavage of HLA-I) and plated in 96-well flat bottom plates at 1 x 105, 2 x 105, and 5 x IO3cells in 100 pL of organoid complete media. TCR-T cells were added at 1 x 105cells in a total of 200 pL media containing IL-2 (50 U / mL) and anti-CD28 antibody (clone CD28.2; 100 ng / mL). Antigen-specific recognition was analyzed after 24 hours co-culture by flow cytometric staining of live (Zombie Aqua), CD8 (clone SKI; BV711; 1:20), and CD137 (clone 4B4-1; APC; 1:20).In vivo evaluation of adoptive cellular therapy (ACT) targeting ncHLAp

[0544] All animal studies described in this study were approved by the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (#22-009). NOD-scid IL2Rgammanull(NSG) mice were purchased from Jackson Labs (J AX# 005557) and CrTac: NCr-Foxnlnu (Nu / Nu; athymic) mice were purchased from Taconic. 6-8 week old NSG or Nu / Nu mice were subcutaneously implanted with 0.75 - 1 x 106P0071_eGFP-ffLuc cells (resuspended in 50% Matrigel + 50% PBS), following brief TrypLE dissociation of established organoids. Patient-derived organoid xenograft (PODX) animals were palpated every other day for tumor engraftment beginning on day +4 to +5 and tumor volumes were measured via calipers once tumors reached sufficient size for quantification. All animals were confirmed to have palpable tumor engraftment immediately prior to ACT. TCR-T cells were expanded via REP for 10-12 days prior to adoptive transfer. Animals were block randomized to experimental or control arms prior to intraveneous delivery of TCR-T cells and received ncHLAp-directed TCR-T (TCR001, TCR005, TCR012) or irrelevant TCR-T (TCR008; non-HLA-A*02:01 restriction), respectively. TCR-T cells (doses indicated in figures) were intravenously delivered via tail vein beginning on day +5 to +8, with repeat dosing every 10 days in indicated experiments. Tumor volumes were measured every 2-3 days with calipers and volumes were calculated using W2x L / 2. Caliper measurements were performed independently by two researchers. No mice were excluded from the in vivo studies, and experimenters were not blinded. Sample sizes for in vivo experiments were not determined by power analysis but were determined by prior experience with preclinical mouse models. Number of animals per arm and statistical tests are described in the figure legends.

[0545] Flow cytometry of pancreatic organoids

[0546] PDOs were grown as described above. Where indicated, organoids were treated with IFNy (100 ng / mL; PeproTech) for 36-40 hours prior to analysis. Organoids were dissociated using TrypLE (8-10 min to minimize cleavage of surface proteins) washed with PBS, and then stained with viability dyes for 20 min on ice. Surface staining was performed using HLA-A / B / C (clone W6 / 32; FITC or PE; 1: 100) in PBS with 1% HI-FBS on ice for 30 min in the dark.Mutation calls and HLA typing for bulk tumor datasets

[0547] HLA typing, somatic mutation calls, and variant effect annotation for TCGA PAAD and DFCI-PancSeq were performed as previously reported (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)).

[0548] Mutation calls and HLA typing for PDOs

[0549] Somatic single nucleotide variants called by Mutect (described in an earlier section) were merged with somatic indels called using a combination of the variant callers, Strelka2, version 2.9.2 (Kim et al., Nat Methods 15, 591-594 (2018)), and Scalpel, version 0.5.4 (Narzisi et al., Nat Methods 11, 1033-1036 (2014)). Somatic indel calls for organoids were made as previously reported for bulk tumors (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)), with the following exceptions: the variant caller, Manta (Chen et al., Bioinformatics 32, 1220-1222 (2016)) (Chen et al., Bioinformatics 32, 1220-1222 (2016)), was not used, and both Scalpel and Strelka2 were run with parameters prescribed for whole genome sequencing datasets. In the case of Scalpel, indel calls were restricted to the exome intervals with a bed file derived from the CGHub bitbucket account (available on the world-wide web at / / cghub. ucsc.edu; whole_exome_agilent_l.l_refseqj31us_3_boosters.targetlntervals.bed). Variant effect annotation was performed with Ensembl Variant Effect Predictor (VEP), version 99 (McLaren et al., Genome Biol 17, 122 (2016)). The corresponding VEP cache for both GRCh37 was downloaded and used to run the software offline. VEP was run using the Wildtype and Frameshift plugins to annotate the effects of indels. The following parameters were employed: —symbol, — terms=SO, --cache, —offline, -transcript version, -pick. The —pick parameter was reordered from the default to report the transcript with the most extreme consequence for each variant: rank, canonical, appris, tsl, biotype, ccds, length, mane (Freed-Pastor et al., Cancer Cell 39, 1342- 1360. el4 (2021)).

[0550] For somatic mutational burden comparisons between organoids and bulk tumors, only the variants overlapping these intervals in bulk samples and organoid samples were considered. HLA typing was performed as reported previously for bulk tumors (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)), with any relevant parameters updated to account for whole-genome sequencing.Construction of patient-specific proteome database for mass spectrometry analysis Somatic mutations, germline mutations, and variant phasing. Somatic mutations (SNVs and indels) were called as described in previous sections. All exonic somatic mutations were included in patient-specific proteome search spaces regardless of potential HLA binding affinity. Germline SNVs were called using Strelka with default parameters and the reference genome file from human assembly 19. Only variants marked PASS were considered for downstream analysis. These were merged with somatic variants into single VCF files using the Genome Analysis Toolkit (GATK), version 4.1.2.0 (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)). Two sets of VCF files for each sample were then processed in parallel: one with germline variants only and one with somatic and germline variants combined. VCF files were then coordinate-sorted, followed by read-backed variant phasing performed using the ReadBackedPhasing function from GATK, version 3.7. Variant effect annotation was performed with Ensembl Variant Effect Predictor (VEP), version 99 (McLaren et al., Genome Biol 17, 122 (2016)). The corresponding VEP cache for GRCh37 was downloaded and used to run the software offline. VEP was run using the Wildtype and Frameshift plugins to annotate the effects of indels. The following parameters were employed: —symbol, — terms=SO, —cache, —offline, — transcript version, —pick. The -pick parameter was reordered from the default to report the transcript with the most extreme consequence for each variant: rank, canonical, appris, tsl, biotype, ccds, length, mane (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)). Finally, variant-recoded protein sequences were retrieved using pVACtools’ generate_protein_fasta function (from version 3.1.1), with flanking sequence length set to 30 and the “-d” parameter set to “full.” These settings ensure that the full downstream sequence of frameshift mutations is included, as well as 30 amino acids on either side of missense mutations. pVACtools was run with and without phasing enabled for combined somatic and germline variant files. Phasing was included to account for proximal protein-altering variants that could co-occur on the same HLA-bound peptide. Phased and unphased protein sequences were then added to patient-specific protein search databases.

[0551] Novel unannotated open reading frame (nuORF) mutations. Sequences of non-canonical ORFs with previously demonstrated Ribo-seq evidence of translation were updated with patientspecific variants. Coordinates corresponding to nuORFs were retrieved from the

[0552] nuORF db vl. O.bed file, downloaded from the NCBI Gene Expression Omnibus (GSE143263)(6). We then took somatic SNVs and indels intersecting these coordinates and generated mutation-encoded nuORF protein sequences using a custom python script modified from one published by Ouspenskaia etal. (Nat Biotechnol 40, 209-217 (2022)). Full-length mutant protein sequences were then aggregated into a single FASTA file for each organoid sample.

[0553] Gene fusions. Novel protein sequences stemming from gene fusions were predicted using the computational pipeline, EasyFuse, version 1.3.6 (56). Protein sequences were extracted from the “fusRank_l.pred.csv” output file and reassembled into a FASTA file for each organoid sample. No peptides mapping to gene fusion-derived protein sequences were detected.

[0554] Retained introns. Raw RNA-seq FASTQ files from the original organoid characterization were pseudoaligned to a transcriptome augmented to contain both exonic and intronic sequences, with intronic sequences set to extend 25 base pairs into adjacent exons, as described by Smart et al. (2018) (Nat Biotechnol 36, 1056-1063 (2018)). TPM values were calculated using Kallisto, version 0.45.0 (Bray et al., Nat Biotechnol 34, 525-527 (2016)).

[0555] Retained introns were called using the KeepMeAround algorithm (Pimentel et al., Nucleic Acids Res 44, 838-851 (2016)), as implemented by Smart et al. (2018) (Nat Biotechnol 36, 1056-1063 (2018)). Retained introns were called for all PDAC organoid samples. Introns were considered retained if they passed the following thresholds: intron retention ratio > 0.05, intron TPM > 1, exonic TPM > 1, and > 5 unique reads mapping to the intron. For the organoid samples profiled with immunopeptidomics, we modified python scripts published by Smart et al. (2018) (57) to retrieve the open reading frames and coordinates associated with each sample’s retained introns. We then used bedtools, version 2.29.1, to retrieve all germline and somatic SNVs intersecting retained intron coordinates from VCF files established in the mutation calling procedures described in the previous section (Quinlan et al. Bioinformatics 26, 841-842 (2010)). We then employed BCFtools in consensus mode to recode intronic sequences with all variants for both haplotypes separately and combined (to account for potential errors in variant phasing) (Danecek et al., Gigascience 10, gia008 (2021)). The resulting sequences were then all translated across all three reading frames, accounting for potential indels, to generate the retained intron-encoded protein sequence search space.

[0556] A second intron retention pipeline, IRFinder, version 1.3.0 (Middleton et al., Genome Biol 18, 51 (2017)), was also used to account for additional introns that could have been missed by KeepMeAround. IRFinder was run with default parameters and with GRCh37 as reference.Introns were kept for further analysis if they passed the following thresholds: coverage > 0.5, IntronDepth > 3, ExonToIntronReadsLeft > 2, ExonToIntronReadsRight > 2, SpliceExact > 4, and IRatio > 0.03. The remaining introns were then passed into the custom python script described above to translate protein sequences across the canonical reading frame using the reference genome sequence.

[0557] Final patient-specific database assembly. Fasta files containing protein sequences for somatic mutations, germline mutations, variant-recoded and wild-type introns, gene fusions, and somatic variant-recoded nuORFs were aggregated into a single fasta file for each patient sample. These FASTA files were then combined with the canonical human proteome and other databases for mass spectrometry analysis (see later section for additional detail).

