DNTT 250-258 off-target peptides and uses thereof

Abstract

The present disclosure relates to compositions that involve isolated peptides, e.g., DNTT250-258 off-target peptides, and the use of such compositions in methods for assessing off-target effects of antigen-recognition molecules that target a DNTT250-258 peptide, as well as for selecting antigen-recognition molecules and enriching samples for antigen-recognition molecules that specifically bind the DNTT250-258 peptide.

Description

Attorney Docket No.250298.000720 DNTT250-258OFF-TARGET PEPTIDES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Application No. 63 / 584,577, filed September 22, 2023, the disclosure of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 6, 2024, is named 250298_000720_SL.xml and is 117,776 bytes in size. FIELD

[0003] The present disclosure relates to compositions that involve isolated peptides, e.g., DNTT250-258off-target peptides, and the use of such compositions in methods for assessing off- target effects of antigen-recognition molecules that target a DNTT250-258 peptide, as well as for selecting antigen-recognition molecules and enriching samples for antigen-recognition molecules that specifically bind the DNTT250-258peptide. BACKGROUND

[0004] Antigen-recognition molecules, such as T-cell receptors (TCRs) and antibodies, are capable of identifying antigens, which include agents recognized by the immune system of a host as defined herein. Antigen-recognition molecules can help the immune system neutralize an antigen and / or initiate an antigen-specific immune response by binding to an antigenic peptide presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-peptide complex) on the surface of an antigen-presenting cell.

[0005] The MHC-peptide complex is presented on the surface of the antigen-presenting cell as a result of a cellular process in which an MHC gene in the antigen-presenting cell encodes an MHC molecule; the MHC molecule subsequently binds to the antigenic peptide thereby creating the MHC-peptide complex; and the resulting MHC-peptide complex is positioned on the cellAttorney Docket No.250298.000720 surface so that a portion of the peptide is presented for binding with an antigen-recognition molecule. Each peptide is made up of a short chain of amino acids, and some of the amino acids of a peptide in an MHC-peptide complex are bound to the MHC molecule while at least some of the remaining amino acids are presented as available for binding with an antigen-recognition molecule. Antigen-recognition molecules are able to bind to a peptide of an MHC-peptide complex on the surface an antigen-presenting cell to help the immune system neutralize the antigen and / or initiate an antigen-specific immune response. This process is an important component of the immune system’s ability to recognize and mount a response against foreign agents.

[0006] This cellular process is also carried out on peptides native to the body. Generally, however, antigen-recognition molecules are able to distinguish between native peptides and non- native peptides presented on antigen-presenting cells so that normal cells are not attacked by the immune system.

[0007] Research is underway with a goal of isolating (e.g., screening / selecting) and / or engineering antigen-recognition molecules to target cells that would otherwise not be targeted through the above-described mechanism. For instance, cancer cells are native cells that are typically not effectively suppressed by the immune system, and research has shown that it can be possible to use TCRs, antibodies, and other antigen-recognition molecules to target the cancer- specific MHC-peptide complexes on cancer cells. While targeted treatments with antigen- recognition molecules may be effective to neutralize intended target cells, side effects of the treatment may be severe if antigen-recognition molecules bind off-target native cells in addition to the intended target cells. Side effects are often identified during clinical trials, which can result in patient death, other adverse effects on patients, and expenditure of time and resources in research and development. Accordingly, there is a need in the art for methods and systems that allow for accurate and efficient prediction of off-targets to the target peptide of interest which helps evaluate the risk associated with the target peptide at the target selection step as well as help in screening the most specific antigen-recognition molecules. SUMMARY

[0008] In one aspect, provided herein is an isolated peptide comprising an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is 8-12 amino acids in length.Attorney Docket No.250298.000720

[0009] In some embodiments, the isolated peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0010] In some embodiments, the isolated peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0011] In some embodiments, the isolated peptide comprises one or more non-proteogenic amino acids, one or more non-naturally occurring amino acids, one or more non-native peptide bonds, or any combination thereof, optionally wherein the isolated peptide comprises one or more reverse peptide bonds, one or more D-isomers of amino acids, one or more chemical modifications, or any combination thereof.

[0012] In some embodiments, the isolated peptide is produced by expression in a heterologous host cell.

[0013] In one embodiment, the isolated peptide is produced synthetically.

[0014] In one aspect, provided herein is a fusion protein comprising one or more peptides as described herein, or fragments or derivatives thereof, fused to one or more heterologous molecules.

[0015] In some embodiments, the one or more heterologous molecules comprise an MHC molecule, or a fragment or derivative thereof.

[0016] In some embodiments, the MHC molecule is a class I MHC molecule.

[0017] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0018] In some embodiments, the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

[0019] In one embodiment, the HLA-A molecule is an HLA-A*02:01 molecule.

[0020] In one aspect, provided herein is a conjugate comprising one or more peptides as described herein, or fragments or derivatives thereof, conjugated to one or more heterologous molecules.

[0021] In some embodiments, the one or more heterologous molecules comprise an MHC molecule, or a fragment or derivative thereof.

[0022] In some embodiments, the MHC molecule is a class I MHC molecule.

[0023] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.Attorney Docket No.250298.000720

[0024] In some embodiments, the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

[0025] In one embodiment, the HLA-A molecule is an HLA-A*02:01 molecule.

[0026] In some embodiments, the one or more peptides or fragments or derivatives thereof are conjugated to a particle or a solid support.

[0027] In one aspect, provided herein is an oligomeric complex comprising two or more isolated peptides as described herein or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0028] In another aspect, provided herein is a non-covalent complex comprising (i) the isolated peptide as described herein or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, and (ii) an MHC molecule, or a fragment or derivative thereof.

[0029] In some embodiments, the MHC molecule is a class I MHC molecule.

[0030] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0031] In some embodiments, the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

[0032] In one embodiment, the HLA-A molecule is an HLA-A*02:01 molecule.

[0033] In one aspect, provided herein is a composition comprising (i) one or more isolated peptides as described herein, one or more fusion proteins as described herein, one or more conjugates as described herein, one or more oligomeric complexes as described herein, or one or more non-covalent complexes as described herein, or any combination thereof; and (ii) a carrier or excipient.

[0034] In another aspect, provided herein is an isolated cell comprising the one or more fusion proteins as described herein, one or more conjugates as described herein, one or more oligomeric complexes as described herein, or one or more non-covalent complexes as described herein, or any combination thereof.

[0035] In some embodiments, the isolated cell is an immune cell.

[0036] In one embodiment, the isolated cell is an antigen-presenting cell (APC).

[0037] In one aspect, provided herein is an isolated polynucleotide comprising a nucleotide sequence encoding one or more isolated peptides as described herein or one or more fusion proteins as described herein.Attorney Docket No.250298.000720

[0038] In some embodiments, the nucleotide sequence is operably linked to a promoter.

[0039] In some embodiments, the isolated polynucleotide comprises DNA.

[0040] In some embodiments, the isolated polynucleotide comprises RNA.

[0041] In one embodiment, the RNA is mRNA.

[0042] In one embodiment, the RNA is self-replicating RNA.

[0043] In one aspect, provided herein is a vector comprising the isolated polynucleotide as described herein.

[0044] In some embodiments, the vector is an expression vector.

[0045] In one embodiment, the vector is a viral vector.

[0046] In one aspect, provided herein is a host cell comprising the isolated polynucleotide as described herein or the vector as described herein.

[0047] In one embodiment, the host cell is a prokaryotic cell.

[0048] In one embodiment, the host cell is a eukaryotic cell.

[0049] In one embodiment, the host cell is an immune cell.

[0050] In one embodiment, the host cell is an antigen-presenting cell (APC).

[0051] In one aspect, provided herein is a composition comprising (i) the isolated polynucleotide as described herein or the vector as described herein; and (ii) a carrier or excipient.

[0052] In one embodiment, the carrier is a lipid nanoparticle carrier.

[0053] In one aspect, provided herein is a composition comprising: one or more isolated peptides as described herein, one or more fusion proteins as described herein, one or more conjugates as described herein, one or more oligomeric complexes as described herein, one or more non-covalent complexes as described herein, or one or more cells as described herein, or any combination thereof, conjugated to a solid support.

[0054] In some embodiments, the solid support is a multi-well plate.

[0055] In one aspect, provided herein is a kit comprising: (i) a) one or more isolated peptides as described herein; b) one or more fusion proteins as described herein; c) one or more conjugates as described herein; d) one or more oligomeric complexes as described herein; e) one or more non-covalent complexes as described herein; f) one or more compositions as described herein; g) one or more cells as described herein; h) one or more polynucleotides as described herein; and / or i) one or more vectors as described herein;Attorney Docket No.250298.000720 (ii) optionally, an antigen-recognition molecule that targets DNTT250-258peptide; and / or (iii) optionally, packaging and / or instructions for use for the same.

[0056] In some embodiments, the antigen-recognition molecule(s) comprises an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0057] In another aspect, provided herein is an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258peptide, comprising: a) contacting the antigen-recognition molecule with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258peptide complex); b) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of the off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) is presented in a complex with the same kind of MHC molecule, or the fragment or derivative thereof, as in (a) (MHC-off-target peptide complex); and c) determining the level of binding of the antigen-recognition molecule to the MHC- DNTT250-258 peptide complex and each of the MHC-off-target peptide complexes.

[0058] In a further aspect, provided herein is an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258peptide, comprising: a) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of the off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and wherein each of the off-target peptides is presented in one of the following forms: (1) as a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising the peptide; and b) determining the level of binding of the antigen-recognition molecule to the one or more MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising the off-target peptides.Attorney Docket No.250298.000720

[0059] In some embodiments, the method further comprises determining that the antigen- recognition molecule is likely to have off-target effects if it detectably binds to at least one MHC- off-target peptide complex, off-target peptide or protein or fragment thereof comprising the off- target peptide.

[0060] In one aspect, provided herein is a method for selecting an antigen-recognition molecule that targets DNTT250-258 peptide, comprising: a) contacting a plurality of antigen-recognition molecules with DNTT250-258peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC- DNTT250-258 G12D peptide complex); b) contacting the same plurality of antigen-recognition molecules with one or more off-target peptides, (i) wherein each of the off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) wherein each of the off-target peptides is presented in one of the following forms: (1) as a complex with the same kind of MHC molecule, or the fragment or derivative thereof as in (a) (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising the off-target peptide; c) selecting one or more antigen-recognition molecules based on their level of binding to MHC-DNTT250-258peptide complex and based at least in part on the number of different MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising the off-target peptides detectably bound by each of the antigen-recognition molecules; and d) optionally, repeating steps (a)-(c) using the one or more selected antigen- recognition molecules.

[0061] In some embodiments, the one or more selected antigen-recognition molecules detectably bind no more than five MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising the off-target peptides.

[0062] In some embodiments, the one or more selected antigen-recognition molecules do not detectably bind to any of the tested MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising the off-target peptides.Attorney Docket No.250298.000720

[0063] In some embodiments, the plurality of antigen-recognition molecules is in a library.

[0064] In one embodiment, the library is a phage display library or a yeast library.

[0065] In some embodiments, one or more of the MHC-DNTT250-258 peptide complexes, MHC- off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising the off-target peptides are immobilized on a solid support.

[0066] In some embodiments, one or more of the MHC-DNTT250-258 peptide complexes, MHC- off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising the off-target peptides are present on cells.

[0067] In some embodiments, one or more of the MHC-DNTT250-258 peptide complexes, MHC- off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising the off-target peptides are present in a soluble form.

[0068] In some embodiments, the level of binding is determined by detecting the amount of antigen-recognition molecules bound to the MHC-DNTT250-258 peptide complexes, MHC-off- target peptide complexes, off-target peptides, or proteins or fragments thereof comprising the off- target peptides.

[0069] In some embodiments, the method is performed in a high-throughput format.

[0070] In one embodiment, the method is performed in a multi-well plate.

[0071] In one aspect, provided herein is a method of enriching a sample for antigen-recognition molecules that target DNTT250-258 peptide, comprising: a) contacting a sample comprising a plurality of antigen-recognition molecules with DNTT250-258peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258 peptide complex) in the presence of one or more off-target peptides, wherein each of the off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, wherein each of the one or more off-target peptides is presented in one of the following forms: (1) as a complex with an MHC molecule, or a fragment or derivative thereof (MHC- off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising the off-target peptide;Attorney Docket No.250298.000720 b) enriching the sample by isolating the antigen-recognition molecules that are detectably bound to the MHC-DNTT250-258 peptide complex; and c) optionally, repeating steps (a)-(b) using the enriched sample.

[0072] In some embodiments, the MHC-DNTT250-258peptide complex is present on a cell or solid support and the off-target peptides or proteins or fragments thereof comprising the off-target peptides or MHC-off-target peptide complexes are present in solution.

[0073] In some embodiments, the MHC-DNTT250-258peptide complex is labeled and the off- target peptides or proteins or fragments thereof comprising the off-target peptides or MHC-off- target peptide complexes are not labeled or are labeled differently.

[0074] In one embodiment, the DNTT250-258peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75).

[0075] In one embodiment, the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75).

[0076] In one embodiment, the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0077] In one embodiment, the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0078] In some embodiments, each of the off-target peptides is presented as an MHC-off-target peptide complex.

[0079] In some embodiments, the MHC molecule is a class I MHC molecule.

[0080] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0081] In some embodiments, the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

[0082] In one embodiment, the HLA-A molecule is an HLA-A*02:01 molecule.

[0083] In some embodiments, the peptide within the MHC-DNTT250-258 peptide complex and / or MHC-off-target peptide complex is covalently bound to the MHC.

[0084] In some embodiments, the peptide within the MHC-DNTT250-258 peptide complex and / or MHC-off-target peptide complex is non-covalently bound to the MHC.

[0085] In some embodiments, the antigen-recognition molecule(s) comprises an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).Attorney Docket No.250298.000720

[0086] In one aspect, provided herein is a library of off-target peptides, the library comprising two or more peptides each selected from the amino acid sequences of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is 8-12 amino acids in length.

[0087] In some embodiments, the at least two off-target peptides are each present in a complex with a major histocompatibility complex (MHC) molecule.

[0088] In some embodiments, the MHC molecule is a class I MHC molecule.

[0089] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0090] In some embodiments, the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

[0091] In some embodiments, the HLA-A molecule is an HLA-A*02:01 molecule.

[0092] In some embodiments, the peptide within the MHC-off-target peptide complex is covalently bound to the MHC.

[0093] In some embodiments, the peptide within the MHC-off-target peptide complex is non- covalently bound to the MHC.

[0094] In one aspect, provided herein is a library comprising two or more proteins or fragments thereof each comprising one or more off-target peptides, each off-target peptide being selected from the amino acid sequences of SEQ ID NOs: 76-82.

[0095] In one aspect, provided herein is a library comprising: a) two or more isolated peptides as described herein; b) two or more fusion proteins as described herein; c) two or more conjugates as described herein; d) two or more oligomeric complexes as described herein; e) two or more non- covalent complexes as described herein; f) two or more as described herein; g) two or more cells as described herein; h) two or more polynucleotides as described herein; and / or i) two or more vectors as described herein, optionally wherein each element of the library exists in a separate container. BRIEF DESCRIPTION OF THE DRAWINGS

[0001] The above and further aspects of this invention should be read with reference to the drawings, in which like elements in different drawings are identically numbered. The drawings, which are not necessarily to scale, depict selected embodiments and are not intended to limit theAttorney Docket No.250298.000720 scope of the invention. The detailed description illustrates by way of example, not by way of limitation, the principles of the invention. This description will clearly enable one skilled in the art to make and use the invention, and describes several embodiments, adaptations, variations, alternatives and uses of the invention, including what is presently believed to be the best mode of carrying out the invention.

[0002] FIG. 1A illustrates a flow diagram of an exemplary embodiment of a Peptide in Groove Similarity Prediction (PIGSPRED) method.

[0003] FIG. 1B illustrates a flow diagram of an exemplary embodiment of a method for predicting amino acid position(s) within a target peptide presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-target peptide complex) that can be performed as part of the PIGSPRED method.

[0004] FIG. 2 illustrates a flow diagram of an embodiment of a method to compute expected off-target toxicity associated with an MHC-target peptide complex.

[0005] FIG. 3 illustrates a flow diagram of an embodiment of a method for ranking potential target peptides to mitigate off-target toxicity.

[0006] FIG.4 illustrates a block diagram of an exemplary embodiment of a computational system configured to output an off-target peptide list and / or an off-target toxicity metric, the system including an exemplary embodiment of a PIGSPRED engine.

[0007] FIG.5 illustrates a block diagram of an exemplary embodiment of a computations system configured to output a list of low risk peptide targets, off-target peptide list(s) for potential target(s), and / or off-target toxicity metric(s) for potential target(s), the system including an exemplary embodiment of a target ranking engine.

[0008] FIG.6 illustrates a block diagram of an exemplary embodiment of a target toxicity database.

[0009] FIG.7 illustrates a block diagram of an embodiment of a computing device.

[0010] FIG.8 illustrates a block diagram of an embodiment of a computing network.

[0011] FIG.9 illustrates cellular functions related to the example embodiments presented herein.

[0012] FIG.10 shows peptide binding signal as a ratio of peptide-pulsed cells to unpulsed cells.Attorney Docket No.250298.000720 DEFINITIONS

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the pertinent art.

[0014] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0015] The term “about” or “approximately” includes being within a meaningful range of a value. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one skilled in the pertinent art.

[0016] The terms “major histocompatibility complex,” and “MHC” encompass the terms “human leukocyte antigen” or “HLA” (the latter two of which are generally reserved for human MHC molecules), naturally occurring MHC molecules (e.g., MHC class I molecule comprising MHC class I α (heavy) chain and β2 microglobulin; MHC class II molecule comprising MHC class II α chain and MHC class II β chain), individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of MHC class I α chain, α1 and / or α2 subunits of MHC class II α chain, β1 and / or β2 subunits of MHC class II β chain) as well as portions (e.g., the peptide-binding portions, e.g., the peptide-binding grooves), mutants and various derivatives thereof (including fusions proteins), wherein such portion, mutants and derivatives retain the ability to display an antigenic peptide for recognition by a T-cell receptor (TCR), e.g., an antigen-specific TCR. An MHC class I molecule comprises a peptide binding groove formed by the α1 and α2 domains of the heavy a chain that can stow a peptide of around 8-10 amino acids. Despite the fact that both classes of MHC bind a core of about 9 amino acids (e.g., 5 to 17 amino acids) within peptides, the open-ended nature of MHC class II peptide binding groove (the α1 domain of a class II MHC α polypeptide in association with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides binding MHC class II usually vary between 13 and 17 amino acids in length, though shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide binding groove, changing which 9-mer sits directly within the groove at any given time. In some embodiments, the MHC-peptideAttorney Docket No.250298.000720 complex described herein may be an MHC-peptide complex from a non-human animal. In other embodiments, the MHC-peptide complex described herein may include an HLA-peptide complex, i.e., an MHC-peptide complex from a human.

[0017] The term “non-human animal” and the like refers to any vertebrate organism that is not a human. In some embodiments, a non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, a non-human animal is a mammal. In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent such as a rat or a mouse.

[0018] The term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that, when introduced into an immunocompetent host can be recognized by the immune system of the host and elicit an immune response by the host. A T-cell receptor (TCR) can recognize a peptide presented in the context of a major histocompatibility complex (MHC) as part of an immunological synapse. The peptide-MHC (pMHC) complex is recognized by the TCR, with the peptide (antigenic determinant) and the TCR idiotype providing the specificity of the interaction. Accordingly, the term “antigen” encompasses peptides presented in the context of MHCs, e.g., peptide-MHC complexes. The peptide displayed on MHC may also be referred to as an “epitope” or an “antigenic determinant”. The terms “peptide,” “antigenic determinant,” “epitopes,” etc., encompass not only those presented naturally by antigen-presenting cells (APCs), but may be any desired peptide so long as it is recognized by an immune cell of an animal, e.g., when presented appropriately to the cells of an immune system. For example, a peptide having an artificially prepared amino acid sequence may also be used as the epitope.

[0019] The term “antigen-recognition molecule” refers to any molecule that is capable of recognizing an antigen as defined above. Antigen-recognition molecules can include, but are not limited to, T cell receptors (TCRs), antibodies, or chimeric antigen receptors (CARs).

[0020] The term “antigen presenting cell” or “APC” refers to any cell that presents on the surface of the cell an antigen in association with a major histocompatibility complex molecule, e.g., either MHC class I or MHC class II molecule, or both.

[0021] “MHC-peptide complex,” “peptide-MHC complex,” “pMHC complex,” “peptide- in-groove,” and the like includesAttorney Docket No.250298.000720

[0022] (i) an MHC molecule, e.g., a human and / or non-human animal MHC molecule, or portion thereof (e.g., the peptide-binding groove thereof, or e.g., the extracellular portion thereof), and

[0023] (ii) an antigenic peptide, where the MHC molecule and the antigenic peptide are complexed in such a manner that the pMHC complex can specifically bind a T-cell receptor. pMHC complexes encompass cell surface expressed pMHC complexes and soluble pMHC complexes.

[0024] “HLA-peptide complex,” “peptide-HLA complex,” “pHLA complex,” and the like refers to an MHC-peptide complex wherein the MHC molecule is a Human Leukocyte Antigen (HLA) molecule.

[0025] The terms “protein” and “polypeptide”, used interchangeably herein, encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as “peptides”.

[0026] The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA- RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0027] In general, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter may be operably linked to other expression control sequences, including enhancer and repressor sequences and / or with a polynucleotide described herein.

[0028] The term “operably linked” or the like refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the controlAttorney Docket No.250298.000720 sequences. “Operably linked” sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest (or sequence of interest). The term “expression control sequence” includes polynucleotide sequences, which are necessary to affect the expression and processing of coding sequences to which they are ligated. “Expression control sequences” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosomal binding sites and transcription termination sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0029] The term “isolated” refers to a homogenous population of molecules (such as polynucleotides or polypeptides) which have been substantially separated and / or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. In certain aspects, “isolated” refers to a molecule that is substantially free of other cellular material and / or chemicals and encompasses molecules that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity. The term “isolated” as used herein may also refer to a cell, or homogenous population of cells, that has been removed from its natural environment and substantially separated from other cellular components with which it is naturally associated. This includes cells that have been cultured in vitro or otherwise manipulated outside of their native biological context. The term encompasses cells that are part of a purified population, free from significant contamination by other cell types, and may include, e.g., genetically modified cells, stem cells, or cells derived from tissues or organs. In some embodiments, an isolated cell of the present disclosure is an immune cell, e.g., an antigen-presenting cell (APC).Attorney Docket No.250298.000720

[0030] The term “derivative” as used herein refers to a peptide, polypeptide, or polynucleotide, or a variant or analog thereof, comprising one or more mutations and / or chemical modifications as compared to a reference peptide, polypeptide or polynucleotide. Mutations and / or chemical modifications are further detailed below and can include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more locations in the amino acid or nucleotide sequence.

[0031] The terms “antibody,” “immunoglobulin,” “binding protein” and the like refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti- Id) antibodies (including, e.g., anti-Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U.S. Pat. Appl. Pub. 20070004909, incorporated herein by reference in its entirety, and Ig-DARTS such as those disclosed in U.S. Pat. Appl. Pub. 20090060910, incorporated herein by reference in its entirety. In some instances, an immunoglobulin or antibody may be a membrane-bound immunoglobulin or antibody, such as a B Cell Receptor (BCR).

[0032] A “pMHC-binding protein” refers to an antigen-binding protein (e.g., an immunoglobulin, antibody, TCR, CAR, or the like) that specifically binds a pMHC complex

[0033] An “individual” or “subject” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In one embodiment, the subject is a human.

[0034] The term “library” refers to an isolated collection of at least two elements that differ from one another in at least one aspect. For example, a “peptide library” is a collection of at least two peptides that may differ from one another by at least one amino acid. As another example, a “pMHC complex library” is a collection of pMHC complexes that may differ from one another by at least one amino acid in the peptide or at least one MHC polypeptide. The elements of the library are isolated from like type of elements that are not part of the library (e.g., peptides of a peptide library are isolated from peptides that are not part of the library). The library may exist in vitro or ex vivo. One or more elements (e.g., each element) of the library may be isolated from one or more other elements (e.g., each other element) of the library. A library may be “sorted” such that eachAttorney Docket No.250298.000720 of the one or more isolated elements exists in an identifiable and accessible physical space, such as a cell or a container, so that each set of the one or more isolated elements may be selectively accessed or pulled from the library for use according to the disclosure elsewhere herein. Each container may be separable (e.g., a vial) or part of an integral solid support (e.g., an individual well of a multi-well plate). One or more elements of a library (including partial portions or the entirety of the library) may be used according to the disclosure herein. Various elements of a library may be used contemporaneously or consecutively, such as for screening antigen-recognition molecules for off-target cross-reactivity.

[0035] The term “administration” and the like refers to and includes the administration of a composition (e.g., antigen-recognition molecule) to a subject or system (e.g., to a cell, organ, tissue, organism, or relevant component or set of components thereof). The skilled artisan will appreciate that route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or a rodent) may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and / or vitreal. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0036] The term “essential, normal tissues” refers to tissues of a patient where side-effects of a given antigen-recognition molecule administered for treating a disease may be deemed unacceptable. The list of tissues considered essential, normal would vary depending on the disease being treated and on the risks associated with the disease itself (e.g., the list would be smaller for life-threatening diseases than for non-life-threatening diseases). For example but not by way of limitation, when treating life-threatening cancers, tissue types that may be considered non-essential may include breast, ovary and testes. The list of tissues considered essential and normal would also vary depending on the likelihood for a given antigen-recognition molecule to reach such tissues. For example, brain may not be included in the list of essential, normal tissues in cases ofAttorney Docket No.250298.000720 antigen-recognition molecules which do not permeate blood-brain-barrier of patients with the disease being treated.

[0037] The terms “component,” “engine,” “module,” “system,” “server,” “processor,” “memory,” and the like are intended to include one or more computer-related units, such as but not limited to hardware, firmware, a combination of hardware and software, software, or software in execution. For example, a component may be, but is not limited to being, a process running on a processor, an object, an executable, a thread of execution, a program, and / or a computer. By way of illustration, both an application running on a computing device and the computing device can be a component. One or more components can reside within a process and / or thread of execution and a component may be localized on one computer and / or distributed between two or more computers. In addition, these components can execute from various computer readable media having various data structures stored thereon. The components may communicate by way of local and / or remote processes such as in accordance with a signal having one or more data packets, such as data from one component interacting with another component in a local system, distributed system, and / or across a network such as the Internet with other systems by way of the signal.

[0038] The term “connected” means that one function, feature, structure, or characteristic is directly joined to or in communication with another function, feature, structure, or characteristic.

[0039] The term “coupled” means that one function, feature, structure, or characteristic is directly or indirectly joined to or in communication with another function, feature, structure, or characteristic.

[0040] The terms “comprising” or “containing” or “including” are meant that at least the named element, or method step is present in article or method, but does not exclude the presence of other elements or method steps, even if the other such elements or method steps have the same function as what is named.

[0041] As used herein, unless otherwise specified, the use of the ordinal adjectives “first,” “second,” “third,” etc., to describe a common object, merely indicate that different instances of like objects are being referred to, and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

[0042] In this description, numerous specific details are set forth. It is to be understood, however, that implementations of the disclosed technology may be practiced without these specificAttorney Docket No.250298.000720 details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description. References to “one embodiment,” “an embodiment,” “some embodiments,” “example embodiment,” “various embodiments,” “one implementation,” “an implementation,” “example implementation,” “various implementations,” “some implementations,” etc., indicate that the implementation(s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every implementation necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one implementation” does not necessarily refer to the same implementation, although it may.

[0043] In accordance with the disclosure herein, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488- 492 (1985), U. S. Patent No.5,071, 743, Fukuoka et al., Biochem. Biophys. Res. Commun.263: 357-360 (1999); Kim and Maas, BioTech.28: 196- 198 (2000); Parikh and Guengerich, BioTech.24: 428-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech.19: 556-559 (1995); Wang and Malcolm, BioTech.26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies l4.3: 74-75 (2001), U. S. Patents Nos.5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech.20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nucl. Acids. Res.26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995),Attorney Docket No.250298.000720 Barrenttino et al., Nuc. Acids. Res.22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol.67: 209-218. DETAILED DESCRIPTION

[0044] Some embodiments presented herein relate to identification of off-target peptide(s) that are similar to an intended target peptide of an MHC-target peptide complex, such that an antigen-recognition molecule that is engineered for the intended target MHC-target peptide complex is likely to also recognize the off-target peptide(s) (e.g., peptide-MHC (pMHC) complexes comprising an off-target peptide). As will be understood herein, an antigen-recognition molecule which is said to “target” a specific peptide (i.e., a “target peptide”) is intended or purposed for (i.e., designed, engineered, and / or screened / selected for) preferentially recognizing and binding a peptide-MHC (pMHC) complex comprising the target peptide. In some examples, identification of the off-target peptide(s) can include predicting amino acid position(s) within the target peptide presented in the MHC-target peptide complex that are available to be involved in interacting with an antigen-recognition molecule. In some embodiments, the identified off-target peptide(s) can be used to predict probability of in vivo toxicity of a treatment targeting the target peptide. In some embodiments, off-target peptides can be identified for several potential target peptides, and the target peptides can be ranked based, at least in part, on their associated off-target peptides. In some embodiments, the identification of the off-target peptide(s) can be agnostic of the antigen-recognition molecule so that the identified off-target peptides, predicted in vivo toxicity, and / or ranking of potential target peptides can be used to guide development of an antigen-recognition molecule for targeting a target peptide having a low number of identified off- target peptides, a low predicted probability of in vivo toxicity, and / or a preferred ranking. Some embodiments disclosed herein include computational systems, engines, modules, devices, and / or networks configured to carry out a majority of steps associated with the above embodiments. The output of such computational systems, etc. can be used to inform antigen-recognition molecule development, screening of patients for clinical trials, individual patient treatment, and other such applications as understood by a person skilled in the pertinent art according to the teachings herein. One aim of some embodiments presented herein is to avoid side effects that would otherwise be identified during clinical trials, thereby reducing patient death, reducing other adverse effects on patients, and reducing expenditure of time and resources in research and development.Attorney Docket No.250298.000720

[0045] MHC molecules are generally classified into two categories: class I and class II MHC molecules. An MHC class I molecule is an integral membrane protein comprising a glycoprotein heavy chain, also referred to herein as the α chain, which has three extracellular domains (i.e., α1, α2 and α3) and two intracellular domains (i.e., a transmembrane domain (TM) and a cytoplasmic domain (CYT)). The heavy chain is noncovalently associated with a soluble subunit called β2 microglobulin (β2m or β2M). An MHC class II molecule or MHC class II protein is a heterodimeric integral membrane protein comprising one α chain and one β chain in noncovalent association. The α chain has two extracellular domains (α1 and α2), and two intracellular domains (a TM domain and a CYT domain). The β chain contains two extracellular domains (β1 and β2), and two intracellular domains (a TM domain and CYT domain).

[0046] The domain organization of class I and class II MHC molecules forms the antigenic determinant binding site, e.g., the peptide-binding portion or peptide binding groove, of the MHC molecule. A peptide binding groove refers to a portion of an MHC protein that forms a cavity in which a peptide, e.g., antigenic determinant, can bind. The conformation of a peptide binding groove is capable of being altered upon binding of a peptide to enable proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex.