[0558] HLA-I Immunoprecipitation from organoid samples

[0559] 60-72 million organoid cells went through the FILA-I immunoprecipitation. Each organoid was lysed with 4°C lysis buffer [20 mM Tris pH 8.0, 100 mM NaCl, 6 mM MgC12, 1 mM EDTA, 60 mM Octyl P-d-glucopyranoside, 0.2 mM lodoacetamide, 1.5% Triton X-100, lx Complete Protease Inhibitor Tablet-EDTA free, 1 mM PMSF, 10 mM NaF, 1: 100 dilution of Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, P5726), 1:100 dilution of Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, P0044), 50 pM PR-619 (Lifesensors, SI9619: PR-619), 10 mM Sodium Butyrate (Sigma, B5887), 2 pM SAHA (Sigma-Aldrich, SML0061), 10 mM Nicotinamide (Sigma-Aldrich, N3376)] obtaining a total of 2 x 1.2 mL lysate per organoid. Each lysate was moved into an Eppendorf tube, incubated on ice for 30 min with 2 x 1 pL Benzonase (Thomas Scientific, E1014-25KU) to degrade nucleic acid and inverted every 5 min. The lysates were then centrifuged at 15,000 x g for 20 min at 4°C.

[0560] Supernatants were transferred to two tubes, each containing -37.5 pL pre-washed Gammabind Plus Sepharose beads (Millipore Sigma, GE17-0886-01) and 15 pg HLA-I antibody (W6 / 32) (Abeam, ab22432) (PANFR0071, PANFR0123, PANFR0151, PANFR0177, PANFR0290, PANFR0402, PANFR0413), or 18 pg HLA-I antibody (W6 / 32) (BioXCell, BE0079) (PANFR0069, PANFR0049, PANFR0359, PANFR0087). The HLA-I antibody-bead-lysate mixture rotated for 3 hours at 4°C and was spun at 1,500 x g for 1 min at 4°C.

[0561] During HLA-I complex capture, a 10 pm PE fritted plate (Agilent, S7898A) was placed on a Waters Positive Pressure Manifold and wells were washed using 1 mL acetonitrile and3 x 1 mL room -temperature PBS. After each liquid addition, positive pressure of <5 psi was applied to the plate to achieve liquid movement. Immediately following HLA-I capture, beads were resuspended in 1 mL cold PBS and transferred to the pre-washed 10 pm PE fritted plate. Each sample tube was rinsed with 500 pL cold PBS and remaining beads were transferred to the correct well. In total, eight wash steps were performed to remove nonspecifically bound material: four washes with 1 mL of cold complete wash buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 6 mM Octyl P-d-glucopyranoside, 0.2 mM lodoacetamide) and four washes with 1 mL of 10 mM Tris pH 8.0 buffer.

[0562] In addition to profiling patient-derived organoids, we additionally profiled one of our PDOs (PANFR0413) grown as a monolayer, following short-term adaptation from 3D culture to tissue culture plastic (2D). This PDO-derived 2D sample, 50 million cells, went through HLA-I immunoprecipitation as described above with the following modifications: PANFR0413 was immunoprecipitated with 15 pg W6 / 32 (Abeam, ab22432) with 37.5 pL of pre-washed Gammabind Plus Sepharose beads (Millipore Sigma, GE17-0886-01) in a total of 1.2 mL lysis buffer with 2 pL Benzonase. Cells were lysed and incubated with beads and antibodies in water pre-conditioned 1.5 mL LoBind eppendorf tube. Other than a single mutHLAp (missense mutHLAp from SLC39A3) empirically detected in this PDO-derived 2D sample, HLAp (canonical or noncanonical) derived from this monolayer culture were not included in subsequent analyses or figures.

[0563] HLA-I Immunoprecipitation from primary PDAC samples

[0564] 34-68 mg (wet weight) cryopulversized primary PDAC tumors (Cao et al., Cell 184, 5031 (2021)) went through HLA-I immunoprecipitation as described above with the following modifications: Supernatants were transferred to one tube per sample, each containing -37.5 pL pre-washed Gammabind Plus Sepharose beads (Millipore Sigma, GE17-0886-01) and 15 pg of HLA-I antibody (W6 / 32) (Abeam, ab22432).

[0565] Elution and desalt of HLA peptides using a positive pressure manifold

[0566] HLA-I peptides were eluted and desalted from IP beads as follows: wells of a tC1840 mg Sep-Pak desalting plate (Waters, Milford, MA) were activated with 2 x 1 mL of methanol (MeOH) and 500 pL of 99.9% acetonitrile (ACN) / 0.1% formic acid (FA), then washed with4 x 1 mL of 1% FA. The 10 pm PE fritted filter plate containing the beads was placed on top of the Sep-Pak plate. To dissociate peptides from HLA molecules and facilitate peptides binding to the tC 18 solid phase, 200 pL of 3% ACN / 5% FA was added to the beads in the filter plate. 50 fmol retention time standards (JPT, RTK-l-100pmol) was spiked into each sample as a loading control and pushed through both the filter plate and 40 mg Sep-Pak plate. Following sample loading there was one wash with 400 pL of 1% FA. Beads were then incubated with 500 pL of 10% acetic acid (AcOH) three times for 5 min to further dissociate bound peptides from the HLA molecules. The beads were rinsed once with 1 mL 1% FA and the filter plate was removed. The Sep-Pak desalt plate was rinsed with 1 mL 1% FA an additional three times. The peptides were eluted from the Sep-Pak desalt plate using 250 pL of 15% ACN / 1% FA and 2 x 250 pL of 50% ACN / 1% FA. HLA peptides were transferred into 1.5 mL micro tubes (Sarstedt, Numbrecht, Germany), frozen, and dried via vacuum centrifugation. Dried peptides were stored at -80°C until microscaled basic reverse phase (bRP) separation.

[0567] Microscaled bRP separation was as follows: StageTips were created with two punches of SDB-XC material (Empore 3M) and activated with 2 x 100 pL MeOH and 100 pL of 50% ACN / 0.1% FA, then washed with 3 x 100 pL of 1% FA. Peptides were loaded onto StageTips in 200 pL 3% ACN / 5% FA. Peptides were washed with 3 x 100 pL 1% FA, and then eluted in three fractions with increasing concentrations of ACN (5%, 10%, and 30%) in 0.1% NH40H, pH 10 (Klaeger et al., Mol Cell Proteomics 20, 100133 (2021)), frozen, and dried down via vacuum centrifugation.

[0568] LC-MS / MS immunopeptidome data acquisition

[0569] Peptides were reconstituted in 3% ACN / 5% FA prior to loading onto an analytical column (35 cm, 1.9 pm C18 (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 pm inner diameter, 10 pm emitter (New Objective)). Peptides in all organoid samples and PANFR0413 (2D) samples were eluted with a linear gradient (EasyNanoLC 1200, Thermo Fisher Scientific) ranging from 6-30% Solvent B (0.1% FA in 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% B for 5 min at 200 nl / min. In all cases, MS / MS data were acquired on a Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition. MS2 fill time was set to 100 ms; collision energy was 30 CE.PDAC organoid and tumor immunopeptidome data interpretation

[0570] MS / MS spectra were interpreted using Spectrum Mill (SM) v 7.08 (proteomics.broadinstitute.org) to provide identification at the peptide level.

[0571] Immunopeptidome database construction. For searching LC-MS / MS data of PDAC organoids, the patient-specific protein databases described above were appended to a base proteome consisting of Ensembl v38 hg19 reference proteome (71,704 entries), common laboratory contaminants and retention time standards (642 entries), and curated smORFs (lncRNA and uORFs, 553 entries) (Prensner et al., Nature Biotechnology 39, 697-704 (2021)), 237,427 non-canonical ORFs supported by ribosomal profiling evidence of translation (nuORF DB vl.01) (Ouspenskaia etal., Nat Biotechnol 40, 209-217 (2022)), for a total of 310,326 entries. Using the SM Protein Database utilities (available on the world-wide web at proteomics.broadinstitute.org / millhtml / faindex.htm), the base proteome and individual patient proteomes were combined to create a sequence database specific for each patient-derived organoid and calculate associated statistics. The search space for each organoid in terms of relative number of unique 9-mer peptides was Ensembl hg19 reference proteome + laboratory contaminants (65%), nuORFs + smORFs (32%), patient specific canonical variants + nuORF variants + gene fusions + retained introns (3%). Equivalent versions of the last GRCh37(hg19) genome assembly were used as the reference genome (Broad GRCh37 / b37) for variant calling above and the reference proteome (Ensembl v38),

[0572] ftp. ensembl. org / pub / grch37 / current / fasta / homo_sapiens / pep / Homo_sapiens. GRCh37. pep. all. fa.g z. The Ensembl v38 protein fasta file was filtered to retain non-redundant entries from standard chromosomes (not GL, H, LRG) with transcript biotype - protein coding, and minimum length 7.

[0573] For the PDAC primary tumors, the same base proteome as above consisting of Ensembl v38 hg19 reference proteome (71,704 entries), common laboratory contaminants and retention time standards (642 entries), and curated smORFs (lncRNA and uORFs, 553 entries) (Prensner et al., Nature Biotechnology 39, 697-704 (2021)), 237,427 non-canonical ORFs supported by ribosomal profiling evidence of translation (nuORF DB vl.01) (Ouspenskaia et al., Nat Biotechnol 40, 209-217 (2022)), for a total of 310,326 entries was used. The search space for all primary tumors included Ensembl hg19 reference proteome + laboratory contaminants, nuORFs+ smORFs, combined organoid patient specific canonical variants + nuORF variants + gene fusions + retained introns.

[0574] Spectrum quality filtering. Using the SM Data Extractor module for HLA-I immunopepti domes, similar MS / MS spectra in the same chromatographic peak with the same precursor mass were merged, the precursor MH + inclusion range was 600-4000, and the spectral quality filter was a sequence tag length >1 (i.e., minimum of three peaks separated by the in-chain masses of two consecutive amino acids).

[0575] MS / MS search conditions. Parameters for the SM MS / MS search module for HLA-I immunopepti domes included: no enzyme specificity; precursor and product mass tolerance of ±10 ppm; minimum matched peak intensity of 30%; ESI-QEXACTIVE-HCD-HLA-v3 scoring; fixed modification: carbamidomethylation of cysteine; variable modifications: cysteinylation of cysteine, oxidation of methionine, deamidation of asparagine, acetylation of protein N-termini, and pyroglutamic acid at peptide N-terminal glutamine; and precursor mass shift range of -18 to 81 Da.