[0047] In some embodiments, MHC molecules include fragments of MHC chains that are sufficient to form a peptide binding groove. For example, a peptide binding groove of a class I protein can comprise portions of the α1 and α2 domains of the heavy chain capable of forming two β-pleated sheets and two α helices. Inclusion of a portion of the β2 microglobulin chain stabilizes the MHC class I molecule. While for most versions of MHC Class II molecules, interaction of the α and β chains can occur in the absence of a peptide, the two-chain molecule of MHC Class II is unstable until the binding groove is filled with a peptide. A peptide binding groove of a class II protein can comprise portions of the α1 and β1 domains capable of forming two β-pleated sheets and two α helices. A first portion of the α1 domain forms a first β-pleated sheet and a second portion of the α1 domain forms a first a helix. A first portion of the β1 domain forms a second β- pleated sheet and a second portion of the β1 domain forms a second a helix. The X-ray crystallographic structure of class II protein with a peptide engaged in the binding groove of the protein shows that one or both ends of the engaged peptide can project beyond the MHC protein (Brown et al., pp.33-39, 1993, Nature, Vol.364; incorporated herein in its entirety by reference). Thus, the ends of the α1 and β1 α helices of class II form an open cavity such that the ends of theAttorney Docket No.250298.000720 peptide bound to the binding groove are not buried in the cavity. Moreover, the X-ray crystallographic structure of class II proteins shows that the N-terminal end of the MHC β chain apparently projects from the side of the MHC protein in an unstructured manner since the first 4 amino acid residues of the β chain could not be assigned by X-ray crystallography. Many human and other mammalian MHCs are well known in the art.

[0048] In some embodiments, the MHC molecule may be a human HLA molecule selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. A list of commonly used HLA alleles is described in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), incorporated herein in its entirety by reference. Shankarkumar et al. also present a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1 / 3 / 4 / 5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75:291-455, each of which publications is incorporated herein in its entirety by reference. In some embodiments, the MHC I or MHC II polypeptides may be derived from any functional human HLA-A, B, C, DR, or DQ molecules. In one embodiment, the HLA molecule is encoded by HLA-A2, such as an HLA-A*02:01 allele. In another embodiment, the HLA molecule is encoded by HLA-A1, such as an HLA-A*01:01 allele.

[0049] Targeting peptide-MHC (pMHC) complexes specifically expressed on cells such as cancer cells via, e.g., antibody-based or cell-based therapeutics approaches, can be an effective way of destroying such cells. However, the potential off-targets associated with these pMHC complexes can often lead to off-target toxicity. The present disclosure provides, among other things, a method named PIGSPRED (Peptide in Groove Similarity Prediction) useful in the prediction of such off-targets.

[0050] Authors have disclosed methods useful in prediction of such off-targets are presented in WO2023122621A2, published June 29, 2023, which is incorporated by reference herein in its entirety. In certain embodiments, WO2023122621A2 presents a method, referred to herein as the “PIGSPRED method” in which, for a target peptide, amino acid positions are distinguished as either being bound in an MHC-target peptide complex or available to bind to an antigen recognition molecule. In certain embodiments, the PIGSPRED method identifies potentialAttorney Docket No.250298.000720 off-target peptides based on binding affinity to the MHC molecule and the similarity of the amino acid sequence of a potential off-target peptide to the available positions of the target peptide. WO2023122621A2 at paragraphs

[0272] -

[0274] and Table 1B presents one example in which the PIGSPRED method is applied to a MAGEA4 target peptide GVYDGREHTV (SEQ ID NO: 1). In this example, positions 4 and 6-9 of said target peptide are identified as available positions, and potential off-target GLADGRTHTV (SEQ ID NO: 2) is determined a higher degree of similarity to said target peptide than other potential off-target peptides GVPDCRIFTV (SEQ ID NO: 3), SVYDAREFSV (SEQ ID NO: 4), GLSDGQWHTV (SEQ ID NO: 5), GVFDNCSHTV (SEQ ID NO: 6), and KVSDGHFHTV (SEQ ID NO: 7).

[0051] In some embodiments, the PIGSPRED method calculates a DoS score, optionally wherein only positions of the target peptide identified as not involved in interacting with the MHC molecule are considered in calculating the DoS score, and the PIGSPRED method calculates a probability of in vivo toxicity of each potential target peptide based at least in part on the number of potential off-target peptide(s), which may be determined at least in part by DoS. Once cancer- specific pHLAs are identified, PIGSPRED can be used to calculate the number of potential off- targets having sequence similarity associated with each cancer-specific pHLA. In certain embodiments of the PIGSPRED method, the number of potential off-targets is representative of the likelihood of off-target toxicity associated with a target and is used to rank the list of pHLA targets and to prioritize the targets for therapeutic development. In certain embodiments of the PIGSPRED method, the off-targets predicted by PIGSPRED can be used in experimental screening of therapeutic molecules to confirm that the therapeutic molecules do not bind the off- targets. The most specific therapeutic molecules can thus be selected for further development.

[0052] FIG. 1A illustrates a flow diagram of an exemplary embodiment of a Peptide in Groove Similarity Prediction (PIGSPRED) method 100.

[0053] At step 102, an MHC-target peptide complex is given as an input to the PIGSPRED method. The MHC-target peptide complex includes a target peptide and an MHC molecule.

[0054] At step 104, peptide positions (amino acids) important for antigen-recognition molecule binding are identified. The target peptide includes some amino acids which are bound to the MHC molecule and some amino acids which are available for binding to an antigen-recognition molecule. When evaluating similarity / homology of a peptide to the target peptide, the similarity can be evaluated only at peptide positions (i.e., positions of amino acids within the peptide) thatAttorney Docket No.250298.000720 are available to bind with an antigen-recognition molecule. These available positions can be ascertained by analyzing the experimental structures of the MHC-target peptide complex with a specific antigen-recognition molecule, wherein such experimental structures are typically derived using crystallography or cryoEM techniques. Therefore, peptide positions important for antigen- recognition molecule binding can be identified through analysis of experimental structures; however, the specific antigen-recognition molecule must be known and such experimental structures can be difficult to obtain. When developing a targeted therapy, at the initial target selection stage, in which the potential risks associated with the target are evaluated, a specific antigen-recognition molecule against the target is unavailable. The Immune Epitope Database (IEDB) is a freely available resource that catalogs experimental data on antibody and T cell epitopes studied in humans, non-human primates, and other animal species in the context of infectious disease, allergy, autoimmunity and transplantation. The IEDB also hosts tools to assist in the prediction and analysis of epitopes. Similar databases and methodologies to characterize epitopes in such databases can be used to obtain experimental structures of MHC peptide complexes.

[0055] FIG. 1B illustrates steps of the PIGSPRED method 100 that can be performed at step 104. The steps illustrated in FIG.1B illustrate an exemplary embodiment of a method 104 for predicting amino acid position(s) within the target peptide presented in the MHC-target peptide complex. The method 104 can be performed agnostic to structure of an antigen-recognition molecule, and therefore presents an alternative to analysis of the experimental structures of the MHC-target peptide complex with a specific antigen-recognition molecule when the specific antigen-recognition molecule is unknown. Positions identified computationally at step 104 can be verified by experimental data such as mass-spectroscopy data or experimental data otherwise available in a database similar to the IEDB.

[0056] At step 110, binding affinity of the MHC molecule to the target peptide is predicted. In some embodiments, the binding affinity can be calculated using a computer-based model. As a non-limiting example, a commercial tool called NetMHCpan, which uses a machine learning model, utilizes a computer-based binding affinity model that may be suitable for computationally calculating the binding affinity. Any version of NetMHCpan may be used, for example, NetMHCpan4.0 or NetMHCpan4.1. Other non-limiting examples include MHCflurry (see, e.g., O'Donnell et al., Cell Syst.2018 Jul 25;7(1):129-132.e4, which is incorporated herein by referenceAttorney Docket No.250298.000720 in its entirety) and MixMHCPred (see, e.g., Boehm et al., BMC Bioinformatics volume 20, Article number: 7 (2019), which is incorporated herein by reference in its entirety).

[0057] Predicting binding affinity estimates how tightly a peptide will bind to a specific MHC molecule. It is measured as either a predicted IC50value in nanomolar (nM) range or a predicted percentile rank value. The predicted IC50 value attempts to approximate experimental IC50 value which measures the concentration of a competing peptide required to displace 50% of the binding of the peptide to the MHC molecule. In some embodiments, if the predicted binding affinity ≤ 500nM, a peptide is considered a binder. If it is ≤ 50nM, it is considered a strong binder. The percentile rank value (e.g., %Rank_BA in NetMHCpan) normalizes the IC50 values across different MHC molecules. The rank is computed by comparing the predicted IC50value of the peptide against the predicted IC50values for a set of (on the order of 105) naturally occurring random peptides (see e.g., Jurtz V et al., J Immunol. 2017, which is incorporated herein by reference in its entirety). In some embodiments, if the predicted percentile rank ≤ 2, a peptide is considered a binder. If it is ≤ 0.5, it is considered a strong binder.

[0058] At steps 112 and 114, computational mutagenesis can be performed in which, at step 112, the target peptide is mutated at a single amino acid position, resulting in several mutated peptides each having a single amino acid mutation from the target peptide. The mutated peptides are each associated with a respective peptide position (i.e., position of mutation). Preferably, the target peptide is mutated at every amino acid position, resulting in a number of mutated peptides equal to the number of amino acids in the target peptide. In some embodiments, mutated peptides can include a single amino acid mutation to glycine or an alanine amino acid. In some embodiments, the target peptide is mutated at single amino acid positions with the amino acid replaced by glycine, and positions of the target peptide having glycine are skipped, resulting in several mutated peptides corresponding to mutations of the target peptide at non-glycine positions.

[0059] At step 114, the binding affinity of each mutated peptide to the MHC molecule is predicted. In some embodiments, the binding affinity can be calculated using a computer-based model identical or similar to the computer-based model utilized at step 110.

[0060] At step 116, the binding affinity of the target peptide to the MHC molecule is compared to each binding affinity of the mutated peptides to the MHC molecule. The peptide positions associated with mutated peptides that do not result in the loss of binding affinity to the MHC molecule (compared to the target peptide binding affinity to the MHC molecule) are flagged.Attorney Docket No.250298.000720 These flagged peptide positions are identified as not involved in binding to the MHC molecule and are therefore free to interact with an antigen-recognition molecule.

[0061] In some embodiments, when the binding affinity of a mutated peptide with a mutation associated with the respective position is approximately equal to the binding affinity of the target peptide to the MHC molecule, the respective position associated with the mutated peptide is predicted to be involved in interacting with an antigen-recognition molecule that recognizes the MHC-target peptide complex or otherwise flagged as free positions. Percentile rank values from NetMHCpan (e.g., NetMHCpan4.0 or NetMHCpan4.1) may be used. A mutated position is deemed as one resulting in loss of binding affinity to MHC molecule, if the percentile rank of the mutated peptide is less than a specific threshold, wherein the lower the percentile rank, the higher the binding affinity. Utilizing predicted percentile rank to quantify binding affinity, in some embodiments, if the rank of the target peptide is ≤ 0.5, then the threshold is 1.0. If the rank of the target peptide is > 0.5 and ≤ 2.0, then the threshold is 2.0. If the rank of the target peptide is > 2.0, then the threshold is 4.0. In other embodiments, a difference in binding affinity (e.g., percentile rank) between the mutated peptide and target peptide may be used to identify positions as involved in binding to the MHC molecule or free to interact with an antigen-recognition molecule. Positions where mutations result in a significant loss of binding affinity (e.g., a loss greater than a threshold) may be deemed to be involved in binding to the MHC molecule and positions where mutations do not result in a significant loss of binding affinity may be deemed to be free to interact with an antigen-recognition molecule. For example, if the rank of the target peptide is ≤ 0.5, then the difference in rank (loss) threshold may be 1.0. If the rank of the target peptide is > 0.5 and ≤ 2.0, then the difference in rank threshold may be 1.5. If the rank of the target peptide is > 2.0, then the difference in rank threshold may be 2.0.

[0062] At step 118, out of these flagged free positions, the ones that contain a non-glycine amino acid are identified as positions important for antigen-recognition molecule binding. In some embodiments, if a mutated position does not result in a significant loss of MHC molecule binding and the amino acid at the position is a non-glycine amino acid, then that position is considered important for antigen-recognition molecule binding. In embodiments in which, at step 112, the target peptide is mutated at single amino acid positions with the amino acid replaced by glycine and positions of the target peptide having glycine are skipped, step 118 can be omitted because allAttorney Docket No.250298.000720 of the mutated peptides resulting from step 112, by definition, correspond to positions containing a non-glycine amino acid.

[0063] In some embodiments, when the structure of an MHC-target peptide complex bound to an antigen-recognition molecule is known, the binding motifs in the peptide sequence involved in antigen-recognition molecule interactions can be used as a comparison to verify that important positions for antigen-recognition molecule binding are correctly identified. Using immunopeptidomics data, predicted off-target peptides that have also been observed in mass- spectrometry experiments can provide additional indication that the predicted off-targets may be bona fide off-targets.

[0064] At step 106, similar peptides are identified. The similar peptides can be identified based on presence in an organism (e.g., human), and similarities to the target peptide at positions important for antigen-recognition molecule binding. Similar peptides may be selected such that they all be the same length as the target peptide. A similar peptide may be identical to the target peptide, although from a different protein source as the target peptide. However, in some instances, a target peptide may have already been screened such that non-unique candidate target peptides (candidate target peptides sharing an identical sequence with a peptide elsewhere in the proteome) are not selected as a target.

[0065] A working list of peptide sequences can be assembled to include peptide sequences in an organism (e.g., human). In some embodiments, the working list can be assembled based on canonical human protein sequences in a medical research database such as, as a non-limiting example, UniprotKB. A working list of peptide sequences can be filtered by comparing peptide sequences in the database to important positions of the target peptide identified in step 104 and keeping only peptide sequences having identical amino acids to the target peptide at a threshold number of important positions. The threshold number of positions can be two positions, three positions, four positions, or five positions. In embodiments in which a specific antigen-recognition molecule is not known, i.e. the PIGSPRED method 100 is agnostic to antigen-recognition molecule structure, three positions is preferred. While there may be an antigen-recognition molecule that binds at only two positions, such antigen-recognition molecule likely will have an impractically large number of off-target peptides. Increasing the threshold number of positions may result in underestimation of off-targets.Attorney Docket No.250298.000720

[0066] The working list of peptides can be further filtered to retain only peptides present in essential, normal tissues or essential cell types. Disrupting cell function in essential, normal tissues or essential cell types is more likely to cause more severe off-target effects, and therefore poses greater risk compared to disruption of cell function in non-essential tissues. In some embodiments, a medical research database can be utilized that includes gene expression in healthy donors. The Gene Tissue Expression database or GTEx is a non-limiting example of a suitable database, see gtexportal.org. For example, GTEx version 6 (v6), based on genome build GRCh38, contains gene expression data across 51 tissue types from 549 healthy donors. GTEx version 8 (v8), based on genome build GRCh38, contains gene expression data across 54 tissue types from 948 healthy donors; it is understood that additional donor and tissue samples are being sequenced for newer releases. In some embodiments, an alternative database to the GTEx database, and / or a different version of the GTEx database can be utilized to determine potential high risk off-target peptides in step 106, which may result in identifying additional or fewer potential high risk peptides, compared to specific examples disclosed herein, as understood by a person skilled in the pertinent art.

[0067] In some embodiments, gene expression values are queried in only those tissue types that are considered essential. Tissue types that may be considered non-essential may be those which may be sacrificed for the purpose of saving a patient’s life, such as breast, ovary, testes, etc. In some embodiments, for each gene, the 95-percentile expression value in each essential tissue type is calculated, where 95-percentile expression value means 95% of all expression measurements of a gene in a tissue type falls at or below that value. Then, the maximum of the 95- percent expression value across all essential tissue types is calculated. If the maximum of the 95- percent expression value of a gene is greater than 0.5 transcripts per million (TPM) (which may be adjusted based on transcriptional noise), then it can be assumed that any similar peptide that is derived from the gene will be a potential high risk off-target peptide. High TPM means high expression and vice versa. To calculate TPM, divide read counts for a gene by the length of each gene in kilobases, resulting in reads per kilobase (RPK); next all RPK values in a sample are counted and divided by 1,000,000, resulting in a “per million” scaling factor; and finally divide each RPK value by the “per million” scaling factor, resulting in TPM for each gene. See, e.g., Li et al. (2010) Bioinformatics, 26(4):493-500; Varabyou et al. (2021) Genome Res.31(2):301-308. The working list of peptide sequences can be filtered to remove peptides not identified as potentialAttorney Docket No.250298.000720 high risk. In some embodiments, an alternative database to the GTEx database, and / or a different version of the GTEx database can be utilized to determine potential high risk off-target peptides in step 106, which may result in identifying additional or fewer potential high risk peptides, compared to specific examples disclosed herein, as understood by a person skilled in the pertinent art.

[0068] In some embodiments, peptides that are filtered from the working list of peptide sequences at step 106 may be separately tracked as a list of potential secondary target peptides. A secondary target peptide may be derived from a secondary target gene that is distinct from the target gene of the target peptide, and cross-reactivity with the secondary target peptide may be therapeutically beneficial. If a potential secondary peptide is identical to the target peptide or highly similar, it is likely to cross-react with an antigen-recognition molecule targeting the target peptide (e.g., when the potential secondary peptide is complexed to an MHC molecule, particularly when complexed to an MHC molecule that is the same as that comprised by a pMHC that the antigen-recognition molecule preferentially recognizes). Additional testing (e.g., antigen- recognition molecule screening) may optionally be performed on one or more of the potential secondary target peptides to assess whether cross-reactivity with those potential secondary target peptides could provide a beneficial therapeutic effect. Similar peptides derived from disease- associated genes or disease-associated antigens, such as cancer-specific genes or peptides that have been identified as or are derived from proteins that have been identified as cancer-specific or tumor-specific antigens, may exhibit beneficial cross-reactivity, and therefore may be a secondary target peptide. For example, cancer / testis (CT) antigens which are not expressed in normal, essential tissue may be tracked if similar to a target peptide, particularly where the target peptide is also a CT antigen, as exemplified in Example 1. Target peptides which share sequence identify (e.g., identical peptides or highly similar) with multiple suitable antigens for therapeutic targeting may be preferable.

[0069] At step 108, the working list of peptide sequences can be further filtered to remove peptides that do not bind to the MHC molecule and a degree of similarity (DoS) score can be calculated for remaining peptides in the working list. Binding affinity of peptides in the list of peptide sequences can be determined by utilizing a computer-based binding affinity model. Peptides that are not predicted to bind to the MHC molecule are removed from the working list of peptide sequences. In some embodiments, a peptide with percentile rank value ≤ 2.0 is consideredAttorney Docket No.250298.000720 a binder. Optionally, a list of potential secondary target peptides, if being tracked, may likewise be filtered to remove peptides that do not bind to the MHC molecule in the same manner as described with respect to the working list of similar peptides in step 108. Also optionally, a degree of similarity (DoS) score can be calculated for potential secondary target peptides.

[0070] The DoS score can be based at least in part on number of amino acids of a respective peptide in the working list that is identical to amino acids at corresponding positions of the target peptide that are identified, at step 104, as important for binding to an antigen-recognition molecule. In some embodiments, the DoS score only considers those positions that are identified, at step 104, as important for binding to an antigen-recognition molecule. Alternatively, the DoS may consider at least some positions that are not identified, at step 104, as important for binding to an antigen- recognition molecule. For instance, in some embodiments, the DoS score can be based on the number of amino acids identified (at step 116) as not involved in binding to the MHC molecule, or the DoS score can be based on number of identical amino acids at every amino acid position of the respective peptide.

[0071] FIG. 2 illustrates a flow diagram of an embodiment of a method 200 to compute expected off-target toxicity associated with an MHC-target peptide complex. The method 200 is preferably executed by computational systems, engines, modules, devices, and / or networks. The method 200 is preferably stored as instructions on non-transitory computer-readable medium.

[0072] At step 202, an MHC-target peptide complex is provided as an input.

[0073] At step 204, peptide positions (amino acids) important for antigen-recognition molecule binding are identified. In preferred embodiments, the peptide positions are identified according to the method 104 illustrated in FIG.1B. In some embodiments, the peptide positions are identified by analyzing the experimental structures of the MHC-target peptide complex with a specific antigen-recognition molecule.

[0074] At step 206, similar peptides can be identified. The similar peptides can be identified based on presence in an organism (e.g., human), similarities to the target peptide at positions important for antigen-recognition molecule binding, and ability to bind to the MHC molecule in the MHC-target peptide complex. Similar peptides may be selected such that they all are the same length as the target peptide. A similar peptide may be identical to the target peptide, although from a different protein source as the target peptide. However, in some instances, a target peptide may have already been screened such that non-unique candidate target peptides (candidateAttorney Docket No.250298.000720 peptides sharing an identical sequence with a peptide elsewhere in the proteome) are not selected as a target.

[0075] A working list of peptide sequences can be assembled to include peptide sequences in an organism (e.g., human). The working list can be represented by an appropriate data structure such as a list, matrix, and / or other appropriate data structure as understood by a person skilled in the pertinent art. In some embodiments, the working list can be assembled based on canonical human protein sequences in a medical research database such as, as a non-limiting example, UniprotKB. In some embodiments, step 206 can include receiving the peptide sequences of an organism (e.g., human) as an input and utilizing the peptide sequences to generate the working list of peptide sequences.

[0076] The working list of peptide sequences can be filtered by comparing peptide sequences in the database to important positions of the target peptide identified in step 204 and keeping only peptide sequences having identical amino acids of the target peptide at or greater than a threshold number of important positions. The threshold number of positions can be two positions, three positions, four positions, or five positions. In embodiments in which a specific antigen-recognition molecule is not known, i.e. the PIGSPRED method 100 is agnostic to antigen- recognition molecule structure, three or four positions is preferred, and three positions is most preferred. The threshold number of positions is preferably determined based on the number of binding positions for an antigen-recognition molecule that is practical in a clinical setting. While there may be an antigen-recognition molecule that binds at only two positions, such antigen- recognition molecule likely will have an impractically large number of off-target peptides which may result in adverse side effects in a clinical setting, therefore such antigen-recognition molecule is impractical in a clinical setting. Increasing the threshold number of positions may result in underestimation of off-targets for antigen-recognition molecules that bind to fewer positions than the threshold number of positions; therefore the resulting expected off-target toxicity computed at step 210 may not be accurate for antigen-recognition molecules which bind to fewer than the threshold number of positions.

[0077] At step 208, expression of the similar peptides in non-target cells is analyzed. The risk associated with an antigen-recognition molecule binding to a respective peptide of the similar peptides identified in step 206 is greater if the respective peptide is present in essential, normal tissues or essential cell types. Disrupting cell function in essential, normal tissues or essential cellAttorney Docket No.250298.000720 types is more likely to cause more severe off-target effects, and therefore poses greater risk compared to disruption of cell function in non-essential tissues. The working list of peptides can be further filtered to retain only peptides present in essential, normal tissues or essential cell types.

[0078] In some embodiments, a medical research database can be utilized that includes gene expression in healthy donors. The Gene Tissue Expression database or GTEx is a non- limiting example of a suitable database, see gtexportal.org. For example, GTEx version 6 (v6), based on genome build GRCh38, contains gene expression data across 51 tissue types from 549 healthy donors. GTEx version 8 (v8), based on genome build GRCh38, contains gene expression data across 54 tissue types from 948 healthy donors; it is understood that additional donor and tissue samples are being sequenced for newer releases. In some embodiments, gene expression values are queried in only those tissue types that are considered essential. Tissue types that may be considered non-essential may be those which may be sacrificed for the purpose of saving a patient’s life, such as breast, ovary, testes, etc. In some embodiments, for each gene, the 95- percentile expression value in each essential tissue type is calculated and then the maximum of that value across all essential tissue types is calculated. If the maximum expression value of a gene is greater than 0.5 TPM (which may be adjusted based on transcriptional noise), then it can be assumed that any similar peptide that is derived from the gene will be a potential high risk off- target peptide. The working list of peptide sequences can be filtered to remove peptides not identified as potential high risk. However, in some embodiments, peptides not identified as potential high risk may not need to be discarded. As discussed above, such peptides may form “secondary target peptides.” Accordingly, another working list comprising the potential secondary target peptides may be prepared, and additional testing (e.g., antigen-recognition molecule screening) may optionally be performed on one or more potential secondary target peptides to assess whether cross-reactivity with those peptides could provide a beneficial therapeutic effect. In some embodiments, an alternative database to the GTEx database, and / or a different version of the GTEx database can be utilized to determine potential high risk off-target peptides in step 208, which may result in identifying additional or fewer potential high risk peptides, compared to specific examples disclosed herein, as understood by a person skilled in the pertinent art.

[0079] At step 210, expected off-target toxicity associated with the MHC-target peptide complex is computed. Once the potential high risk off-target peptides have been identified, a Degree of Similarity (DoS) score is calculated for each of the high risk off-target peptides. TheAttorney Docket No.250298.000720 DoS score quantifies the homology between the high risk off-target peptide and the target peptide. DoS represents the number of identical amino acids at identical positions or hamming distance between peptides. Using immunopeptidomics data, off-target peptides that have been observed in mass-spectrometry experiments can provide additional evidence for the potential off-targets.

[0080] The working list of peptide sequences can be filtered to remove peptides which have a DoS score below a given threshold. The number of peptide sequences remaining in the working list can be used as a metric indicating off-target toxicity of the MHC-target peptide complex which was input at step 202 to the method 200 illustrated in FIG.2.

[0081] FIG. 3 illustrates a flow diagram of an embodiment of a method 300 for ranking potential target peptides to mitigate off-target toxicity.

[0082] At step 302, two or more potential peptides are selected among disease-associated peptides that are predicted to bind to an MHC molecule. Disease-specific MHC-target peptide complexes can be identified by ascertaining genes that are specifically expressed in diseased tissue. In some embodiments, the disease-specific MHC-target peptide complex can be identified based on a medical database such as The Cancer Genome Atlas (TCGA) and Genome Tissue Expression Database (GTEx), which include human gene sequences. In some embodiments, a gene that is expressed in a cancer type at 75-percentile TPM value > 2 and is negligibly expressed in all essential, normal tissues or essential cell types in the GTEx is considered a cancer-specific gene. The canonical protein sequence corresponding to the cancer-specific gene can be derived from a medical research database including canonical human protein sequences such as the UniProtKB database.

[0083] The derived protein sequences can be used to predict the potential 8-25 mer peptide sequences that are predicted to bind the MHC of interest. When the MHC molecule is a class I MHC molecule, the predicted or detected peptides in step 302 can be 8-12 amino acids in length. When the MHC molecule is a class II MHC molecule, the predicted or detected peptides in step 302 can be 13-17 amino acids in length. The predictions can be done using a binding affinity calculation tool such as the NetMHCpan tool. In some embodiments, if the predicted binding affinity ≤ 500nM, a peptide is considered a binder. If it is ≤ 50nM, it is considered a strong binder. In some embodiments, if the predicted percentile rank ≤ 2, a peptide is considered a binder. If it is ≤ 0.5, it is considered a strong binder.Attorney Docket No.250298.000720

[0084] At step 304, a number of off-target peptide(s) associated with each of the potential target peptides can be estimated. In some embodiments, the number of off-target peptide(s) can be estimated utilizing steps of methods 100, 200 in FIGs.1A and / or FIG.2.

[0085] At step 306, the potential target peptides are ranked based at least in part on the number of off-target peptide(s) associated with each of the potential target peptides. In some embodiments, the number of potential off-targets estimated in step 304 is representative of the likelihood of off-target toxicity that might be associated with the MHC-target peptide complex, and thus this number can be used to rank the list of potential target peptides and to prioritize them for therapeutic development.

[0086] After the target selection and the generation of therapeutic molecules that bind the target, the off-targets predicted by PIGSPRED play an important role in experimental screening of therapeutic molecules for molecules that do not bind the off-targets. The most specific therapeutic molecules can be selected for further development.

[0087] In some embodiments, the method 300 illustrated in FIG. 3 can be executed by repeating the method 200 in FIG.2, providing differing target MHC-target peptide complexes as input at step 202 and resulting in an off-target toxicity metric at step 210 for each of the input MHC-target peptide complexes. The off-target toxicity metrics can be compared to rank the MHC- target peptide complexes. The MHC-target peptide complex ranked as having lower off-target toxicity can be selected for further analysis, can be used for generation of antigen-recognition molecule(s), and / or can be used for testing of antigen-recognition molecule(s).

[0088] FIG.4 illustrates a block diagram of an exemplary embodiment of a computational system 400 configured to output an off-target peptide list and / or an off-target toxicity metric. The system 400 includes an exemplary embodiment of a PIGSPRED engine 410. The PIGSPRED engine 410 is configured to receive, as an input, a computational representation of a target peptide presented in an MHC-target peptide complex 402. In the illustrated embodiment, the PIGSPRED engine 410 provides as an output an off-target peptide list 426 and / or an off-target toxicity metric 428.

[0089] The illustrated embodiment of the PIGSPRED engine 410 includes a peptide position identification module 412, a working peptide list builder 414, a binding affinity filter 416, a risk severity assessment module 418, a risk severity filter 420, a risk likelihood assessment module 422, and a risk likelihood filter 424. The PIGSPRED engine 410 includes one or moreAttorney Docket No.250298.000720 processor(s) and non-transitory computer-readable medium with instructions thereon, that when executed by the one or more processor(s) cause the PIGSPRED engine 410 to perform functions associated with each of the aforementioned features 412, 414, 416, 418, 420, 422, 424. In some embodiments, the PIGSPRED engine can be configured to provide as an output intermediate values determined by features 412, 414, 416, 418, 420, 422, 424 of the PIGSPRED engine 410. The features 412, 414, 416, 418, 420, 422, 424 are illustrated separately and in a specific order for illustrative purposes. As understood by a person skilled in the pertinent art, the features 412, 414, 416, 418, 420, 422, 424 can be combined, reordered, or otherwise implemented in numerous configurations to achieve the functionality disclosed in relation to the illustrated embodiment of the PIGSPRED engine 410.

[0090] The illustrated system 400 further includes a binding affinity calculation engine 404, a canonical protein sequence database 406, and a tissue expression database 408 in communication with the PIGSPRED engine 410. In a preferred embodiment, the binding affinity calculation engine 404, the canonical protein sequence database 406, and the tissue expression database 408 are separate from the PIGSPRED engine 410, and the PIGSPRED engine 410 is in communication with each. Alternatively, one or more of the ancillary features 404, 406, 408, 410 can be integral to the PIGSPRED engine 400 as understood by a person skilled in the pertinent art. The binding affinity calculation engine 404 includes a computer-based model for calculating binding affinity of a peptide to an MHC molecule. In some embodiments, the binding affinity calculation engine includes a commercial tool such as NetMHCpan or similar product. The canonical protein sequence database 406 includes information from which healthy tissue protein sequences can be derived. In some embodiments, the canonical protein sequence database 406 includes the UniProtKB database or similar medical database. As a non-limiting example, the tissue expression database 408 can include gene expression data across approximately 30 tissue types from approximately 549 healthy donors; in another non-limiting example, the tissue expression database 408 can include gene expression data across approximately 54 tissue types from approximately 948 healthy donors. In some embodiments, the tissue expression database 408 includes the Gene Tissue Expression database, GTEx, or similar medical database.

[0091] The peptide position identification module 412 of the PIGSPRED engine 410 can receive the computational representation of the MHC-target peptide complex 402 as an input and identify positions important for binding to an antigen-recognition molecule.Attorney Docket No.250298.000720

[0092] In a preferred embodiment, the peptide position identification module 412 is configured to perform the following steps: (a) receive, as an input, a computational representation of a target peptide in an MHC-target peptide complex; (b) determine binding affinity of the target peptide to the MHC molecule; (c) generate sequences of a plurality mutated peptides each associated with a mutation at a respective amino acid position of the target peptide; (d) determine binding affinity of each mutated peptide of the plurality of mutated peptides of the MHC molecule; (e) predict the amino acid position(s) involved in interacting with an antigen-recognition molecule that recognizes said MHC-target peptide complex based at least in part on a comparison of the binding affinity for each mutated peptide to the binding affinity of the target peptide; and (f) provide, as an output, an indication of the amino acid position(s) likely to be involved in interacting with the antigen-recognition molecule.