[0576] Peptide-spectrum match filtering and false discovery rates. Using the SM Autovalidation module, peptide-spectrum matches (PSMs) for individual spectra were confidently assigned by applying target-decoy based FDR estimation to achieve <1.0% FDR at the PSM level. PSM-level thresholding was done with a minimum peptide length of 7, minimum backbone cleavage score of 5, and <1.0% FDR across all three fractions. Allowed precursor charges were 1-4. Allowed peptide length was 8-11 amino acids. Immunopeptidomics data were further filtered to remove non-human contaminants, peptides that match peptides identified in blank bead negative control IPs (15, 65), and tryptic contaminant peptides. For one PDO sample (P0413), only 2 of 3 fractions were included in downstream analyses.

[0577] FDR filtering for mutation-derived HLAp, nuORFs, and retained introns. All MS / MS spectra of mutation-derived HLAp were manually inspected. While the aggregate FDR for each dataset was set to <1%, as described above, FDR for certain subsets of rarely observed classes (<5% of total) of peptides required more stringent score thresholding to reach a suitable subsetspecific FDR< 1.0%. To this end, we devised and applied subset-specific filtering approaches.

[0578] From the cumulative list of nuORFs, peptides matched in any organoid subsets of nuORF types were thresholded independently using a two-step approach. First, PSM scoring metric thresholds were tightened in a fixed manner for all nuORF PSMs so that nuORF distributions foreach metric improved to meet or exceed the aggregate distributions. The fixed thresholds were: minimum score: 7, minimum percent scored peak intensity: 50%, minimum backbone cleavage score (BCS): 5. Second, individual nuORF type subsets with FDR estimates remaining above 1% were further subject to a grid search to determine the lowest values of BCS (sequence coverage metric) and score (fragment ion assignment metric) that improved FDR to <1% for each ORF type in the dataset. Peptide sequences remaining after filtering steps were included in the dataset in all samples in which that sequence had passed aggregate FDR filtering.

[0579] HLA allele prediction for HLA-I-bound peptides

[0580] HLAthena was used to predict HLA allele restriction for each empirically identified peptide using the set of patient-specific HLA alleles established through HLA typing (Sarkizova et al., Nature Biotechnology 2019 38:2 38, 199–209 (2019)). Peptides were assigned to an allele using a percentile rank cutoff < 2.0. Strong binders were reported when the percentile rank cutoff is < 0.5. For post hoc analysis of HLA-I-bound mutation-derived peptides, we employed a combination of NetMHC-4.0, NetMHCpan-4.0, SMM, and SMMPMBEC as previously described (Freed-Pastor et al., Cancer Cell 39, 1342–1360.e14 (2021)). We applied this same method to estimate the predicted binding affinity of theoretical mutation-derived neoepitopes depicted that were not detected by LC-MS / MS.

[0581] Synthetic peptide analysis of LC-MS / MS detected mutHLAp and ncHLAp

[0582] Synthetic peptides (GenScript) were analyzed at 5, 10, 50, and 100 fmol / µL without background. The synthetic peptide data were collected on a Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a NanoSpray Flex NG ion source using the same method and collision energy as the experimental sample. LC-MS / MS data were searched as previously described against a database containing the synthetic peptide sequences. Any peptides not found in the LC-MS / MS data were analyzed using direct infusion on the Orbitrap Exploris 480 mass spectrometer.

[0583] To validate their initial peptide spectrum matches (PSMs) in the organoid immunopeptidome LC-MS / MS datasets, MS / MS spectra of the synthetic peptides (ncHLAp, mutHLAp, or canonical HLAp) were compared to the organoid MS / MS spectra for spectrum similarity using a standard dot-product measure of the peak masses and intensities. FIG. 12 andFIG. 13 plot the metrics for the best representative organoid MS / MS spectrum of each peptide for each organoid compared to its best representative synthetic MS / MS spectrum, shown with select mirror plots. The best representative spectrum in the organoid dataset was selected simply as the one with the highest PSM score. Since each synthetic peptide was measured several times across its chromatographic peak and in separate LC-MS / MS runs at different concentrations, a custom python script was written to select the best representative synthetic peptide spectrum. All spectra of a synthetic peptide with the same precursor mass and charge as the corresponding best representative organoid spectrum were scored for similarity to the organoid spectrum. The best representative was designated as the one with the highest spectrum similarity score from amongst the subset of spectra tied with the highest sequence coverage according to the SM backbone cleavage score metric. While a perfect dot product spectrum similarity score is 1.0, we applied a threshold of > 0.7 to validate a peptide’s identification.

[0584] Retention Time Prediction using DeepLC

[0585] The retention times of HLA-I peptides were predicted using DeepLC (Bouwmeester et al., Nature Methods 2021 18:11 18, 1363-1369 (2021)) and compared to the measured retention times. DeepLC was run separately for each individual LC run and calibrated on the entire set of validated peptides passing the 1% FDR threshold from the given LC run.

[0586] Reprocessing normal autopsy HLA Ligand Atlas data

[0587] Raw mass spectrometry data from Marcu et al. (Marcu et al., J Immunother Cancer 9, 2071 (2021)) were downloaded from ProteomeXchange Consortium via the PRIDE partner repository using the dataset identifier PXD019643 and reanalyzed using Spectrum Mill (SM) v 7.08 (proteomics.broadinstitute.org) to provide identification at the peptide level. The same personalized database used for the organoid immunopeptidome searches was leveraged with the following parameters.

[0588] Spectrum quality filtering. Using the SM Data Extractor module for HLA-I immunopepti domes, spectral merging was disabled, the precursor MH + inclusion range was 600-2500, and the spectral quality filter was a sequence tag length >1 (i.e., minimum of three peaks separated by the in-chain masses of two consecutive amino acids).MS / MS search conditions. Parameters for the SM MS / MS search module for HLA-I immunopepti domes included: no enzyme specificity; precursor and product mass tolerance of ±10 ppm; minimum matched peak intensity of 30%; ESI-ORBITRAP-CID-HLA-v3 scoring; variable modifications: cysteinylation of cysteine, oxidation of methionine, deamidation of asparagine, acetylation of protein N-termini, and pyroglutamic acid at peptide N-terminal glutamine; and precursor mass shift range of -18 to 136 Da.

[0589] Peptide-spectrum match filtering and false discovery rates. Using the SM Autovalidation module, peptide-spectrum matches (PSMs) for individual spectra were confidently assigned by applying target-decoy based FDR estimation to achieve <1.0% FDR at the PSM level. PSM-level thresholding was done with a minimum peptide length of 7, minimum backbone cleavage score of 5, and <1.0% FDR across all three fractions. Allowed precursor charges were 1-4. Allowed peptide length was 8-11 amino acids. Immunopeptidomics data were further filtered to remove non-human contaminants, peptides that match peptides identified in blank bead negative control Ips (Sarkizova et al., Nature Biotechnology 2019 38:2 38, 199–209 (2019); and Abelin et al., Immunity 46, 315 (2017)), and tryptic contaminant peptides.

[0590] Single-cell RNA-seq and gene module analysis

[0591] Single-cell Gene Expression Flex (10X Genomics) was performed on two fresh primary PDAC resection specimens obtained from IRB-consented patients at Dana-Farber Cancer Institute. Briefly, samples were divided into 25 mg sections and minced finely with razor blades before overnight fixation in 4% formaldehyde (Fisher Scientific) at 4°C. Twenty-four hours post fixation, samples were dissociated using Liberase TH (Roche) in a heated gentleMACs Octodissociator (Miltenyi), following recommended parameters. Probe hybridization and library preparation were conducted following manufacturer instructions. Dual-index, paired-end reads were sequenced on a NovaSeq S2 (Illumina) at 20,000 read pairs per cell following recommended sequencing parameters. Data analysis was conducted with CellRanger (7.1), using 10X Genomics provided probeset reference and human GRCh38 reference transcriptome. Count matrices were produced with the CellRanger / ^ / / / / -function and used to create a Seurat object. Genes expressed in less than 10 cells were filtered out.

[0592] Human metastatic PDAC scRNA-seq (Raghavan, Cell 184, 6119-6137. e26 (2021)) data containing 23,042 cells was downloaded from the Broad Institute’s Single Cell Portal (SCP1644)and were subsequently used to create a Seurat object. Human primary PDAC scRNA-seq data (Peng et al., Cell Res 29, 725-738 (2019)) was downloaded from the Genome Sequencing Archive (accession: CRA001160; downloaded on April 27, 2020). A count matrix of 41,986 pre-processed cells was retrieved from these data and used to create a Seurat object. Genes expressed in less than 10 cells were filtered out.

[0593] For all three datasets (separately), data normalization and scaling, variable feature selection, cell clustering, and differential gene expression analysis was performed using Seurat (Butler et al., Nature Biotechnology 36, 411-420 (2018)). Data were normalized by total expression per cell and scaled using a factor of 10,000 and log transformed (natural scale). The top 2,000 variable genes were selected using Seurat’s default “vsf ’ method. The expression of these genes was then scaled and centered, and these genes were then used for downstream analysis. Principal component analysis (PCA) was performed for dimensionality reduction. The first 15 principal components were used for the construction of the k-nearest neighbor graph and the UMAP plot (metric parameter = Euclidean). Clusters were then assigned using the Louvain method with a resolution of 1. Clusters were re-annotated by cell type according to gene expression patterns as previously described (Peng et al., Cell Res 29, 725-738 (2019)). UMAP feature plots were generated using min. cutoff = "qOl", max.cutoff = "q99" as parameters.

[0594] Seurat’s AddModule Score function (control parameter = 8) was used to calculate gene module scores for all cells. Peptide source gene module (PSGM) scores were derived from immunopeptidomics data for each bulk tumor sample and each patient-derived organoid sample. For each individual sample, the gene symbol was retrieved (using the “entry jiame” column) for every peptide that was mapped to a single canonical Ensembl protein ID (accession number) (i.e., peptides that could be mapped to multiple canonical genes or any non-canonical ORFs were not included). A small number of peptides, whose parental gene symbol was not available in the Spectrum Mill output, were not included. Peptide source gene module (PSGM) scores were then computed with AddModule Score for the genes in each sample’s list that were present in the Seurat data frame (i.e., genes that were not detected in the scRNA-Seq datasets were not included).