[0093] Steps (b) and (d) are executed by utilizing the binding affinity calculation engine 404. In some embodiments, at step (e) a threshold value of the binding affinity for a mutated peptide can be used to determine whether the amino acid position associate with the mutation is involved in interacting with an antigen-recognition molecule. In some embodiments, predicted percentile rank is utilized to quantify binding affinity, such that, if the rank of the target peptide is ≤ 0.5, then the threshold value of the binding affinity for the mutated peptide is 1.0; if the rank of the target peptide is > 0.5 and ≤ 2.0, then the threshold value of the binding affinity for the mutated peptide is 2.0; and if the rank of the target peptide is > 2.0, then the threshold value of the binding affinity for the mutated peptide is 4.0. A mutated position is deemed as one resulting in loss of binding affinity to MHC molecule. Percentile rank is inversely related to binding affinity, therefore when the threshold value of the binding affinity for a mutated peptide is based on percentile rank, a percentile rank less than the threshold value indicates the amino acid position associate with the mutation is involved in interacting with an antigen-recognition molecule.

[0094] The working list builder 414 is configured to generate a working list of peptides, within a total pool of predicted or detected peptides of suitable length, such that peptides listed in the working list each include at least two amino acids that (i) are located at positions corresponding to positions within the target peptide that are involved in interacting with the antigen-recognition molecule and (ii) are identical to the corresponding amino acids of the target peptide. In some embodiments, the working peptide list builder 414 can derive the total pool of predicted or detected peptides of suitable length from the canonical protein sequence database 406. When the MHCAttorney Docket No.250298.000720 molecule is a class I MHC molecule, a peptide of suitable length may be 8-12 amino acids in length. A peptide of suitable length may be the same length as the target peptide. When the MHC molecule is a class II MHC molecule, a peptide of suitable length may be 13-17 amino acids in length. A peptide of suitable length may be the same length as the target peptide.

[0095] The binding affinity filter 414 is configured to determine binding affinity, of each of the peptides listed in the working list, to the MHC molecule and filter the working list to include only peptide(s) having a calculated binding affinity to the MHC molecule indicating a greater likelihood to bind compared a threshold value. The binding affinity filter 416 can utilize the binding affinity calculation engine 404 to determine the binding affinity of each of the peptides in the working list to the MHC molecule. In some embodiments, a peptide with percentile rank value ≤ 2.0 is considered a binder by the binding affinity filter 414, the percentile rank value being used as the threshold value. The threshold value used by the binding affinity filter 414 need not be equal to the threshold value used by the position identification module 412.

[0096] The risk severity assessment module 418 is configured to estimate the number of peptide(s) in the working list of off-target peptides which are expressed in essential, normal tissues or essential cell types. Disrupting cell function in essential, normal tissues or essential cell types is more likely to cause more severe off-target effects, and therefore poses greater risk compared to disruption of cell function in non-essential tissues. The risk severity assessment module 418 can utilize the tissue expression database 408 to determine which peptide(s) in the working list are expressed in essential, normal tissues or essential cell types. In some embodiments, gene expression values are queried in only those tissue types that are considered essential. Tissue types that may be considered non-essential may be those which may be sacrificed for the purpose of saving a patient’s life, such as breast, ovary, testes, etc. In some embodiments, for each gene, the 95-percentile expression value in each essential tissue type is calculated and then the maximum of that value across all essential tissue types is calculated. If the maximum expression value of a gene is greater than 0.5 TPM (which may be adjusted based on transcriptional noise), then it can be assumed that any similar peptide that is derived from the gene will be a potential high risk off- target peptide. A similar peptide may be identical to the target peptide, although from a different protein source as the target peptide. However, in some instances, a target peptide may have already been screened such that non-unique candidate target peptides (candidate target peptides sharing an identical sequence with a peptide elsewhere in the proteome) are not selected as a target.Attorney Docket No.250298.000720

[0097] The risk severity filter 420 filters the working list to remove peptides not identified as potential high risk by the risk likelihood assessment module 418.

[0098] The risk likelihood assessment module 422 calculates a Degree of Similarity (DoS) score for the peptide(s) in the working list of peptides. The DoS score is based at least in part on a number of amino acids identical to amino acids at corresponding positions of the target peptide, which amino acids of the target peptide are involved in interacting with the antigen-recognition molecule. In some embodiments, the DoS score only considers those positions that are identified, by the peptide position identification module 412, as important for binding to an antigen- recognition molecule. Alternatively, the DoS may consider at least some positions that are not identified as important for binding to an antigen-recognition molecule. For instance, in some embodiments, the DoS score can be based on the number of amino acids not involved in binding to the MHC molecule, or the DoS score can be based on number of identical amino acids at every amino acid position of the respective peptide. A greater DoS score is utilized as a predictor of the likelihood that an antigen-recognition molecule which binds to the MHC-target peptide complex will also bind with a peptide in the working list of peptides. A greater likelihood of an off-target peptide binding to the antigen-recognition molecule therefore reflects a greater risk likelihood. In some embodiments, for off-target peptides for which immunopeptidomics data is available, off- target peptides that have been observed in mass-spectrometry experiments can provide additional evidence for the similarity of potential off-targets. For example, a similar peptide observed in the immunopeptidome (via mass-spectrometry) may be identified as an off-target peptide based on having a DoS score satisfying a DoS threshold that may be lower than a DoS threshold that would otherwise be used to identify a similar peptide as an off-target peptide when it has not been observed in the immunopeptidome (e.g., in some implementations a DoS score of 5 or greater may qualify a similar peptide as an off-target peptide if observed in the immunopeptidome, whereas a DoS score of 6 or greater may qualify a similar peptide that has not been observed in the immunopeptidome as an off-target peptide).

[0099] The risk likelihood filter 424 filters the working list to include only peptide(s) having a DoS score greater than a threshold value.

[0100] In the illustrated embodiment, the resulting working list represents a list of off- target peptides associated with the input MHC-target peptide complex 402. In the illustratedAttorney Docket No.250298.000720 embodiment, the PIGSPRED engine 410 provides an off-target peptide list, which is the resulting working list, as an output.

[0101] In the illustrated embodiment, a total count of off-target peptides in the off-target peptide list 426 is utilized as an off-target toxicity metric 428, which can be provided as a second output to the PIGSPRED engine 410. In some embodiments, the off-target toxicity metric 428 can be based at least in part on other factors such as DoS scores of off-target peptides (e.g. average DoS score of all off-target peptides), a numerical quantification of how “essential” the tissues and / or cell types are that are associated with the off-target peptides, alignment of protein amino acid sequences, and / or a probability score related to off-target likelihood. The toxicity metric 428 can therefore be a mathematical function of one or more factors related to the off-target peptides and the potential risks associated with the off-target peptides as understood by a person skilled in the pertinent art informed by the teachings herein.

[0102] In the illustrated embodiment, the PIGSPRED engine further provides a list of verified off-target peptides 430 including peptides for which immunopeptidomics data is available and has been used to verify similarity of the off-target peptides to the target peptide.

[0103] In some embodiments, the PIGSPRED engine can be configured to output results of intermediate steps including, but not limited to binding affinity, DoS scores, the working list at various stages of being filtered.

[0104] FIG.5 illustrates a block diagram of an exemplary embodiment of a computations system 500 configured to output a list of low risk peptide targets 558, off-target peptide list(s) for potential target(s) 526, and / or off-target toxicity metric(s) for potential target(s) 528. The system 500 includes an exemplary embodiment of a target ranking engine 550.

[0105] The illustrated target ranking engine 550 includes a potential target selection module 552, a PIGSPRED module 510, and a target ranking module 554.The target ranking engine 550 includes one or more processor(s) and non-transitory computer-readable medium with instructions thereon, that when executed by the one or more processor(s) cause the target ranking engine 550 to perform functions associated with each of the aforementioned features 552, 510, 554. In some embodiments, the target ranking engine 550 can be configured to provide as an output intermediate values determined by features 552, 510, 554 of the target ranking engine 550. The features 552, 510, 554 are illustrated separately and in a specific order for illustrative purposes. As understood by a person skilled in the pertinent art, the features 552, 510, 554 can be combined,Attorney Docket No.250298.000720 reordered, or otherwise implemented in numerous configurations to achieve the functionality disclosed in relation to the illustrated embodiment of the target ranking engine 550.

[0106] The illustrated system 500 further includes a binding affinity calculation engine 504, a disease expressing database 556, a canonical protein sequence database 506, and a tissue expression database 508. The binding affinity calculation engine 504, canonical protein sequence database 506, and tissue expression database 508 can be configured similarly to corresponding features 404, 406, 408 in FIG. 4. The disease expression database can include information to identify disease-specific MHC-target peptide complexes. In some examples, the disease expression database 556 can include TCGA database which include human gene sequences related to cancer. In a preferred embodiment, the binding affinity calculation engine 504, the disease expressing database 556, the canonical protein sequence database 506, and the tissue expression database 508 are separate from the target ranking engine 550, and the target ranking engine 550 is in communication with each. Alternatively, one or more of the ancillary features 504, 556, 506, 508 can be integral to the target ranking engine 550 as understood by a person skilled in the pertinent art.

[0107] The potential target selection module 552 is configured to be in communication with the disease expression database 556. The potential target selection module 552 is configured to determine a plurality of potential target peptides and associated MHC-target peptide complexes for each of the potential target peptides. The potential target selection module 552 can utilize the disease expression database 556 to identify potential target peptides based on type of disease under investigation, prevalence of the potential target peptide, etc. The potential target selection module 552 can determine binding affinity of the potential target peptides to one or more MHC molecules. The resulting MHC-target peptide complexes can be provided as input to the PIGSPRED module 510.

[0108] In the illustrated embodiment, the PIGSPRED module 510 functions similarly to the PIGSPRED engine 410 in FIG. 4, wherein MHC-target peptide complexes provided by the potential target selection module 552 each are provided as an input MHC-target peptide complex 402 to the system 400 in FIG. 4, and resulting off-target peptide list(s) 426, off-target toxicity metric(s) 428, and verified off-target peptide list 430 are provided as outputs for each MHC-target peptide complex respectively. In the illustrated embodiment, the off-target peptide list(s) forAttorney Docket No.250298.000720 potential target(s) 526, off-target toxicity metric(s) for potential target(s) 528, and verified off- target peptide list(s) 530 are provided as outputs to the target ranking engine 550.

[0109] The target ranking module 554 is configured to rank the MHC-target peptide complexes according to risk. In some examples, the target ranking module 554 can provide a list of low risk target(s) 558 as an output to the target ranking engine 550.

[0110] FIG.6 illustrates a block diagram of an exemplary embodiment of a target toxicity database 600 including MHC-target peptide complexes, a list of their respective associated off- target peptides, and an associated risk metric. In a preferred embodiment, the off-target lists and risk metrics can be determined for each MHC-target peptide complex using the system 400 illustrated in FIG.4. In a preferred embodiment, the MHC-target peptide complexes can be ranked using the system illustrated in FIG.5.

[0111] FIG. 7 illustrates a block diagram of an embodiment of a computing device 700. As shown, computing device 700 may include one or more processor(s) 710, an I / O device 720, a memory 730 containing an operating system ("OS") 740, a database 750, and a program 760. In a preferred embodiment, the PIGSPRED engine 410 illustrated in FIG. 4 is realized by the computing device 700. In another preferred embodiment, the target ranking engine 550 can be realized by the computing device 700. In these preferred embodiments, instructions are stored in the memory 730 that are executable by the processor 710 to perform functions of the respective features 412, 414, 416, 418, 420, 422, 424, 552, 510, 554 of the respective engines 410, 510. In these preferred embodiments, the I / O device 720 is configured to communicate with the respective ancillary features 404, 406, 408, 504, 556, 506, 508 of the respective systems 400, 500 illustrated in FIGs.4 and 5.

[0112] In some embodiments, the computing device 700 can include some or all of the features of the PIGSPRED engine 410 illustrated in FIG. 4 and / or the target ranking engine 550 illustrated in FIG.5 and additional features, which can be realized as instructions in the memory 730. In one example embodiment, the instructions, when executed by the processor(s) 710, cause the computational device 700 to provide, as an input, a computational representation of the antigen-recognition molecule, the antigen-recognition molecule being capable of binding to the MHC-target peptide complex; determine binding affinity of the antigen-recognition molecule to a plurality of MHC-peptide complexes each including a respective likely off-target peptide from the working list and the MHC molecule; and filter the working list to include only likely off-targetAttorney Docket No.250298.000720 peptides for which the respective MHC-peptide complex has a binding affinity to the antigen- recognition molecule. The example embodiment may be utilized to screen specific therapeutic antigen-recognition molecules. Assuming there is a list of off-targets that is agnostic to the antigen- recognition molecule being used, this example embodiment finds off-targets for a specific antigen- recognition molecule by removing off-targets from the agnostic list that do not bind to the antigen- recognition molecule.

[0113] In another example embodiment, the instructions, when executed by the processor(s) 710, cause the computational device 700 to provide, as an input, off-target peptide expression in essential, normal tissues or essential cell types of a specific patient; and provide, as an output, an indication of the off-target effects for said patient. The example embodiment may be utilized to screen patients for clinical trial or other treatment.

[0114] Computing device 700 may be a single server or may be configured as a distributed computer system including multiple servers or computers that interoperate to perform one or more of the processes and functionalities associated with the disclosed embodiments. In some embodiments, computing device 700 may further include a peripheral interface, a transceiver, a mobile network interface in communication with processor 710, a bus configured to facilitate communication between the various components of computing device 700, and a power source configured to power one or more components of computing device 700. A peripheral interface may include the hardware, firmware and / or software that enables communication with various peripheral devices, such as media drives (e.g., magnetic disk, solid state, or optical disk drives), other processing devices, or any other input source used in connection with the instant techniques. In some embodiments, a peripheral interface may include a serial port, a parallel port, a general- purpose input and output (GPIO) port, a game port, a universal serial bus (USB), a micro-USB port, a high definition multimedia (HDMI) port, a video port, an audio port, a BluetoothTMport, an NFC port, another like communication interface, or any combination thereof.

[0115] In some embodiments, a transceiver may be configured to communicate with compatible devices and ID tags when they are within a predetermined range. A transceiver may be compatible with one or more of: RFID, NFC, BluetoothTM, low-energy Bluetooth™ (BLE), WiFi™, ZigBee™, ABC protocols or similar technologies.

[0116] A mobile network interface may provide access to a cellular network, the Internet, or another wide-area network. In some embodiments, a mobile network interface may includeAttorney Docket No.250298.000720 hardware, firmware, and / or software that allows processor 710 to communicate with other devices via wired or wireless networks, whether local or wide area, private or public, as known in the art. A power source may be configured to provide an appropriate alternating current (AC) or direct current (DC) to power components.

[0117] Processor 710 may include one or more of a microprocessor, microcontroller, digital signal processor, co-processor or the like or combinations thereof capable of executing stored instructions and operating upon stored data. Memory 730 may include, in some implementations, one or more suitable types of memory (e.g., volatile or non-volatile memory, random access memory (RAM), read only memory (ROM), programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), magnetic disks, optical disks, floppy disks, hard disks, removable cartridges, flash memory, a redundant array of independent disks (RAID), and the like) for storing files, including an operating system, application programs (including, e.g., a web browser application, a widget or gadget engine, or other applications, as necessary), executable instructions, and data. In one embodiment, the processing techniques described herein are implemented as a combination of executable instructions and data within memory 730.

[0118] Processor 710 may be one or more known processing devices, such as a microprocessor from the PentiumTMfamily manufactured by IntelTMor the TurionTMfamily manufactured by AMDTM. Processor 710 may constitute a single core or multiple core processor that executes parallel processes simultaneously. For example, processor 710 may be a single core processor that is configured with virtual processing technologies. In certain embodiments, processor 710 may use logical processors to simultaneously execute and control multiple processes. Processor 710 may implement virtual machine technologies, or other similar known technologies to provide the ability to execute, control, run, manipulate, store, etc. multiple software processes, applications, programs, etc. Other types of processor arrangements could be implemented that provide for the capabilities disclosed herein as understood by a person skilled in the pertinent art.

[0119] Computing device 700 may include one or more storage devices configured to store information used by processor 710 (or other components) to perform certain functions related to the disclosed embodiments. In one example, computing device 700 may include memory 730 that includes instructions to enable processor 710 to execute one or more applications, such as serverAttorney Docket No.250298.000720 applications, network communication processes, and any other type of application or software known to be available on computer systems. Alternatively, the instructions, application programs, etc., may be stored in an external storage or available from a memory over a network. The one or more storage devices may be a volatile or non-volatile, magnetic, semiconductor, tape, optical, removable, non-removable, or other type of storage device or tangible computer-readable medium.

[0120] In one embodiment, computing device 700 may include memory 730 that includes instructions that, when executed by processor 710, perform one or more processes consistent with the functionalities disclosed herein. Methods, systems, and articles of manufacture consistent with disclosed embodiments are not limited to separate programs or computers configured to perform dedicated tasks. For example, computing device 700 may include memory 730 that may include one or more programs 760 to perform one or more functions of the disclosed embodiments. Moreover, processor 710 may execute one or more programs 760 located remotely from computing device 700. For example, computing device 700 may access one or more remote programs 760, that, when executed, perform functions related to disclosed embodiments.

[0121] Memory 730 may include one or more memory devices that store data and instructions used to perform one or more features of the disclosed embodiments. Memory 730 may also include any combination of one or more databases controlled by memory controller devices (e.g., server(s), etc.) or software, such as document management systems, MicrosoftTMSQL databases, SharePointTMdatabases, OracleTMdatabases, SybaseTMdatabases, or other relational databases. Memory 730 may include software components that, when executed by processor 710, perform one or more processes consistent with the disclosed embodiments. In some embodiments, memory 730 may include database 750 for storing related data to enable computing device 700 to perform one or more of the processes and functionalities associated with the disclosed embodiments.

[0122] Computing device 700 may also be communicatively connected to one or more memory devices (e.g., databases (not shown)) locally or through a network. The remote memory devices may be configured to store information and may be accessed and / or managed by computing device 700. By way of example, the remote memory devices may be document management systems, MicrosoftTMSQL database, SharePointTMdatabases, OracleTMdatabases, SybaseTMdatabases, or other relational databases. Systems and methods consistent with disclosed embodiments, however, are not limited to separate databases or even to the use of a database.Attorney Docket No.250298.000720

[0123] Computing device 700 may also include one or more I / O devices 720 that may include one or more interfaces for receiving signals or input from devices and providing signals or output to one or more devices that allow data to be received and / or transmitted by computing device 700. For example, computing device 700 may include interface components, which may provide interfaces to one or more input devices, such as one or more keyboards, mouse devices, touch screens, track pads, trackballs, scroll wheels, digital cameras, microphones, sensors, and the like, that enable computing device 700 to receive data from one or more users (such as via user device 130).

[0124] In example embodiments of the disclosed technology, computing device 700 may include any number of hardware and / or software applications that are executed to facilitate any of the operations. The one or more I / O interfaces may be utilized to receive or collect data and / or user instructions from a wide variety of input devices. Received data may be processed by one or more computer processors as desired in various implementations of the disclosed technology and / or stored in one or more memory devices.

[0125] While computing device 700 has been described as one form for implementing the techniques described herein, other, functionally equivalent techniques may be employed as understood by a person skilled in the pertinent art. For example, as known in the art, some or all of the functionality implemented via executable instructions may also be implemented using firmware and / or hardware devices such as application specific integrated circuits (ASICs), programmable logic arrays, state machines, etc. Furthermore, other implementations may include a greater or lesser number of components than those illustrated.

[0126] FIG.8 illustrates a block diagram of an embodiment of a computing network 800 including computing device(s) 810, server(s) 820, memory store(s) 840, and a network 830 facilitating communication between each. In a preferred embodiment, computing device(s) 810 includes a computing device configured as illustrated in FIG. 7 and including the PIGSPRED engine 410 in FIG.4 and / or the target ranking engine 550 in FIG.5. In some embodiments, the computing device(s) 810 communicate with some or all of the ancillary features 404, 406, 408, 504, 556, 506, 508 via the network 830, wherein said ancillary features are hosted on the server(s) 820 and / or accessible from the memory store(s) 840.

[0127] Network 830 may be of any suitable type, including individual connections via the internet such as cellular or WiFiTMnetworks. In some embodiments, network 830 may connectAttorney Docket No.250298.000720 terminals, services, and mobile devices using direct connections such as radio-frequency identification (RFID), near-field communication (NFC), BluetoothTM, low-energy BluetoothTM(BLE), WiFiTM, ZigBeeTM, ambient backscatter communications (ABC) protocols, USB, WAN, or LAN. Because the information transmitted may be personal or confidential, security concerns may dictate one or more of these types of connections be encrypted or otherwise secured. In some embodiments, however, the information being transmitted may be less personal, and therefore the network connections may be selected for convenience over security.

[0128] FIG.9 illustrates cellular functions related to the example embodiments presented herein. A cell 910 includes MHC-peptide complexes 920, 930 presented on a surface 912 of the cell 910. The cell 910 includes a class I MHC-peptide complex 920 and a class II MHC-peptide complex 930. Each MHC-peptide complex 920, 930 includes a respective MHC molecule 922, 932 and a respective peptide 924, 934. Each peptide 924, 934 includes amino acids that are bound to the respective MHC molecule 922, 932 (illustrated as shaded shapes) and amino acids that are unbound to the respective MHC molecule 922, 932 (illustrated as white shapes). An antigen- recognition molecule 940 includes a receptor 942 that can bind to amino acids of a peptide in an MHC-target peptide complex that are unbound to the MHC molecule. The antigen-recognition molecule 940 may also bind to off-target peptides which have a similar configuration of amino acids (compared to the MHC-target peptide complex) that are unbound to the respective MHC molecule in an MHC-peptide complex.

[0129] Various methods described herein may relate to identifying (e.g., ranking / selecting) target peptides and / or to identifying (e.g., ranking / selecting) off-target peptides for a given target peptide. Any of these methods may further comprise synthesizing a target peptide and / or one or more off-target peptides identified. Methods for peptide synthesis include those known in the art and described herein. Any of these methods may further comprise isolating (e.g., purifying) a target peptide and / or one or more off-target peptides identified. Methods for peptide isolation and / or purification include those known in the art and described herein. Any of these methods may further comprise loading a target peptide and / or one or more off-target peptides to an MHC molecule or any suitable component thereof to form a pMHC complex as described elsewhere herein. Any of these methods may further comprise binding a target peptide-MHC complex and / or one or more off-target peptide-MHC complexes to an antigen-recognition molecule (e.g., an antibody, TCR, or CAR). For example, any of these methods may comprise screening a targetAttorney Docket No.250298.000720 peptide-MHC complex and / or one or more off-target peptide MHC complexes for binding to an antigen-recognition molecule. Any of these methods may comprise incubating and / or contacting a target peptide and / or one or more off-target peptides with one or more cells (e.g., pulsing cells with peptide as described elsewhere herein).

[0130] In a further aspect, provided herein are off-target peptides identified using the methods described herein. Accordingly, the present disclosure also provides libraries (e.g., target- specific libraries) comprising one or more of the off-target peptides identified using the methods described herein. In some embodiments, the libraries of the present disclosure may include the target peptides associated with the off-target peptides as well. In some embodiments, the libraries of the present disclosure may include off-target peptides identified by analyzing experimental structures of a pHLA in complex with an antigen-recognition molecule.

[0131] An exemplary peptide library of the present disclosure may comprise one or more off-target peptides identified for MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) as described herein. In some embodiments, off-target peptides associated with MAGEA4230-239target GVYDGREHTV (SEQ ID NO: 1) include those listed in Tables 2B, 3B, and 4B herein. In some embodiments, an off-target peptide associated with MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) comprises an amino acid sequence of any of SEQ ID NOs: 2-7, 9, 11-28, and 73-74 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) consists essentially of an amino acid sequence of any of SEQ ID NOs: 2-7, 9, 11-28, and 73-74. In some embodiments, an off-target peptide associated with MAGEA4230-239target GVYDGREHTV (SEQ ID NO: 1) consists of an amino acid sequence of any one of SEQ ID NOs: 2-7, 9, 11-28, and 73-74.

[0132] Another exemplary peptide library of the present disclosure may comprise one or more off-target peptides identified for MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) as described herein. In some embodiments, off-target peptides associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) include those listed in Table 5B herein. In some embodiments, an off-target peptide associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) comprises an amino acid sequence of any of SEQ ID NOs: 49-72 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with MAGEA4286-294target KVLEHVVRV (SEQ ID NO: 48) consists essentially of anAttorney Docket No.250298.000720 amino acid sequence of any of SEQ ID NOs: 49-72. In some embodiments, an off-target peptide associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) consists of an amino acid sequence of any one of SEQ ID NOs: 49-72.

[0133] Another exemplary peptide library of the present disclosure may comprise one or more off-target peptides identified for MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) as described herein. In some embodiments, off-target peptides associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) include those listed in Table 6B herein. In some embodiments, an off-target peptide associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) comprises an amino acid sequence of any of SEQ ID NOs: 30-47 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with MAGEA3168-176target EVDPIGHLY (SEQ ID NO: 29) consists essentially of an amino acid sequence of any of SEQ ID NOs: 30-47. In some embodiments, an off-target peptide associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) consists of an amino acid sequence of any one of SEQ ID NOs: 30-47.

[0134] A yet further exemplary peptide library of the present disclosure may comprise one or more peptides selected from: MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1), SEQ ID NOs: 2-7, 9, 11-28, and 73-74; MAGEA4286-294target KVLEHVVRV (SEQ ID NO: 48), SEQ ID NOs: 49-72; MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29), and SEQ ID NOs: 30-47, or any combination thereof.

[0135] In some embodiments, off-target peptides identified using the methods described herein may be identified for a DNA nucleotidylexotransferase (DNTT)) target peptide, e.g., for DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75). DNA nucleotidylexotransferase (DNTT), also known as terminal deoxynucleotidyltransferase (TdT), terminal addition enzyme, and terminal transferase, is a DNA polymerase of the type-X family that catalyzes template- independent addition of nucleotides during junction formation in V(D)J recombination, which leads to an increase in antigen-receptor diversity in B and T lymphocytes. The DNTT protein is expressed in a population of both normal and malignant pre-T and pre-B lymphocytes during early differentiation. This generates diversity in antigen receptors through synthesizing non-germline elements at the junctions of T cell receptor gene segments and rearranged immunoglobulin heavy chain. Template-independent additions, non-template additions, and non-germline additions all refer to nucleotide additions catalyzed by DNTT, and these terms may be used interchangeably. InAttorney Docket No.250298.000720 some embodiments, the nucleic acid sequence is the genomic DNTT sequence (i.e., including exons and introns). In some embodiments, the nucleic acid sequence encodes DNTT mRNA / cDNA (i.e., the exons of one or more DNTT isoforms).

[0136] Human DNTT is encoded by the DNTT gene, which is located on human chromosome 10. An exemplary genomic DNA sequences of DNTT can be found at position 96304328 to 96338564 of NCBI accession number NC_000010.11, which is hereby incorporated by reference. Exemplary mRNA sequence of isoforms of DNTT is provided by NCBI accession numbers NM_001017520.1 and NM_004088.3, each of which is hereby incorporated by reference. The protein sequences encoded by these isoforms is provided by NCBI accession numbers NP_001017520.1 and NP_004079.3, respectively, each of which is hereby incorporated by reference. Among the TdT isoforms is a short isoform (TdTS) and two long isoforms (TdTL1 and TdTL2). The sequences of the three isoforms are provided, for example, in Thai and Kearney, Adv. Immunol.86:113-36 (2005), which is hereby incorporated by reference.

[0137] Without wishing to be bound by theory, DNTT is associated with diseases such as B-lymphoblastic leukemia / lymphoma, myeloid sarcoma, acute leukemia, lymphoblastic lymphoma, and thymoma. Related pathways of DNTT are DNA-PK in nonhomologous end joining and C-MYC transcriptional repression.

[0138] In some embodiments, DNTT is associated with cancer. In one embodiment, the cancer is a gastrointestinal cancer. In one embodiment, the cancer is a pancreatic cancer. In one embodiment, the cancer is a colorectal cancer. In one embodiment, the cancer is a lung cancer. In one embodiment, the cancer is a glioma cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is a head and neck cancer. In one embodiment, the cancer is a bladder cancer. In one embodiment, the cancer is a prostate cancer. In some embodiments, the cancer is a hematologic malignancy, for example, a lymphoma, a leukemia, or a myeloma. In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin’s lymphoma. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), or acute myeloid leukemia (AML). In some embodiments, the cancer is a solid cancer. In one embodiment, the cancer is a head and neck cancer. In one embodiment, the cancer is a renal cell carcinoma. In one embodiment, the cancer is a breast cancer, e.g., triple-negative breast cancer (TNBC). In one embodiment, the cancer is a non-small cell lung cancer (NSCLC), for example,Attorney Docket No.250298.000720 lung adenocarcinoma. In one embodiment, the cancer is liver hepatocellular carcinoma (HCC). In one embodiment, the cancer is lung squamous cell carcinoma (SCC). In one embodiment, the cancer is bladder cancer. In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is uveal melanoma. In one embodiment, the cancer is nasopharyngeal cancer. In one embodiment, the cancer is synovial sarcoma. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is uterine cancer. In one embodiment, the cancer is endometrial cancer. In one embodiment, the cancer is melanoma.

[0139] In various embodiments, Authors contemplate using a variety of platforms for targeting a DNTT, such as, but not limited to, alternative format multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, CAR T-cells (e.g., scFv-based CAR T cells) and engineered (T-cell receptor) TCR-T cells, and cancer vaccines. In one embodiment, anti-tumor TCRs may be engineered into T cells for cell-based therapy. In one embodiment, an anti-tumor TCR targeting DNTT250-258 peptide target KLFTSVFGV (SEQ ID NO: 75)- HLA-A*02:01 can be developed for a HLA-A*02:01 / DNTT250-258-targeted autologous TCR T cell therapy.

[0140] Without wishing to be bound by theory, TCRs can be a promising therapeutic modality for the treatment of patients with solid tumors. TCRs greatly expand the repertoire of tumor antigens that can be targeted using immunotherapy, compared to CARs and traditional antibodies. Over 75% of proteins reside exclusively within intracellular compartments. Tumor- associated antigens (TAAs) and tumor-specific antigens (TSAs) typically consist of peptides derived from intracellular proteins that are loaded onto MHC. TCRs recognize tumor antigens via interactions with peptide-MHC (pMHC) complexes. Intracellular antigens undergo proteasomal cleavage to generate antigenic peptides. Peptides are transported into the ER, where they are further processed and loaded onto MHC. pMHC complexes are subsequently trafficked to the cell surface to enable extracellular presentation of intracellular antigens. TCRs are therefore a powerful tool that which can use to leverage the adaptive immune system in targeting and eliminating solid tumors. Ensuring tumor specificity of a target p-MHC is not enough for therapeutic TCR / antibody development; off-target toxicity remains a challenge.

[0141] In certain embodiments, the DNTT peptide described herein is wild type. In certain embodiments, the DNTT peptide described herein comprises a mutation. Non-limiting examples of amino acid mutations comprise amino acid insertions, substitutions, and / or deletions. Inserted amino acid residues may be inserted at any position and inserted amino acid residues may beAttorney Docket No.250298.000720 inserted in a way such that some of or all the inserted amino acid residues are immediately adjacent to one another or such that none of the inserted amino acid residues are immediately adjacent to one another. Amino acid substitution means exchanging an amino acid residue for a replacement amino acid residue at the same position.

[0142] In some embodiments, the DNTT peptide described herein comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 amino acid residues of a DNTT protein. In some embodiments, the peptide comprises 9 amino acid residues.

[0143] In various embodiments, a peptide library of the present disclosure may comprise one or more off-target peptides identified for DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) as described herein. In some embodiments, off-target peptides associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75) include those listed in Table 10 herein. In some embodiments, an off-target peptide associated with DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) comprises an amino acid sequence of any of SEQ ID NOs: 76-82 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) consists essentially of an amino acid sequence of any of SEQ ID NOs: 76-82. In some embodiments, an off-target peptide associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75) consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0144] In various embodiments, a peptide library of one or more off-target peptides for an identified target peptide may further comprise the target peptide itself (e.g., DNTT250-258target KLFTSVFGV (SEQ ID NO: 75)). In some embodiments, the target peptide comprises an amino acid sequence of SEQ ID NO: 75 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, the target peptide consists essentially of an amino acid sequence of SEQ ID NO: 75. In some embodiments, the target peptide consists of an amino acid sequence of SEQ ID NO: 75. In various embodiments, a peptide library of one or more off-target peptides for an identified target peptide, optionally including the target peptide itself (e.g., DNTT250-258target KLFTSVFGV (SEQ ID NO: 75) may further include other variants of the target peptide, such as a wild-type / non-mutated version of the target peptide. The target peptide and / or wild-type version of the target peptide may be used, for example, in comparison studies with the one or more off-target peptides, such as to assess antigen-recognition molecule cross-reactivity with off-target peptides.Attorney Docket No.250298.000720

[0145] In some embodiments, an off-target peptide associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75) may be cross-reactive to an antigen-recognition molecule targeting DNTT250-258 such as an antibody or TCR. In some embodiments, an antigen-recognition molecule described herein, such as an antibody or TCR, may react to cells that present DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) in complex with HLA-A*02:01 on a cell surface. In some embodiments, an antigen-recognition molecule described herein, such as an antibody or TCR, may be cross-reactive with an off-target peptide associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75).