[0595] Stromal and immune gene modules were comprised of the subset of stromal and immune genes marked as unexpressed in PDAC organoids (see bulk RNA-sequencing methods section describing the Estimation of STromal and Immune cells in MAlignant Tumors using Expressiondata (ESTIMATE) package). These subsets of unexpressed genes are derived from ESTIMATE’S stromal and immune modules.

[0596] After cluster annotation and gene module scoring, the Seurat object metadata table was exported for analysis using the XL-mHG package in Python (Wagner, “The XL-mHG Test For Enrichment: ATechnical Report,” arXiv: 1507.07905 [stat. OT] (2015)). The XL-mHG hypergeometric test was used to determine whether gene modules were significantly enriched within each cell type.

[0597] Normal tissue translation filtering pipeline

[0598] Ribo-Seq analysis. Human ribosome profiling data were downloaded from the Sequencing Read Archive (accession: GSE182377, GSE59820) and 49 untreated samples not marked as low quality were retained for further processing. Assayed tissues included primary cells or tissue samples from kidney, brain, fat, fibroblasts, human aortic endothelial cells, human coronary artery endothelial cells, hepatocytes, and vascular smooth muscle cells. A custom transcriptome annotation was constructed in line with Ouspenskaia et al. 2022 and was comprised of GENCODE v26lift37, transcripts annotated as status ‘unannotated’ from MiTranscriptome annotation (Iyer et al., Nat Genet 47, 199–208 (2015)), and a cohort of all IncRNAs reported by Cabili and colleagues (Cabili et al., Genes Dev 25, 1915-1927 (2011)). Sequencing reads were trimmed using Trimmomatic (Bolger et al., Bioinformatics 30, 2114-2120 (2014)) (version 0.36) with the following parameters: -phred33, ILLUMINACLIP: All_TruSeqForTrimmomatic.fa:2:30:10, MAXINFO:20:0.5, and MINLEN:20. Trimmed reads were then aligned against a contaminant database of tRNA, ribosomal RNA, and mitochondrial RNA using bowtie2 and the parameter, -L 20 (Langmead et al., Nat Methods 9, 357-359 (2012)). Unaligned sequenced reads were then aligned against the human genome with STAR, version 2.5.3 (— alignSJDBoverhangMin 1 — alignSJoverhangMin 51 — outFilterMismatchNmax 2 — alignEndsType EndToEnd — alignlntronMin 20 — alignlntronMax 100000 — outFilterType BySJout — outFilterMismatchNoverLmax 0.04 — twopassMode Basic — outSAMattributes MD NH) (Dobin et al., Bioinformatics 29, 15-21 (2013)). The resulting BAM files were then sorted and indexed and merged for each tissue type with samtools, version 1.10 (Danecek et al., Gigascience 10, gia008 (2021)). Ribosome profiling quality was confirmed using Ribo-TISH’s quality function (Zhang et al., Nature Communications 8: 1749 (2017)).Finally, translated ORFs were empirically defined using Ribo-TISH’s prediction function, the augmented nuORF transcriptome annotation described above, and the following parameters: —alt — seq — aaseq — minaalen 6 -blocks. All ORFs passing with a RiboPvalue < 0.05 and a FrameQvalue <0.05 were considered translated in healthy tissues.

[0599] Filtering process. Cancer-restricted HLAp were designated using custom R scripts assessing their translation in healthy tissues. Reference datasets included healthy tissue immunopeptidomics (n = 229, 28 tissues, excluding testis and thymus (Marcu et al., J Immunother Cancer 9, 2071 (2021)), healthy tissue Ribo-seq (n = 49, 8 tissues (Chothani et al., Mol Cell 82, 2885-2899. e8 (2022); and Loayza-Puch et al., Nature 530, 490-494 (2016)), and normal thymus immunopeptidomics (n = 5 (Marcu et al., J Immunother Cancer 9, 2071 (2021)). Input PDO-derived ncHLAp data and healthy tissue reference immunopeptidomics data were fdtered to only include peptides with definitive source genes / ORFs (i.e., peptides that were annotated as originating from only one ORF / accession number, which we refer to throughout the text as “uniquely mapping”). We did not include PDAC retained intron-derived ncHLAp (n = 10) in the translation-centric filtering process or follow-up analyses.

[0600] To determine cancer-restriction, each HLAp was searched for exact matches (i.e., the identical peptide) in immunopeptidomics data, and its parent ORF was compared to all ORFs detected in these datasets to identify potential translation. ncHLAp were classified as non-cancer restricted if: 1) the exact peptide sequence was found in any normal tissue or thymus, or 2) it originated from a nuORF overlapping (i.e., by genomic coordinates) with a translated nuORF in any healthy tissue or thymus, regardless of frame. In other words, for immunopeptidomics data, a nuORF was considered translated if any peptides encoded by it were detected, or if it has any genomic overlap with another nuORF that encodes detected peptides. For analysis of canonical HLAp, we used analogous filtering parameters, querying for the presence of the exact canonical HLAp and / or translation of the canonical ORF. Finally, for analysis of Ribo-Seq data, all HLAp were considered non-cancer-restricted if that sequence was translated (i.e., in-frame) in any healthy tissue. Cancer-restricted HLAp were defined by the absence of translation evidence in all analyzed datasets. Pipeline scripts are available on the GitHub link in the Code Availability section. In general, only HLAp that were mapped to a single canonical protein or nuORF (i.e., accession number) were considered when enumerating HLAp in the analyses depicted in figures.Quantification of tumor mutational burden

[0601] We defined tumor mutational burden as the number of all somatic mutations per megabase across coding regions of the genome. Coding regions were classified as the set of genomic regions in final_whole_exome_agilent_l.l_refseq_plus_3_boosters. targetintervals. bed (totaling 32,950,014 bases or 32.950014 Mb). To make a fair comparison between patient-derived organoids and bulk tumor samples, we considered only the subset of mutations that overlapped the Agilent file for calculating TMB. TMB was then calculated as the number of variants in the VCF (grep -v “#” | wc -1) divided by 32.950014 Mb.

[0602] Bulk RNA-Sequencing of PDOs with or without IFNy

[0603] PDOs were expanded as described above and treated with vehicle (PBS) or IFNy (100 ng / mL; Peprotech) for 36-40 hours. Total RNA was harvested using the Pure Link RNA Mini Kit (Thermo Fisher) according to manufacturer instructions using the Trizol Plus Whole Transcriptome protocol. cDNA was generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to manufacturer instructions. Samples were sequenced on a NovaSeq (Illumina) with 150nt PE reads at 20M reads per sample.

[0604] Analysis. Raw sequencing reads from the IFNY experiment were sorted and aligned to reference human genome GRCh37 using STAR, version 2.6.1 (Dobin et al., Bioinformatics 29, 15-21 (2013)), coupled with a transcriptome annotation comprised of GENCODE v26lift37, transcripts annotated as tstatus ‘unannotated’ from MiTranscriptome annotation (Iyer et al., Nat Genet 47, 199-208 (2015), and a cohort of all IncRNAs reported by Cabili and colleagues (Cabili et al., Genes Dev 25, 1915-1927 (2011)). Only entries with an annotated strand were retained. Isoform and gene expression were then quantified with RSEM, version 1.3.1 (Li and Dewey, BMC Bioinformatics 12, 323 (2011)). RSEM-based counts were then converted into a count matrix for all samples and Ensembl genes (i.e., excluding source genes underlying the MiTranscriptome and Cabili datasets). The raw count matrix was loaded into R (version 4.0.2) and incorporated along with sample information into a DESeq2 (version 1.28.1) object (Love et al., Genome Biol 15, 550 (2014)). Ensembl gene IDs were converted to HGNC symbols using the biomaRt package where possible. Only genes among a list of 17,165 canonical proteincoding genes (see next section) were considered.Differential gene expression was then assessed between treated and untreated samples using default DeSeq2 parameters. Genes significantly upregulated by IFNy treatment were then identified using a log-2 fold-change > 1 and an adjusted E- value < 0.05. Of the 380 genes in the IFNy-upregulated module, 3 did not have a HGNC symbol and were excluded from subsequent analysis: ENSG00000261884, ENSG00000284554, and ENSG00000244255. The remaining 377 genes were used as inputs for the Broad Institute’s Molecular Signatures Database to identify significantly overlapping gene modules. From this, five representative immune response modules were chosen to quantify overlap with immune response genes (BLANCO_MELO_BRONCHIAL_EPITHELIAL_CELLS_INFLUENZA_A_DEL_NS1_INFE CTION UP, OSMAN_BLOOD_CHAD63_KH_AGE_18_50YO_HIGH_DOSE_ SUBJECTS 24HR UP, ZAK PBMC MRKAD5 HIV 1 GAG POL NEF AGE _20_50YO_1DY_UP, GOBP IMMUNE RESPONSE, and HALLMARK INTERFERON GAMMA RESPONSE).

[0605] Data were prepared for principal components analysis (PCA) using DESeq2’s implementation of the variance stabilization transformation (VST) without blinding to the experimental design. After observing that PCI separated replicates by the presence or absence of IFNy, the top genes with the most positive loadings and the top genes with the most negative loadings in PCI were used to construct heatmaps.

[0606] Quantification of stromal gene signature programs in bulk tumors and organoids RNA-sequencing data were processed and analyzed as described for the IFNy experiment. Expected counts calculated by RSEM were extracted from gene-level output files; only features annotated with “ENSG” Ensembl codes were retained for downstream analysis of stromal signatures. We further filtered genes down to a list of 17,165 canonical protein-coding genes by intersecting this list with all Ensembl gene IDs designated as protein-coding and “canonical” in the nuORFdb annotation file. Raw counts for pancreatic cancer organoids and 49 bulk tumor samples from PancSeq (Aguirre et al., Cancer Discov 8, 1096-1111 (2018)) with more than 50 million RNA-Seq reads were then aggregated within a single matrix for downstream analysis. We then took a subset of only the genes expressed in bulk tumor samples, defined as genes with a mean log2(count +1) > 2 across bulk tumor samples.Raw counts were normalized using the estimateSizeFactors function from DESeq2 and then log2 -transformed (Love et al., Genome Biol 15, 550 (2014)). The resulting transformed expression matrix was used as an input for the Estimation of STromal and Immune cells in MAlignant Tumors using Expression data (ESTIMATE) package (Yoshihara et al., Nat Commun 4 (2013)), using default parameters outlined in the package vignette. The computed stromal, immune, and ESTIMATE scores were then extracted to compare differences between bulk and organoid samples. Different normalization methods were tested, including the variance stabilizing transformation from DESeq2, and expression data for the two cohorts was also normalized separately as well as together. All these methods robustly and uniformly indicated higher stromal and immune signatures in bulk tumor samples compared to organoids. We proceeded with the results obtained from log2 -transformed expression data (normalized for both cohorts together) for computing the results described above. For this analysis and RNA-Seq analyses described above, we excluded bulk tumor samples with a library size less than 50 million reads, including two samples with matched bulk tumor datasets (PANFR0151 and PANFR0181). The median tumor purity score across ten solid tumors was calculated from the RNASeqV2 supplementary table reported by Yoshihara et al. (Yoshihara et al., Nat Commun 4 (2013)).