[0146] In some embodiments, an off-target peptide associated with DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) comprises an amino acid sequence of any of SEQ ID NOs: 76- 82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75) consists essentially of an amino acid sequence of any of SEQ ID NOs: 76-82. In some embodiments, an off-target peptide associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75) consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0147] In some embodiments, a peptide library comprising off-target peptides described herein may comprise two or more of any of various off-target peptides described herein. As a non- limiting example, a peptide library comprising off-target peptides may comprise two or more peptides each selected from the amino acid sequences of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative. In a specific embodiment, the isolated peptide can be, e.g., 8-12 amino acids in length. In a specific embodiment, the isolated peptide can be 9-12 amino acids in length. In a specific embodiment, the isolated peptide is 9 amino acids in length. In some embodiments, the at least two off-target peptides can each present in a complex with a major histocompatibility complex (MHC) molecule described herein. In certain embodiments, the MHC molecule is a class I MHC molecule such as, but not limited to, a class I human leukocyte antigen (HLA) molecule. The class I human leukocyte antigen (HLA) molecule can be an HLA-A molecule. In some embodiments, the HLA-A molecule is an HLA- A*02 molecule. In some specific embodiments, the HLA-A molecule is an HLA-A*02:01 molecule.

[0148] In some embodiments, a peptide within an MHC-peptide complex described herein, e.g., a DNTT250-258peptide complex, and / or an MHC-off-target peptide complex described herein,Attorney Docket No.250298.000720 e.g., an MHC-off-target peptide complex comprising an off-target peptide associated with DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75), can be covalently bound to an MHC described herein.

[0149] In some embodiments, a peptide within an MHC-peptide complex described herein, e.g., a DNTT250-258 peptide complex, and / or an MHC-off-target peptide complex described herein, e.g., an MHC-off-target peptide complex comprising an off-target peptide associated with DNTT250-258target KLFTSVFGV (SEQ ID NO: 75), can be non-covalently bound to an MHC described herein.

[0150] In various embodiments, the present disclosure provides a library comprising two or more proteins or fragments thereof each comprising one or more off-target peptides described herein. In some embodiments, each off-target peptide can be selected from the amino acid sequences of SEQ ID NOs: 76-82.

[0151] Peptide libraries of present disclosure may comprise any number of a plurality of peptides as desired. For example, a peptide library of the present disclosure may comprise at least 2 peptides, at least 3 peptides, at least 4 peptides, at least 5 peptides, at least 6 peptides, at least 7 peptides, at least 8 peptides, at least 9 peptides, at least 10 peptides, at least 20 peptides, at least 30 peptides, at least 40 peptides, at least 50 peptides, or about 2-5 peptides, about 2-10 peptides, about 5-15 peptides, about 10-20 peptides, about 10-30 peptides, about 12-25 peptides, about 20- 30 peptides, about 25-50 peptides, about 40-80 peptides, about 50-100 peptides, about 60-120 peptides, about 70-140 peptides, about 80-160 peptides, about 90-180 peptides, about 100-200 peptides, about 110-220 peptides, about 120-240 peptides, about 130-260 peptides, about 140-280 peptides, about 150-300 peptides, about 160-320 peptides, about 170-340 peptides, about 180-360 peptides, about 190-380 peptides, about 200-400 peptides, about 210-420 peptides, about 220-440 peptides, about 230-460 peptides, about 240-480 peptides, about 250-500 peptides, about 260-520 peptides, about 270-540 peptides, about 280-560 peptides, about 290-580 peptides, about 300-600 peptides, about 310-620 peptides, about 320-640 peptides, about 330-660 peptides, about 340-680 peptides, about 350-700 peptides, about 360-720 peptides, about 370-740 peptides, about 380-760 peptides, about 390-780 peptides, about 400-800 peptides, about 410-820 peptides, about 420-840 peptides, about 430-860 peptides, about 440-880 peptides, about 450-900 peptides, about 460-920 peptides, about 470-940 peptides, about 480-960 peptides, about 490-980 peptides, about 500- 1000 peptides, etc.Attorney Docket No.250298.000720

[0152] In a further aspect, provided herein are databases including computational representations of the off-target peptides identified using the methods described herein. The database can include information for each of the off-target peptides represented in any one of the exemplary libraries disclosed hereinabove. Accordingly, the databases may include computational representations of the target peptides associated with the off-target peptides as well. The computer- readable representations of the peptides in the database can include a peptide sequence or a computer model of the peptide. For instance, the database can include features of the database 600 illustrated in FIG.6, including one or more lists of off-target peptides. The database 600 may or may not include the associated MHC-target peptide complex, and may or may not include the associated risk metric.

[0153] A peptide of the disclosure may be synthetically produced or produced by hydrolysis. Synthetically produced peptides can include randomly generated peptides, specifically designed peptides, and peptides where at least some of the amino acid positions are conserved among several peptides and the remaining positions are random. Alternatively, a peptide of the present disclosure may be produced by expression in a heterologous host cell.

[0154] In some embodiments, peptides of the disclosure can be synthesized by e.g., solid phase synthesis. As such, the peptides may be immobilized, for example to a solid support such as a bead. Peptides of the disclosure may be synthesized by the Fmoc-polyamide mode of solid- phase peptide synthesis. Temporary N-amino group protection is afforded by the 9- fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is done using 20% piperidine in N, N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6- trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4′-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl- phenoxyacetic acid derivative. All amino acid derivatives are added as their preformedAttorney Docket No.250298.000720 symmetrical anhydride derivatives except for asparagine and glutamine, which are added using a reversed N, N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used include ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Also, a combination of solid phase and solution phase methodologies for the synthesis of peptides is possible.

[0155] Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers.

[0156] Purification may be performed by techniques such as re-crystallization, ion- exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography and reverse-phase high performance liquid chromatography using e.g. acetonitrile / water gradient separation, or a combination thereof.

[0157] Peptides may be analyzed using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.

[0158] Alternatively, the peptide may be produced by recombinant expression in a heterologous host cell. Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the peptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.

[0159] In further embodiments, in vitro cell-free systems may be used. The peptides may be isolated and / or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other peptides or proteins.

[0160] In some embodiments, the peptides of the disclosure may be about 8-25 amino acids in length. In some embodiments, the peptides of the disclosure may be about 8-12 amino acids inAttorney Docket No.250298.000720 length. For example, a peptide disclosed herein may be 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids in length. In some embodiments, the peptides of the disclosure may be about 13-17 amino acids in length. For example, a peptide disclosed herein may be 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, or 17 amino acids in length.

[0161] The peptides of the disclosure may comprise one or more chemical modifications. Non-limiting examples of chemical modifications include, for example, phosphorylation, acetylation, deamidation acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH. Chemical modifications may correspond to those that are not present in vivo.

[0162] For example, modification of, for example, arginyl residues in proteins may be based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. Selective reduction of disulfide bonds in proteins can also be performed. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward’s Reagent K may be used to modify specific glutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intra-molecular crosslinks between a lysine residue and a glutamic acid residue. For example, diethylpyrocarbonate and 4-hydroxy-2-nonenal can be used to modify histidyl residues in proteins. The reaction of lysine residues and other α-amino groups is, for example, useful in binding of peptides to surfaces or the cross-linking of proteins / peptides. Lysine is the site of attachment of poly(ethylene)glycol and the major site of modification in the glycosylation of proteins. Methionine residues in proteins can be modified with e.g. iodoacetamide, bromoethylamine, and chloramine T. Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking via the formation of dityrosine can be accomplished with hydrogen peroxide / copper ions. N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-Attorney Docket No.250298.000720 methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole) have been used in recent studies for the modification of tryptophan.

[0163] Peptides described herein may comprise one or more (e.g., 1, 2, 3, or 4) amino acid substitutions and / or insertions and / or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue. One or more (e.g., 1, 2, 3 or 4) amino acids may be substituted and / or inserted and / or deleted from the sequence of any one of SEQ ID NOs: 1-7, 9, and 11-74. Each substitution and / or insertion and / or deletion can take place at any position of any one of SEQ ID NOs: 1-7, 9, and 11- 74.

[0164] In some embodiments, the peptides of the disclosure may comprise additional amino acids (e.g., 1, 2, 3 or 4) at the C-terminal end and / or at the N-terminal end of the sequence of any one SEQ ID NOs: 1-7, 9, and 11-74. A peptide of the disclosure may comprise the amino acid sequence of any one of SEQ ID NOs: 1-7, 9, and 11-74 except for one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions or deletions.

[0165] Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. For example, the following groups of amino acids share similar chemical properties such as size, charge, and polarity: Group 1 - Ala, Ser, Thr, Pro, Gly; Group 2 - Asp, Asn, Glu, Gln; Group 3 - His, Arg, Lys; Group 4 - Met, Leu, Ile, Val, Cys; Group 5 - Phe, Thy, Trp.

[0166] In another aspect, the disclosure provides a complex of a peptide of the disclosure and an MHC molecule (pMHC complex). Preferably, the peptide is bound to the peptide binding groove of the MHC molecule. In some embodiments, the peptide and the MHC molecule form a non-covalent complex. In other embodiments, the peptide and the MHC molecule may be covalently linked, for example, via a linker. Accordingly, the present disclosure also provides libraries comprising one or more of the pMHC complexes described herein.

[0167] An exemplary pMHC complex library of the present disclosure may comprise one or more pMHC complexes comprising an off-target peptide associated with MAGEA4230-239target GVYDGREHTV (SEQ ID NO: 1) as described herein. In some embodiments, off-target peptidesAttorney Docket No.250298.000720 associated with MAGEA4230-239target GVYDGREHTV (SEQ ID NO: 1) present in the pMHC complexes include those listed in Tables 2B, 3B, and 4B herein. In some embodiments, an off- target peptide associated with MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) present in a pMHC complex comprises an amino acid sequence of any of SEQ ID NOs: 2-7, 9, 11-28, and 73-74 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) present in a pMHC complex consists essentially of an amino acid sequence of any of SEQ ID NOs: 2-7, 9, 11-28, and 73-74. In some embodiments, an off-target peptide associated with MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1) present in a pMHC complex consists of an amino acid sequence of any one of SEQ ID NOs: 2-7, 9, 11-28, and 73-74.

[0168] Another exemplary pMHC complex library of the present disclosure may comprise one or more pMHC complexes comprising an off-target peptide associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) as described herein. In some embodiments, off-target peptides associated with MAGEA4286-294target KVLEHVVRV (SEQ ID NO: 48) present in pMHC complexes include those listed in Table 5B herein. In some embodiments, an off-target peptide associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) present in a pMHC complex comprises an amino acid sequence of any of SEQ ID NOs: 49-72 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48) present in a pMHC complex consists essentially of an amino acid sequence of any of SEQ ID NOs: 49-72. In some embodiments, an off-target peptide associated with MAGEA4286-294target KVLEHVVRV (SEQ ID NO: 48) present in a pMHC complex consists of an amino acid sequence of any one of SEQ ID NOs: 49-72.

[0169] Another exemplary pMHC complex library of the present disclosure may comprise one or more pMHC complexes comprising an off-target peptide associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) as described herein. In some embodiments, off-target peptides associated with MAGEA3168-176target EVDPIGHLY (SEQ ID NO: 29) include those listed in Table 6B herein. In some embodiments, an off-target peptide associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) present in pMHC complexes comprises an amino acid sequence of any of SEQ ID NOs: 30-47 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associatedAttorney Docket No.250298.000720 with MAGEA3168-176target EVDPIGHLY (SEQ ID NO: 29) present in a pMHC complex consists essentially of an amino acid sequence of any of SEQ ID NOs: 30-47. In some embodiments, an off-target peptide associated with MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29) present in a pMHC complex consists of an amino acid sequence of any one of SEQ ID NOs: 30-47.

[0170] A yet further exemplary pMHC complex library of the present disclosure may comprise one or more pMHC complexes comprising a peptide selected from: MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1), SEQ ID NOs: 2-7, 9, 11-28, and 73-74; MAGEA4286-294target KVLEHVVRV (SEQ ID NO: 48), SEQ ID NOs: 49-72; MAGEA3168-176 target EVDPIGHLY (SEQ ID NO: 29), and SEQ ID NOs: 30-47, or any combination thereof.

[0171] In a preferred exemplary pMHC complex library of the present disclosure may comprise one or more pMHC complexes comprising an off-target peptide associated with DNTT250-258 peptide target KLFTSVFGV (SEQ ID NO: 75) as described herein. In some embodiments, off-target peptides associated with DNTT250-258 peptide target KLFTSVFGV (SEQ ID NO: 75) present in the pMHC complexes include those listed in Table 10 herein. In some embodiments, an off-target peptide associated with DNTT250-258 peptide target KLFTSVFGV (SEQ ID NO: 75) present in a pMHC complex comprises an amino acid sequence of any of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an off-target peptide associated with DNTT250-258 peptide target KLFTSVFGV (SEQ ID NO: 75) present in a pMHC complex consists essentially of an amino acid sequence of any of SEQ ID NOs: 76-82. In some embodiments, an off-target peptide associated with DNTT250-258peptide target KLFTSVFGV (SEQ ID NO: 75) present in a pMHC complex consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0172] pMHC complex libraries of present disclosure may comprise any number of a plurality of pMHC complexes as desired. For example, a pMHC complex library of the present disclosure may comprise at least 2 pMHC complexes, at least 3 pMHC complexes, at least 4 pMHC complexes, at least 5 pMHC complexes, at least 6 pMHC complexes, at least 7 pMHC complexes, at least 8 pMHC complexes, at least 9 pMHC complexes, at least 10 pMHC complexes, at least 20 pMHC complexes, at least 30 pMHC complexes, at least 40 pMHC complexes, at least 50 pMHC complexes, or about 2-5 pMHC complexes, about 2-10 pMHC complexes, about 5-15 pMHC complexes, about 10-20 pMHC complexes, about 10-30 pMHC complexes, about 12-25 pMHC complexes, about 20-30 pMHC complexes, about 25-50 pMHC complexes, about 40-80 pMHCAttorney Docket No.250298.000720 complexes, about 50-100 pMHC complexes, about 60-120 pMHC complexes, about 70-140 pMHC complexes, about 80-160 pMHC complexes, about 90-180 pMHC complexes, about 100- 200 pMHC complexes, about 110-220 pMHC complexes, about 120-240 pMHC complexes, about 130-260 pMHC complexes, about 140-280 pMHC complexes, about 150-300 pMHC complexes, about 160-320 pMHC complexes, about 170-340 pMHC complexes, about 180-360 pMHC complexes, about 190-380 pMHC complexes, about 200-400 pMHC complexes, about 210-420 pMHC complexes, about 220-440 pMHC complexes, about 230-460 pMHC complexes, about 240-480 pMHC complexes, about 250-500 pMHC complexes, about 260-520 pMHC complexes, about 270-540 pMHC complexes, about 280-560 pMHC complexes, about 290-580 pMHC complexes, about 300-600 pMHC complexes, about 310-620 pMHC complexes, about 320-640 pMHC complexes, about 330-660 pMHC complexes, about 340-680 pMHC complexes, about 350-700 pMHC complexes, about 360-720 pMHC complexes, about 370-740 pMHC complexes, about 380-760 pMHC complexes, about 390-780 pMHC complexes, about 400-800 pMHC complexes, about 410-820 pMHC complexes, about 420-840 pMHC complexes, about 430-860 pMHC complexes, about 440-880 pMHC complexes, about 450-900 pMHC complexes, about 460-920 pMHC complexes, about 470-940 pMHC complexes, about 480-960 pMHC complexes, about 490-980 pMHC complexes, about 500-1000 pMHC complexes, etc.