[0607] Statistical methods

[0608] Except for XL-mHG, all statistical tests mentioned in this manuscript were conducted in R, PRISM, or SPSS. For each dataset, we evaluated whether they followed a Gaussian distribution using the Anderson-Darling test. Parametric or nonparametric tests were then selected based on this result. Results were considered significant if the computed test statistic was associated with aP-value < 0.05. *P < 0.05, **P < 0.01, ***p < 0.001, ****P < 0.0001.

[0609] RESULTS

[0610] Organotypic culture enables high-resolution characterization of the pancreatic cancer immunopeptidome

[0611] Pancreatic cancer has a low tumor cellularity

[0612] (Cancer Genome Atlas Research Network, Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell 32, 185–203.e13 (2017); Cao et al., Cell 184,5031 (2021); and Aguirre et al., Cancer Discov 8, 1096-1111 (2018)), with malignant cells often accounting for only 20-30% of the overall cellular mass in both primary pancreatic and metastatic PDAC lesions. While prior efforts to query the pancreatic cancer immunopeptidome have focused primarily on profiling bulk tumors (Bradley et al., Nat Commun 11, 5332 (2020)), it was hypothesized that organotypic culture would enrich for the PDAC malignant compartment and enable high-resolution proteogenomic characterization. PDOs were established from patients with histologically confirmed, mismatch repair-proficient PDAC undergoing surgical resection, metastatic biopsy, or paracentesis. PDOs and paired normal tissue samples were subjected to genomic (whole genome sequencing; WGS) and transcriptomic (RNA-sequencing) profiling to enable mutation calling and HLA typing. For a subset of patients, autologous bulk tumors were also subjected to genomic (whole exome sequencing; WES) and transcriptomic sequencing (Aguirre et al., Cancer Discov 8, 1096-1111 (2018)). In line with the hypothesis, a substantial decrease in stromal and immune gene signatures were observed in PDOs compared to matched bulk tumor specimens. Purification of the malignant compartment through organotypic culture also enabled improved sensitivity for mutation detection, in line with prior reports that non-malignant cells in the tumor microenvironment contribute sequencing reads that deflate variant allele frequency and reduce sensitivity for somatic variant detection (Stenzinger et al., Genes Chromosomes Cancer 58, 578 (2019)).

[0613] To investigate the PDAC epitope landscape without stromal contamination, twelve PDAC PDOs were selected for further proteogenomic characterization. Frequent alterations were observed in known PDAC driver genes (KRAS, TP53, CDKN2A, SMAD4) (Aguirre et al., Cancer Discov 8, 1096-1111 (2018)); however, as expected, the majority of other somatic mutations were patient-specific. HLA-I expression was next evaluated using flow cytometric analysis, which confirmed intact HLA-I surface expression and interferon-y (IFNy) responsivity on 92% (11 / 12) of PDOs. This brief treatment with low-dose IFNy could upregulate surface HLA-I without appreciable impacts on cell viability or gene expression beyond canonical IFNy response programs. The eleven HLA-I-expressing PDOs were expanded ex vivo and their immunopepti domes were profiled via liquid chromatography tandem mass spectrometry (LC-MS / MS). Over 91,000 unique PDAC HLAp were identified, with 8,000-18,500 HLAp per patient specimen, eclipsing prior efforts performed using bulk tumors (Bradley et al., NatCommun 11, 5332 (2020)). In line with the known binding properties of HLA-I ( / 5), the majority of peptides were 8-11 amino acids in length.

[0614] Beyond increasing depth, it was hypothesized that PDOs would generate immunopeptidome data more specific to cancer cells, since non-malignant cells within the PDAC TME could contribute HLAp when profiling bulk PDAC tumors (Jaeger et al., Nature 2022 607, 149–155 (2022)). To directly evaluate this hypothesis, HLA-I immunopeptidomics was performed on three unmatched primary PDAC resection specimens (bulk tumors) (Cao et al., Cell 184, 5031 (2021)). Empirically identified HLAp were mapped back to their canonical source genes. These genes were used to define a peptide source gene module (PSGM) for each PDAC bulk tumor or PDO (see Methods). To deconvolute the contribution of various cell types within the PDAC TME, single-cell RNA sequencing (scRNA-seq) was performed on primary PDAC bulk tumors and previously published scRNA-seq data was reprocessed from both primary and metastatic PDAC. It was found that PDO-derived PSGMs were specifically enriched in malignant pancreatic cancer cells from primary bulk tumors and metastatic tumor biopsies (P < 1x10-5; XL-minimum hypergeometric test; see Methods). In contrast, bulk PDAC-derived PSGMs exhibited less restricted expression and were enriched in non-malignant populations like myeloid cells and cancer-associated fibroblasts. These data demonstrate that non-malignant cells in the PDAC TME can contribute HLAp that may cloud interpretation of immunopeptidomic data, whereas organotypic enrichment enables unambiguous identification of HLAp presented by cancer cells.

[0615] Empiric identification of cryptic peptides in the pancreatic cancer immunopeptidome To investigate whether mutation-derived neoepitopes (mutHLAp) were detectable via immunopeptidomics, the proteomic search space was augmented for each PDO sample with patient-specific mutations, including phased germline variants to account for variants that cooccur on the same haplotype (see Methods). Each customized proteomic search space encompassed the full set of non-synonymous variants and were not based on HLA binding predictions. Despite a low-to-intermediate mutational burden, five endogenously expressed mutHLAp were empirically detected across four PDAC biospecimens. These neoepitopes included four peptides derived from missense mutations and one peptide generated from a frameshift mutation in the SMAD4 tumor suppressor gene. However, in line with prior studies inother tumor types (77), only a small percentage of nonsynonymous exonic mutations produced detectable mutHLAp. In fact, the majority (63%; 7 / 11) of profiled PDAC specimens exhibited no detectable mutHLAp. These findings reinforce the need to look beyond somatic mutations to identify novel classes of targetable antigens that may be present in a broader patient population.

[0616] To assess whether ncHLAp are presented in pancreatic cancer, the immunopeptidomic search space was augmented with a set of translated unannotated ORFs (nuORFs) identified through ribosome profiling (Ouspenskaia et al., Nat Biotechnol 40, 209-217 (2022)) and patientspecific retained introns identified with a custom detection pipeline (see Methods). Using stringent nuORF category-specific false discovery rate (FDR) filtering (see Methods), 1722 ncHLAp (1712 mapped uniquely to a single nuORF and 10 derived from retained introns) were detected across the eleven patient-derived organoid lines, with hundreds of ncHLAp (median = 257) found in each PDO. A correlation was observed between predicted (Bouwmeester et al., Nature Methods 2021 18, 1363-1369 (2021)) and observed retention times for ncHLAp that was comparable to the correlation observed for canonical HLAp. ncHLAp accounted for 2.2 - 10.1% (median 5.1%) of the overall immunopeptidome in each PDO. In line with observations in other tumor types (Laumont et al., Nat Commun 7, 10238 (2016)), an overrepresentation of peptides derived from translation of the 5’ UTR (5’ uORFs and 5’ overlap uORFs) in PDAC samples was observed, but also observed ncHLAp from translation of alternative reading frames (intORF), IncRNAs, retained introns, and 3’ UTRs. Peptides harboring mutations in nuORFs were not detected, although included in the customized proteomic search spaces.

[0617] It was also found that many ncHLAp were shared among HLA-matched PDOs, including more than 50 ncHLAp detected in two or more HLA-A*02:01 samples (of n = 7), HLA-B*44:02 samples (of n = 4), or HLA-C*05:01 samples (of n = 4). No appreciable differences were observed between canonical and non-canonical PDAC HLAp in terms of peptide length or sequence motif. In contrast with previous reports (Laumont et al., Nat Commun 7, 10238 (2016)), a slight preference was observed for ncHLAp arising from the C-terminal portion of parent ORFs. However, in line with recent studies (Laumont et al., Nat Commun 7, 10238 (2016); and Lozano-Rabella et al., Clinical Cancer Research 29, 2250-2265 (2023)), an enrichment for ncHLAp predicted to bind to HLA-A *03:01 and HLA-A *11:01 was observed.

[0618] To ensure that aberrant translation, leading to ncHLAp presentation, was not a consequence of organotypic culture conditions, ncHLAp was searched for in theimmunopepti domes of three bulk PDAC tumors. In agreement with data from PDAC organoids, 493 unique ncHLAp (each derived from a single nuORF) were detected across these three tumors, with ncHLAp overall accounting for 3.8% (2.9 - 4.1%) of the bulk PDAC immunopepti dome (FIG. 10A and FIG. 10B). As expected, ncHLAp identified from bulk tumors exhibited peptide lengths and nuORF biotype distributions highly similar to PDOs (FIG. IOC and FIG. 10D). It was also found that individual nuORFs were commonly translated in both PDOs and bulk tumors (FIG. 11), confirming that aberrant translation patterns are present in situ and conserved during organotypic culture.

[0619] Cancer-restriction of ncHLAp

[0620] While ncHLAp have previously been detected in other solid tumors, earlier efforts largely relied upon transcription-based analyses to assess cancer-specificity (Laumont et al, Sci Transl Med 10, eaau5516 (2018); Lozano-Rabella et al., Clinical Cancer Research 29, 2250-2265 (2023); and Ouspenskaia et al., Nat Biotechnol 40, 209-217 (2022)). While a portion of ncHLAp may be the product of cancer-restricted transcription, nuORFs are postulated to be regulated primarily at the level of translation (Dersh et al., Nat Rev Immunol 21, 116-128 (2021); and Sendoel et al., Nature 541,494-499 (2017)). However, it is currently unknown to what extent translation of these unannotated ORFs is truly cancer restricted. Therefore, it was opted to pursue a translation-centric approach to assess cancer restriction. Specifically, immunopepti domic data was leveraged from 28 distinct healthy tissues (n = 229 samples) (Marcu et al., J Immunother Cancer 9, 2071 (2021)), ribosome profiling (Ribo-seq) from eight different healthy tissues (n = 49 samples) (Chothani et al., Mol Cell 82, 2885-2899. e8 (2022)), and immunopeptidomics of healthy thymus (n = 5 samples) (Marcu et al., J Immunother Cancer 9, 2071 (2021)) to assess translation of uniquely mapping ncHLAp (n = 1712) across these healthy tissues (FIG. 1A and FIG. IB). Notably, widespread translation and / or HLA-I presentation of peptides derived from outside the annotated protein-coding genome across many healthy tissues was observed, suggesting that ncHLAp should not be universally considered tumor-specific. Indeed, almost 70% of ncHLAp identified in pancreatic cancer PDOs displayed evidence of translation in one or more healthy tissues, highlighting the need for a translation-centric approach to effectively nominate therapeutic targets.To rigorously identify cancer- restricted ncHLAp (i.e., absent or below the limit of detection in adult healthy tissues), all ncHLAp derived from parental ORFs with any evidence of translation and / or HLA presentation in even a single healthy tissue were filtered out (see Methods). After filtering, 517 (30.2%) cancer-restricted ncHLAp (CR ncHLAp) were identified that were detected in pancreatic cancer PDOs, but not in healthy tissues (FIG. IB). To stress-test the translation-centric pipeline and assess saturation, canonical HLAp detected from the same PDOs were subjected to an equivalent analysis. In line with the hypothesis that most canonical proteins should be expressed by at least one healthy tissue, 99.5% of canonical HLAp exhibited evidence of parental ORF translation in one or more healthy tissues (FIG. 1C). This suggests that one is at or nearing saturation in terms of the ability to detect evidence of translation in healthy tissues.

[0621] Intriguingly, a distinctive nuORF biotype distribution was observed (FIG. 2A) in CR ncHLAp compared to nonCR ncHLAp (FIG. 2B), suggesting that certain categories of nuORFs are preferentially translated in a cancer-restricted fashion. It is also noted that was a much smaller proportion of ncHLAp (10.2%), compared to canonical HLAp (74.6%), with evidence of translation in healthy thymus. In fact, of the 536 ncHLAp not identified in (non-thymus) healthy adult tissues, more than 96% (517 peptides) derive from nuORFs with no evidence of translation in healthy thymus tissues (FIG. 1).

[0622] In contrast to mutation-derived neoepitopes, which were patient-specific in the cohort, a substantial proportion (29%) of CR ncHLAp were shared by two or more PDAC patients. As HLA-A*02:01 was shared between seven PDAC patients profiled as part of this effort, recurrence for CR ncHLAp with predicted restriction to HLA-A *02:01 was examined and it was found that 48% were shared, with 21% being shared by more than half of HLA-A *02:01+PDAC patients (FIG. 2C). At the individual patient level, more detected CR ncHLAp compared to mutHLAp across all PDOs evaluated as observed (FIG. 3).

[0623] PD AC-restricted cryptic epitopes exhibit immunogenic potential

[0624] It is currently unknown how efficiently ncHLAp can be recognized by cytotoxic T cells (Leko and Rosenberg, Cancer Cell 38, 454-472 (2020)). Prior preclinical and clinical studies have suggested that the adaptive immune system frequently fails to recognize potential cancer-restricted epitopes (Stronen et al., Science (1979) 352, 1337-1341 (2016)), particularly in PDAC(Rojas et al., Nature 618, 144-150 (2023); Hegde et al., Cancer Cell 37, 289-307. e9 (2020); and Vonderheide, Cancer Cell 33, 563-569 (2018)) implying that the T cell receptor (TCR) repertoire of circulating memory T cells or tumor-infdtrating lymphocytes (TILs) may not be the most sensitive method for gauging potential immunogenicity of PDAC HLAp. To circumvent this limitation and directly assess the immunogenic potential of PDAC HLAp, a highly sensitive ex vivo platform was employed for priming and expansion of antigen-specific T cells using HLA-matched healthy donor peripheral blood mononuclear cells (PBMCs) (Rollins et al., Curr Protoc Immunol 129, e97 (2020)). Briefly, monocytes were isolated from PBMCs and differentiated into dendritic cells (MoDCs) using a defined cytokine cocktail. Peptide-loaded DCs were then used to prime and expand -8-10 independent lines of autologous CD8+CTLs per healthy donor (2.5 - 5xl06cells per line; 8-41 evaluable CTL lines per candidate peptide) (FIG.

[0625] 4). With this platform, the immunogenic potential of LC-MS / MS-detected ncHLAp (n = 56 total; n = 33 CR; n = 23 nonCR) was evaluated. All assayed peptides were validated by comparing LC-MS / MS spectra to synthetic peptides prior to immunogenicity assays (FIG. 12 and FIG. 13). In total, more than 1,100 CTL:peptide combinations were evaluated to investigate the immunogenic potential of these putative epitopes.

[0626] It was found that a large proportion (36.3%; 12 / 33) of CR ncHLAp were immunogenic, generating antigen-specific T cells that were at undetectable frequencies in precursor populations (FIG. 4 and FIG. 5A). Immunogenic CR ncHLAp derived from a variety of nuORF categories / biotypes, including intORFs (NU11, DDX47; NU49, TMED10; NU53, OAS2; NU92, ATL3; NU46, LDHB; NU47, CTDSPL2),, 5’ overlap uORFs (NU10, INSIG2 3’ dORFs (NU42, RB1CC1,- NU57, NBAS), pseudogenes (NU48, RPL32P3), lincRNAs (NU84, CCATI), and ncRNA Processed Transcripts (NU85, DNAJC11). Intriguingly, positive immunogenicity was observed for two nonCR ncHLAp (NU7, linc-SEPP1-3; NU104, RAB24 5' uORF) (FIG. 4 and FIG. 5); however, CR ncHLAp exhibited a substantially higher immunogenicity rate compared to nonCR ncHLAp (36.3% vs 8.7%) (FIG. 4 and FIG. 5A). Notably, the ORFs encoding the only two immunogenic nonCR ncHLAp (NU7, NU104) were not detected in healthy thymus, whereas no ncHLAp detected in the thymus exhibited immunogenicity, reinforcing the potential predictive power of this translation-centric approach (FIG. 5A).

[0627] To benchmark these results, parallel interrogation of mutHLAp immunogenicity was performed using the same platform. Both LC-MS / MS-detected mutHLAp (n = 4) andcomputationally predicted mutHLAp (n = 22) were evaluated. Computationally predicted mutHLAp were selected based on predicted restriction by HLA-A*02:01 and were detected at the DNA level in PDAC biospecimens (PDOs and / or bulk tumors) (29). To ensure a representative cohort, computationally-predicted mutHLAp were varied across a range of metrics previously reported to predict immunogenicity (Wells et al., Cell 183, 818-834.el3 (2020); Balachandran et al., Nature 551, 512-516 (2017); and Gartner et al., Nature Cancer 2021 2:52, 563-574 (2021)), including mutation class (missense / frameshift / inframe-indel), predicted HLA-I binding affinity, and agretopicity. In line with previous studies (Stronen et al., Science (1979) 352, 1337-1341 (2016); and Rojas et al., Nature 618, 144-150 (2023)), it was observed that a substantial proportion (38.5%; 10 / 26) of mutHLAp demonstrated immunogenicity. Notably, of the four assayed empirically identified (LC-MS / MS-detected) mutHLAp, only one encoded by a frameshift mutation in SMAD4, a common PDAC driver gene, displayed immunogenicity.

[0628] Collectively, the proportion and magnitude of CR ncHLAp immunogenicity was on par with a more traditional class of neoantigens (mutHLAp). This finding suggests that CR ncHLAp may be readily amenable to immunotherapeutic modalities like TCR-T cell therapy, vaccines, or TCR-mimetics. However, this therapeutic potential hinges upon the ability of ncHLAp-reactive TCRs to recognize endogenous levels of CR ncHLAp presented by malignant cells.

[0629] ncHLAp-specific T cells can recognize and kill patient-derived pancreatic cancer organoids expressing endogenous levels of cryptic antigens

[0630] To identify candidate CR ncHLAp-reactive TCRs, the 10X Genomics Barcode Enabled Antigen Mapping (BEAM-T) workflow was adapted for the ex vivo T cell priming and expansion platform. Briefly, we generated custom, DNA-barcoded peptide: HLAmultimers paired with 5’ gene expression and V(D)J sequencing to identify full-length TCRaP sequences and simultaneously deconvolute antigen-specificity at single-cell resolution. 17 candidate CR ncHLAp-reactive TCRαβ clonotypes were identified, spanning multiple nuORF biotypes (FIG.

[0631] 6A). Functional evaluation of candidate TCRs were prioritized based on antigen-specificity scores and clonotype abundance (see Methods).

[0632] CRISPR / Cas9 ribonucleoproteins (RNPs) complexed with guide RNAs targeting TRAC and TRBC1 / TRBC2 were used to simultaneously knockout (KO) the endogenous TCRaP from healthy donor CD8+T cells with >99% efficiency. Lentiviruses encoding gRNA-resistantcandidate CR ncHLAp-reactive TCRs (FIG. 14) were then used to redirect antigen-specificity. Successful TCR redirection (TCR-T) was confirmed using flow cytometry with peptide: HLA multimers. Engineered TCR-T cells were sorted and expanded using a rapid expansion protocol (REP) (Anderson et al., J Immunother Cancer 10, e003959 (2022)) prior to functional evaluation. Binding of each reconstituted ncHLAp TCR in redirected TCR-T cells to its respective soluble peptide-HLA complex demonstrated that 7 / 7 (100%) of tested TCRs recognized the cognate antigen predicted from our BEAM-T pipeline.

[0633] The functional avidity of the HLA-A*02:01-restricted (n = 6) ncHLAp-directed TCRs was next evaluated through co-culture with luciferase-expressing, TAP -deficient T2 cells loaded with cognate peptides. Three candidate ncHLAp-reactive TCRs (TCR004 (NU42), TCR010 (NU47), TCR013 (NU10)) exhibited extremely low functional avidities (EC50105– 108pg / mL) (FIG. 6B). In contrast, TCR001 and TCR012, both reactive to NU11, exhibited high TCR functional avidities (EC503.7 and 4.3 pg / mL, respectively), and a third ncHLAp-reactive TCR, TCR005 (NU57; NRAS 3’ dORF), exhibited an intermediate functional avidity (EC501.2 x103pg / mL) (FIG. 6B).

[0634] It was next assessed whether CR ncHLAp-directed TCRs were truly capable of recognizing endogenous levels of their cognate antigens presented on the surface of PDAC organoids (FIG. 7A). P0071 patient-derived organoids (HLA-A*02:01⁺) were first engineered, which harbor NU11 andNU57 as identified via immunopeptidomics, to stably express eGFP and firefly luciferase (P0071_eGFP-ffLuc) and enable quantitative assessment of live PDAC organoids. As expected, the three low-avidity ncHLAp-reactive TCRs were unable to recognize endogenous levels of cognate antigens (FIG. 15A-B). However, co-culture of PDAC PDOs with each of the three high-avidity, ncHLAp-directed TCR-T cells demonstrated robust antigenspecific recognition, evidenced by effector cytokine secretion and upregulation of surface CD137 / 4-1BB (FIG. 7 and FIG. 15C). High-avidity ncHLAp-directed T cells were also able to effectively kill PDAC PDOs, demonstrating cytotoxicity even at low effectortarget (E: T) ratios, which could be inhibited by sterically blocking HLA-I (FIG. 8A). Robust killing across both NU11 -reactive TCRs (TCR012 and TCR001) was observed, as well as the NU57-reactive TCR (TCR005) (FIG. 8A).

[0635] TCR001, the most potent ncHLAp-directed TCR in the ex vivo co-culture system, was selected for deeper characterization. Alanine scanning (Cameron et al., Sci Transl Med 5,197ral03 (2013)) was performed to dissect the residues within NU11 critical forTCROOl recognition (FIG. 8B). Only substitution at position 6 of NU11 was even mildly tolerated, and even substitution at P6 shifted the functional avidity by >20,000 fold (FIG. 8B). All other substitutions within NU11 effectively abrogated TCR001 recognition. With K-L-F-L-W-X-Y-K-V as a cross-reactive motif, ScanProSite (de Castro et al., Nucleic Acids Res 34, W362-W365 (2006)) was used to search for all possible human peptides with potential cross-reactivity. This did not reveal any predicted cross-reactive peptides, reinforcing the notion that TCR001 is exquisitely specific to the NU11 peptide sequence.

[0636] Lastly, the ability of ncHLAp-directed TCR-T to delay PDAC tumor growth in vivo was investigated. To assess this, PDAC PDOs were first subcutaneously transplanted into athymic mice. It was found that even a single administration of 1 x 107TCR001-T could significantly delay tumor progression (p = 0.022) compared to adoptive transfer of an equivalent dose of TCRin-T (irrelevant TCR), whereas TCR012-T and TCR005-T could not (p = 0.195 and p = 0.26, respectively). However, in all cases, tumors eventually progressed (FIG. 16 and FIG. 17). It was hypothesized that this might represent a defect in T cell persistence and / or T cell function (Baulu et al., Sci Adv 9, eadf3700 (2023)) based on the TCR-T manufacturing process or choice of recipient mouse strain (Chen et al., Front Immunol 13, 1007579 (2022)), and thus this in vivo study was repeated using NOD-scid IL2Rgammanull(NSG) mice with two administrations of TCR001-T or TCRirr-T (FIG. 9 and FIG. 18). Here, a highly significant delay in tumor progression was observed in animals that received adoptive transfer of ncHLAp-specific TCR-redirected T cells (p < 0.001). This in vivo result establishes a key proof-of-concept for targeting cryptic antigens in pancreatic cancer.

[0637] CONCLUSIONS

[0638] Through high-resolution immunopeptidomics and a sensitive ex vivo T cell priming platform, two major gaps in the study of antigenic peptides in cancer were addressed, namely the cancer restriction and immunogenicity of ncHLAp. Using patient-derived organoids, the deepest characterization of the PDAC immunopeptidome to date was performed and a previously unrecognized role for cryptic antigen presentation in this disease was discovered. These cryptic peptides were frequently cancer-restricted, shared across patients, and immunogenic. The latter property enabled us to furnish and characterize ncHLAp-specific TCRs, primed and expandedfrom healthy donor PBMCs. Using barcode-enabled antigen mapping of TCRs (BEAM-T), a suite of TCRαβ clonotypes targeting ncHLAp across a range of nuORF biotypes were identified. It was demonstrated that multiple ncHLAp-directed TCR-T cells were capable of recognizing and killing patient-derived PDAC organoids expressing endogenous levels of CR ncHLAp both ex vivo and in vivo.

[0639] PDAC, like many solid tumors, harbors a low-to-intermediate mutational burden that results in a limited pool of mutation-derived neoantigens. While recent clinical efforts have demonstrated an ability to elicit neoantigen-specific T cell responses in a subset of PDAC patients (Rojas et al., Nature 618, 144-150 (2023)), it is currently unknown to what extent tumor cells directly present these peptides in the context of HLA-I. The findings presented herein align with previous studies detecting few mutation-derived neoepitopes in other solid tumors (Bassani-Sternberg et al., Nat Commun 7, 13404 (2016)) and further underscore the need for novel antigenic targets. Cryptic antigens, stemming from a variety of sources including translation of alternative reading frames (intORFs), long non-coding RNAs (IncRNAs), and untranslated regions (5’ and 3’ UTRs), provide such targets.

[0640] Leveraging immunopeptidomics and ribosome profiling data from healthy tissues, a stringent filtering process was established to identify a subset of ncHLAp that lacked evidence of translation across profiled healthy tissues. With this approach, over 500 cancer-restricted cryptic epitopes were identified. Intriguingly, many ncHLAp (-1,000 detected peptides) displayed evidence of parental ORF translation in one or more healthy tissue.

[0641] Many questions remain regarding the molecular mechanisms that lead to aberrant nuORF translation and subsequent ncHLAp presentation in PDAC. The high degree of recurrence of CR ncHLAp across individual patients and lack of detection in normal tissues suggests that nuORFs may arise as a consequence of oncogenic transformation, rather than simply resulting from stochastic errors in translation. As oncogenic RAS has been previously implicated in altering translational initiation (Dong et al., SciAdv 7, 6927-6944 (2021)), it is possible that this hallmark oncogenic event may lead to alterations in the PDAC immunopeptidome.

[0642] This example has published as Zackery A Ely and Zachary J Kulstad et al., “Pancreatic cancer-restricted cryptic antigens are targets for T cell recognition,” Science. 2025 May 8;388(6747):eadk3487. doi: 10.1126 / science.adk3487. Epub 2025 May 8. This publication, including all supplementary materials referenced in the publication (such as supplementarytables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data), is herein incorporated by reference in its entirety.

[0643] Example 2

[0644] T cell receptors

[0645] As described in Example 1, seventeen candidate CR ncHLAp-reactive TCRαβ clonotypes were identified, spanning multiple nuORF biotypes (FIG. 6A). The amino acid sequences of the variable regions of both the a and P chains of these T cell receptors as well as the amino acid sequences of the CDR1, CDR2, and CDR 3 of the a and P chains of these T cell receptors are shown in Table 4.

[0646] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0647] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0648] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.Sequence Listing Free Text

[0649] SEQ ID NOs: 1-517 CR ncHLAp peptide sequences

[0650] SEQ ID NOs: 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 550 T cell receptor alpha chain variable region amino acid sequences SEQ ID Nos: 519. 521, 523. 525, 527. 529, 531. 533, 535. 537, 539. 541, 543. 545, 547, 549, 551

[0651] T cell receptor beta chain variable region amino acid sequences SEQ ID NOs: 552, 554, 556, 572, 559, 561, 563, 564, 563, 566, 556, 569, 564, 561, 572, 561, 574

[0652] T cell receptor alpha chain CDR1 amino acid sequences SEQ ID NOs: 575, 577, 579, 581, 583, 585, 587, 588, 587, 590, 579, 593, 594, 585, 597, 585, 599

[0653] T cell receptor alpha chain CDR2 amino acid sequences SEQ ID NOs: 600, 602, 604, 606, 608, 610. 612, 614. 616, 618. 620, 622. 624, 626. 628, 610. 631

[0654] T cell receptor alpha chain CDR3 amino acid sequences SEQ ID NOs: 553, 555, 557, 558, 560, 562, 557, 565, 555, 567, 568, 565, 570, 571, 573, 562, 567

[0655] T cell receptor beta chain CDR1 amino acid sequences SEQ ID NOs: 576, 578, 580, 582, 584, 586, 580, 589, 578, 591, 592, 589, 595, 596, 598, 586, 591

[0656] T cell receptor beta chain CDR2 amino acid sequences SEQ ID NOs: 601, 603. 605, 607, 609, 611, 613, 615. 617, 619. 621, 623. 625, 627. 629, 630. 632

[0657] T cell receptor beta chain CDR3 amino acid sequences

Claims

WHAT TS CLAIMED IS:

1. A composition comprising two or more isolated non-canonical HLA-I bound peptides (ncHLAp), each isolated ncHLAp comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

2. The composition of claim 1, comprising any two, any ten, any twenty-five, any fifty, or any hundred isolated ncHLAp selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

3. The composition of claim 2, further comprising at least one more isolated ncHLAp selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

4. The composition of any one of claims 1 to 3, further comprising one or more adjuvants.

5. An immunogenic composition comprising at least one isolated non-canonical HLA-I bound peptide (ncHLAp) and one or more adjuvants, said isolated ncHLAp comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

6. The composition of any one of claims 1 to 4, wherein each said isolated ncHLAp is about 7 to about 12 amino acids in length.

7. The composition of any one of claims 1 to 6, wherein each said isolated ncHLAp consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

8. The composition of any one of claims 1 to 7, comprising about 2 to about 10, about 10 to about 20, about 21 to about 30, about 31 to about 40 or about 41 to about 50 isolated ncHLAp.

9. An expression vector comprising a nucleic acid sequence coding for an isolated non-canonical HLA-I bound peptide (ncHLAp), said isolated ncHLAp comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

10. The expression vector of claim 9, wherein said isolated ncHLAp is about 7 to about 12 amino acids in length.

11. The expression vector of any one of claims 9 or 10, wherein said isolated ncHLAp consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

12. A host cell comprising the expression vector of any one of claims 9 to 11.

13. The cell of claim 12, wherein the host cell is an antigen presenting cell.

14. The cell of claim 13, wherein the antigen presenting cell is a dendritic cell.

15. A T cell receptor (TCR) or functional fragment thereof comprising:a) a T cell receptor alpha chain (TCA) comprising a complementarity determining region (CDR) 1 comprising the sequence of SEQ ID NO:552, a CDR2 comprising the sequence of SEQ ID NO:575, and a CDR3 comprising the sequence of SEQ ID NO:600 and a T cell receptor beta chain (TCB) comprising a CDR1 comprising the sequence of SEQ ID NO:553, a CDR2 comprising the sequence of SEQ ID NO:576, and CDR3 comprising the sequence of SEQ ID NO:601; orb) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:554, a CDR2 comprising the sequence of SEQ ID NO:577, and a CDR3 comprising the sequence of SEQ ID NO:602, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:555, a CDR2 comprising the sequence of SEQ ID NO:578, and a CDR3 comprising the sequence of SEQ ID NO: 603; orc) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:556, a CDR2 comprising the sequence of SEQ ID NO:579, and a CDR3 comprising the sequence of SEQ ID NO:604, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:557, a CDR2 comprising the sequence of SEQ ID NO: 580, and a CDR3 comprising the sequence of SEQ ID NO:605;d) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:572, a CDR2 comprising the sequence of SEQ ID NO:581, and a CDR3 comprising the sequence of SEQ ID NO:606, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:558, a CDR2 comprising the sequence of SEQ ID NO:582, and a CDR3 comprising the sequence of SEQ ID NO:607; ore) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:559, a CDR2 comprising the sequence of SEQ ID NO:583, and a CDR3 comprising the sequence of SEQ ID NO:608, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:560, a CDR2 comprising the sequence of SEQ ID NO:584, and a CDR3 comprising the sequence of SEQ ID NO:609; orf) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:610, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:562, a CDR2 comprising the sequence of SEQ ID NO:586, and a CDR3 comprising the sequence of SEQ ID NO:611; org) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:563, a CDR2 comprising the sequence of SEQ ID NO:587, and a CDR3 comprising the sequence of SEQ ID NO:612, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:557, a CDR2 comprising the sequence of SEQ ID NO: 580, and a CDR3 comprising the sequence of SEQ ID NO:613; orh) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:564, a CDR2 comprising the sequence of SEQ ID NO:588, and a CDR3 comprising the sequence of SEQ ID NO:614, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:565, a CDR2 comprising the sequence of SEQ ID NO: 589, and a CDR3 comprising the sequence of SEQ ID NO:615; ori) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:563, a CDR2 comprising the sequence of SEQ ID NO:587, and a CDR3 comprising the sequence of SEQ ID NO:616, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:555, a CDR2 comprising the sequence of SEQ ID NO: 578, and a CDR3 comprising the sequence of SEQ ID NO:617; orj) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:566, a CDR2 comprising the sequence of SEQ ID NO:590, and a CDR3 comprising the sequence of SEQ ID NO:618, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:567, a CDR2 comprising the sequence of SEQ ID NO:591, and a CDR3 comprising the sequence of SEQ ID NO:619; ork) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:556, a CDR2 comprising the sequence of SEQ ID NO:579, and a CDR3 comprising the sequence of SEQ ID NO:620, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:568, a CDR2 comprising the sequence of SEQ ID NO:592, and a CDR3 comprising the sequence of SEQ ID NO:621; orl) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:569, a CDR2 comprising the sequence of SEQ ID NO:593, and a CDR3 comprising the sequence of SEQ ID NO:622, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:565, a CDR2 comprising the sequence of SEQ ID NO:589, and a CDR3 comprising the sequence of SEQ ID NO: 623; orm) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO: 564, a CDR2 comprising the sequence of SEQ ID NO:594, and a CDR3 comprising the sequence of SEQ ID NO:624, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:570, a CDR2 comprising the sequence of SEQ ID NO:595, and a CDR3 comprising the sequence of SEQ ID NO:625; orn) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:626, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:571, a CDR2 comprising the sequence of SEQ ID NO:596, and a CDR3 comprising the sequence of SEQ ID NO:627; oro) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:572, a CDR2 comprising the sequence of SEQ ID NO:597, and a CDR3 comprising the sequence of SEQ ID NO:628, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:573, a CDR2 comprising the sequence of SEQ ID NO:598, and a CDR3 comprising the sequence of SEQ ID NO:629; orp) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:561, a CDR2 comprising the sequence of SEQ ID NO:585, and a CDR3 comprising the sequence of SEQ ID NO:610, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:562, a CDR2 comprising the sequence of SEQ ID NO:586, and a CDR3 comprising the sequence of SEQ ID NO:630; orq) a TCA comprising a CDR1 comprising the sequence of SEQ ID NO:574, a CDR2 comprising the sequence of SEQ ID NO:599, and a CDR3 comprising the sequence of SEQ ID NO:631, and a TCB comprising a CDR1 comprising the sequence of SEQ ID NO:567, a CDR2 comprising the sequence of SEQ ID NO:591, and a CDR3 comprising the sequence of SEQ ID NO:632.

16. The TCR or functional fragment thereof of claim 15 wherein:a) the TCR of a) is specific for an isolated non-canonical HLA-I bound peptide (ncHLAp) having SEQ ID NO:9; orb) the TCR of b) is specific for an isolated ncHLAp having SEQ ID NO:239; or c) the TCR of c) is specific for an isolated ncHLAp having SEQ ID NO:84; or d) the TCR of d) is specific for an isolated ncHLAp having SEQ ID NO:84; or e) the TCR of e) is specific for an isolated ncHLAp having SEQ ID NO:93; or f) the TCR of f) is specific for an isolated ncHLAp having SEQ ID NO:509; or g) the TCR of g) is specific for an isolated ncHLAp having SEQ ID NO:84; or h) the TCR of h) is specific for an isolated ncHLAp having SEQ ID NO:509; or i) the TCR of i) is specific for an isolated ncHLAp having SEQ ID NO:239; or j) the TCR of j) is specific for an isolated ncHLAp having SEQ ID NO:449; or k) the TCR of k) is specific for an isolated ncHLAp having SEQ ID NO:9; or l) the TCR of l) is specific for an isolated ncHLAp having SEQ ID NO:9; or m) the TCR of m) is specific for an isolated ncHLAp having SEQ ID NO:202; or n) the TCR of n) is specific for an isolated ncHLAp having SEQ ID NO:84; or o) the TCR of o) is specific for an isolated ncHLAp having SEQ ID NO:509; or p) the TCR of p) is specific for an isolated ncHLAp having SEQ ID NO:509; or q) the TCR of q) is specific for an isolated ncHLAp having SEQ ID NO:239.

17. The TCR or functional fragment thereof of claim 15 or 16 comprising a soluble T cell receptor (sTCR).

18. The TCR or functional fragment thereof of claim 15 or 16 comprising a single-chain T cell receptor (scTCR).

19. Abispecific T cell engager (BiTE) comprising a sTCR of claim 17 or a scTCR of claim 18 and a second T cell-specific binding molecule.

20. The BiTE of claim 19, wherein the T cell-specific binding molecule comprises an anti-CD3 immunoglobulin or scFv thereof.

21. A nucleic acid sequence coding for the variable region of the alpha chain and / or the beta chain of the TCR of claim 15 or 16 or a TCR or functional fragment thereof of any one of claims 17 to 20.

22. A vector comprising a nucleic acid sequence of claim 21.

23. A cultured cell comprising the TCR or functional fragment thereof of any one of claims 15 to 20, the nucleotide sequence of claim 21, or the vector of claim 22.

24. A T cell comprising the TCR or functional fragment thereof of any one of claims 15 to 20, the nucleotide sequence of claim 21, or the vector of claim 22.

25. An in vitro method for producing antigen-specific activated T lymphocytes, the method comprising contacting T cells in vitro with antigen presenting cells loaded with one or more isolated non-canonical HLA-I bound peptides (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517 for a period of time sufficient to activate said T cells in an antigen-specific manner to the one or more ncHLAp.

26. An in vitro method for producing antigen-specific activated T lymphocytes, the method comprising contacting T cells in vitro with antigen presenting cells loaded with a composition of any one of claims 1 to 8 for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

27. An in vivo method for producing antigen specific activated T lymphocytes in a subject, the method comprising administering to the subject one or more isolated non-canonical HLA-I bound peptides (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

28. An in vivo method for producing antigen specific activated T lymphocytes in a subject, the method comprising administering to the subject a composition of any one of claims 1 to 8.

29. An antigen-specific activated T lymphocyte produced by the method of any one of claims 25 to 28.

30. The antigen-specific activated T lymphocyte of claim 29, wherein the antigen-specific activated T lymphocyte selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517.

31. A method of producing an antigen-specific T lymphocytes, the method comprising transforming a T lymphocyte with a nucleic acid of claim 21 or a vector of claim 22.

32. A method of treating a subject having a cancer, the method comprising administering to the human subject:a) one or more peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517;b) a composition of any one of claims 1 to 8;c) a nucleic acid sequence coding for an isolated non-canonical HLA-I bound peptide (ncHLAp) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 517;d) an expression vector of any one of claims 9 to 11;e) a host cell of any one of claims 12 to 14;f) a TCR or functional fragment thereof of any one of claims 15 to 20;g) a nucleic acid of claims 21;h) a vector of claim 22;i) a cultured cell of claim 23;j) a T cell of claim 24; and / ork) an antigen-specific activated T lymphocyte of claim 29 or 30.

33. The method of claim 32, wherein the subject is human.

34. The method of claim 32 or 33, wherein the cancer is selected from glioblastoma, melanoma, ovarian cancer, meningioma, lung cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, B-acute lymphoblastic leukemia, lymphoma, T-acute lymphoblastic leukemia, chronic lymphocytic leukemia, colon carcinoma, breast cancer and neuroblastoma.

35. The method of any one of claims 32 to 34, wherein the cancer is pancreatic cancer.

36. The method of any one of claims 32 to 35, wherein the cancer expresses a peptide comprising an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID: 517 or a combination thereof.

37. The method of any one of claims 32 to 36, further comprising administering chemotherapy, immunotherapy, checkpoint inhibitor, radiation or surgery to the subject.

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