[0173] MHC molecules used in pMHC complexes described herein include naturally occurring full-length MHC molecules as well as individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHCs (e.g., α1, α2 and / or α3 subunits of MHC class I α chain, α1 and / or α2 subunits of MHC class II α chain, β1 and / or β2 subunits of MHC class II β chain) as well as fragments, mutants, and various derivatives thereof (including fusion proteins, e.g., fusions with viral envelope proteins or fusogens), wherein such fragments, mutants, and derivatives retain the ability to display an antigenic determinant for recognition by an antigen- recognition molecule.

[0174] Naturally-occurring MHC molecules are encoded by a cluster of genes on human chromosome 6 or mouse chromosome 17. MHCs are also referred to as H-2 in mice and Human Leucocyte Antigen (HLA) in humans. MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes (CD8+ T cells), whereas MHC class II molecules specifically bind CD4 molecules expressed on helper T lymphocytes (CD4+ T cells). MHCsAttorney Docket No.250298.000720 include, but are not limited to, HLA specificities such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), E, G, DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9) and DP (e.g., DP1-DP6).

[0175] In some embodiments, the MHC molecule in a pMHC complex of the present disclosure is a human leukocyte antigen (HLA) molecule. The MHC molecule may be a human HLA molecule selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the MHC class I or MHC II polypeptides may be derived from any functional human HLA-A, B, C, DR, or DQ molecules. Non-limiting examples of HLA-A alleles comprise, without limitation, A*01:01, A*02:01, A*02:02, A*03:01, A*11:01, A*23:01, A*24:02, A*25:01, A*26:01, A*29:01, A*29:02, A*31:01, A*32:01, A*33:01, A*34:01, A*36:01, A*43:01, A*66:01, A*68:01, A*69:01, A*74:01, and A*80:01. Non-limiting examples of HLA-B alleles comprise, without limitation, B*07:02. B*08:01, B*13:01, B*14:01, B*14:02, B*15:01, B*18:01, B*18:02, B*27:01, B*27:02, B*35:01, B*35:02, B*37:01, B*38:01, B*39:01, B*40:01, B*41:01, B*42:01, B*44:02, B*45:01, B*46:01, B*47:01, B*48:01, B*49:01, B*50:01, B*51:01, B*52:01, B*53:01, B*54:01, B*55:01, B*55:02, B*56:01, B*57:01, B*58:01, B*59:01, B*67:01, B*73:01, B*15:17, B*81:01, B*82:01, and B*83:01. Non-limiting examples of HLA-C alleles comprise, without limitation, C*01:01, C*02:02, C*03:03, C*04:01, C*05:01, C*06:02, C*07:01, C*07:02, C*08:02, C*12:03, C*14:01, C*15:02, C*16:01, C*17:01, and. C*18:01. Non- limiting examples of HLA-DR alleles comprise, without limitation, DRB1*01:01, DRB1*01:03, DRB1*15:01, DRB1*15:02, DRB1*16:01, DRB1*16:02, DRB1*03:01, DRB1*04:01, DRB1*04:04, DRB1*11:01, DRB1*12:01, DRB1*13:01, DRB1*13:02, DRB1*14:01, DRB1*14:02, DRB1*07:01, DRB1*08:01, DRB1*08:02, DRB1*08:03, DRB1*09:01, and DRB1*10:01.

[0176] In some embodiments, the MHC class I molecule may be selected from HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-B*07, HLA-B*08, HLA- B*40, HLA-B*44, HLA-B*15, HLA-C*04, HLA*C*03, and HLA-C*07. There are also allelic variants of the above HLA types, all of which are encompassed by the present disclosure.

[0177] In some embodiments, the MHC molecule may be HLA-A*02:01 or HLA- A*01:01.

[0178] Naturally occurring MHC class I molecules consist of an α (heavy) chain associated with β2-microglobulin. The heavy chain consists of subunits α1-α3. The β2-microglobulin proteinAttorney Docket No.250298.000720 and α3 subunit of the heavy chain are associated. In certain embodiments, β2-microglobulin and α3 subunit are covalently bound. In certain embodiments, β2-microglobulin and α3 subunit are non-covalently bound. The α1 and α2 subunits of the heavy chain fold to form a groove for a peptide to be displayed and recognized by TCR.

[0179] In some embodiments, the MHC contained in a pMHC complex of the disclosure comprises (i) a class I MHC polypeptide or a fragment, mutant or derivative thereof, and, optionally, (ii) a β2 microglobulin polypeptide or a fragment, mutant or derivative thereof. In one specific embodiment, the class I MHC polypeptide is linked to the β2 microglobulin polypeptide by a peptide linker.

[0180] pMHC complexes of the disclosure may be isolated and / or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other peptides or proteins. MHC molecules as disclosed herein can include recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained. For example, MHC molecules may be attached to a solid support, in soluble form, attached to a tag, biotinylated and / or in multimeric form. A peptide disclosed herein may be covalently attached to the MHC.

[0181] Methods to produce soluble recombinant MHC molecules with which peptides disclosed herein can form a complex include, but are not limited to, expression and purification from E. coli cells or insect cells. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

[0182] The peptides disclosed herein may be presented on the surface of a cell in complex with MHC. Thus, the present disclosure also provides a cell presenting on its surface a pMHC complex disclosed herein. Such a cell may be a mammalian cell, preferably a cell of the immune system, and a specialized antigen-presenting cell (APC) such as a dendritic cell or a B cell. The cell may be an immortalized cell. The cell may be a cell that does not naturally present the peptide of interest. In certain embodiments, the cell may be a cell that is deficient in presenting endogenous peptides in complex with MHC on the cell surface. For example, the cell may be a cell that has been genetically modified to be deficient in endogenous peptide presentation, such as a cell that has been modified to be transporter associated with antigen processing (TAP) deficient. The cell may be a cell that requires loading exogenous peptide on the cell surface in order to measurablyAttorney Docket No.250298.000720 detect antigen-recognition molecule binding to the peptide. Incubation of the cell with exogenous β2 microglobulin (β2m) may increase the loading / presentation of exogenously administered peptides. Preferred cells may include T2 cells, which are well known in the art. See, e.g., Elliot et al., J Exp Med. 1995 Apr 1;181(4):1481-91, which is herein incorporated by reference in its entirety. Cells presenting a peptide or pMHC complex of the disclosure may be isolated, preferably in the form of a homogenous population, or provided in a substantially pure form. Such cells may not naturally present a pMHC complex of the disclosure, or alternatively said cells may present the pMHC complex at a level higher than they would in nature. Cells presenting pMHC complexes may be obtained by pulsing said cells with one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure, or genetically modifying the cells (via DNA or RNA transfer) to express one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure. Pulsing involves incubating the cells with the peptide for several hours using peptide concentrations typically ranging from 10−5to 10−12M. Pulsing may also involve incubating the cells with exogenous β2m molecules, for example at the same or in similar concentrations as the peptide. Such cells may additionally be transduced with HLA molecules, such as HLA-A*02 or HLA-C*08 to further induce presentation of the peptide(s). Cells may be produced recombinantly. Cells presenting peptides of the disclosure may be used to isolate antigen-binding molecules (e.g., antibodies, T cells, TCRs and CARs) which can bind to the cells.

[0183] Peptides or pMHC complexes disclosed herein may be fused or conjugated to one or more heterologous molecules. Peptides or pMHC complexes of the disclosed herein may also be in multimeric form. Accordingly, the present disclosure also provides fusion proteins, conjugates, and oligomeric complexes comprising a peptide or a pMHC complex of the disclosure.

[0184] In some embodiments, peptides are fused or conjugated to one or more heterologous molecules which can include an MHC molecule (or fragments thereof).

[0185] Heterologous molecules suitable for genetical fusion and / or chemical conjugation with the peptides or the pMHC complexes of the disclosure include, but are not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc. The heterologous molecule(s) may be fused at the N- and / or C-terminus of the peptide and / or another polypeptide chain in the pMHC complex.

[0186] Heterologous peptides and polypeptides include, but are not limited to, an epitope (e.g., FLAG) or a tag sequence (e.g., His6(SEQ ID NO: 133), and the like) to allow for theAttorney Docket No.250298.000720 detection and / or isolation of a fusion protein; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region (e.g., an Fc domain); a half-life-extending sequence comprising a combination of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non-naturally occurring charged and / or uncharged amino acids (e.g., Ser, Gly, Glu or Asp) designed to form a predominantly hydrophilic or predominantly hydrophobic fusion partner for a fusion protein; a functional or non-functional antibody (e.g., an antibody that is specific for dendritic cells), or a heavy or light chain thereof; and a polypeptide which has an activity different from fusion proteins of the present disclosure.

[0187] In some embodiments, fusion proteins of the disclosure may comprise one or more affinity tags, e.g., to allow for affinity purification or coupling to another molecule. Examples of affinity tags include, but are not limited to, a His6 tag (SEQ ID NO: 133), an Avi-tag, a biotin, a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, a GST tag, a MBP tag, a chitin binding protein tag, a calmodulin tag, a V5 tag, a streptavidin binding tag, a green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, SUMO tag, and Ubiquitin tag.

[0188] Peptides or pMHC complexes of the disclosure may be provided in soluble form, or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead (e.g., a magnetic bead), a membrane, sepharose, a plate, a tube, a column, etc. pMHC complexes may be attached, for example, to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching peptides or pMHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens. In some embodiments, peptides or pMHC complexes are labeled with biotin and attached to streptavidin- coated surfaces.

[0189] In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding one or more peptide(s) and / or peptide-based molecules (such as complexes (e.g., pMHC complexes), fusion proteins, or conjugates comprising the described peptides) of the disclosure. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orAttorney Docket No.250298.000720 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.

[0190] In some embodiments, a polynucleotide described herein encodes a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-7, 9, and 11-74, or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide consisting essentially of an amino acid sequence of any one of SEQ ID NOs: 1-7, 9, and 11-74, or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1-7, 9, and 11-74, or a fragment or derivative thereof.

[0191] In some embodiments, a polynucleotide described herein encodes a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 76-82, or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide consisting essentially of an amino acid sequence of any one of SEQ ID NOs: 76-82, or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 76-82, or a fragment or derivative thereof.

[0192] In a further aspect, the disclosure provides a vector comprising a nucleic acid sequence of the disclosure. The vector may include, in addition to a nucleic acid sequence encoding only a peptide of the disclosure, one or more additional nucleic acid sequences encoding one or more additional peptides. Such additional peptides may, once expressed, be fused to the N- terminus or the C-terminus of the peptide of the disclosure. Examples of such additional peptides are detailed in the sections above. In one embodiment, the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC- tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.

[0193] The off-target peptides identified herein may be used for evaluation and / or screening of therapeutic molecules for molecules that have minimal off-target effects. Such therapeutic molecules can include antigen-recognition molecules such as T cell Receptors (TCRs), chimeric antigen receptors (CARs), and antibodies or antigen-binding fragments thereof. In various embodiments of the present disclosure, the antigen-recognition molecule is present in a solution. In various embodiments of the present disclosure, the antigen-recognition molecule isAttorney Docket No.250298.000720 present on a cell (e.g., T cell, B cell or hybridoma). In certain embodiments, the antigen- recognition molecule is present on a heterologous cell. For example, a heterologous host cell may be genetically modified to express the antigen recognition molecule as is known in the art.

[0194] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule, said method comprising a) contacting the antigen- recognition molecule with a target peptide presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-target peptide complex); b) contacting the antigen-recognition molecule with one or more off-target peptides associated with said target peptide, wherein each of said off-target peptides is presented in a complex with the same MHC molecule as in a) (MHC- off-target peptide complex); and c) determining and comparing the level of binding of the antigen- recognition molecule to MHC-target peptide complex and each of the MHC-off-target peptide complexes.

[0195] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule, comprising a) contacting the antigen-recognition molecule with one or more off-target peptides associated with a target peptide that is recognized by the antigen-recognition molecule, wherein each of said off-target peptides is presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-off-target peptide complex); and b) determining the level of binding of the antigen-recognition molecule to each of the MHC-off-target peptide complexes.

[0196] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258peptide, comprising a) contacting the antigen-recognition molecule with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC- DNTT250-258peptide complex); b) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) is presented in a complex with the same kind of MHC molecule, or the fragment or derivative thereof, as in (a) (MHC-off-target peptide complex); and c) determining the level of binding of the antigen- recognition molecule to the MHC-DNTT250-258peptide complex and each of the MHC-off-target peptide complexes.Attorney Docket No.250298.000720

[0197] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258 peptide, comprising a) contacting the antigen-recognition molecule with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC- DNTT250-258 peptide complex); b) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) is presented in a complex with the same kind of MHC molecule, or the fragment or derivative thereof, as in (a) (MHC-off-target peptide complex); and c) determining the binding affinity of the antigen- recognition molecule to the MHC-DNTT250-258peptide complex and each of the MHC-off-target peptide complexes.

[0198] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258, comprising a) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said peptide; and b) determining the level of binding of the antigen-recognition molecule to said one or more MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

[0199] In one aspect, the disclosure provides an in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258, comprising a) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-off-target peptide complex), (2) as a peptide, or (3) as aAttorney Docket No.250298.000720 protein or fragment thereof comprising said peptide; and b) determining the binding affinity of the antigen-recognition molecule to said one or more MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

[0200] In some embodiments of the above-described in vitro methods of assessing off- target effects of an antigen-recognition molecule that targets DNTT250-258, the DNTT250-258 peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75). In some embodiments of the in vitro methods, the DNTT250-258peptide consists essentially of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75). In some embodiments of the in vitro methods, the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75).

[0201] In some embodiments of the above-described in vitro methods of assessing off- target effects of an antigen-recognition molecule that targets DNTT250-258, the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the in vitro methods, the off-target peptide consists essentially of an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the in vitro methods, the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0202] In some embodiments of the in vitro methods described above, the level of binding is determined by detecting the amount of antigen-recognition molecules bound to the MHC- peptide complexes. For example, if a signal correlated to binding of an antigen-recognition molecule to an MHC peptide complex has a S:N ratio (such as that displayed in Example 2, Table 8) of at least about 2.0, 2.5, 3.0, 3.5, 4.0, or 5.0 then the antigen-recognition molecule may be determined to exhibit detectable binding to the MHC peptide complex. Noise levels (N) may be determined, for example, by measuring a signal / level of binding to a cell with no peptide and / or with no primary / capture antibody / agent (i.e., with only a detection antibody / agent). If an antigen- recognition molecule specifically binds to one or more MHC-off-target peptide complexes, then the antigen-recognition molecule may exhibit off-target effects. In some specific embodiments, a S:N ratio of at least about 3 is indicative of detectable binding. In some embodiments, binding affinity may be determined. Binding affinity may be determined by measuring an equilibrium dissociation constant (KD) of the binding reaction. Alternatively, binding affinity may be characterized using other methods.

[0203] In some embodiments, the in vitro methods include determining that the antigen- recognition molecule is likely to have off-target effects if it detectably binds to at least one MHC-Attorney Docket No.250298.000720 off-target peptide complex, wherein the off-target peptide is expressed in essential, normal tissues. In some embodiments, the in vitro methods include determining that the antigen-recognition molecule is likely to have off-target effects if it detectably binds to at least one MHC-off-target peptide complex, wherein the off-target peptide is expressed in diseased tissues.

[0204] In some embodiments, the in vitro methods include determining that an antigen- recognition molecule is likely to have off-target effects if it detectably binds to at least one MHC- off-target peptide complex, off-target peptide or protein or fragment thereof comprising said off- target peptide.

[0205] In another aspect, the disclosure provides a method of selecting an antigen- recognition molecule that binds a target pMHC complex with minimal off-target effects. Such method can comprise a) contacting a plurality of antigen-recognition molecules with a target peptide presented in a complex with a major histocompatibility complex (MHC) molecule (MHC- target peptide complex); b) contacting the same plurality of antigen-recognition molecules with one or more off-target peptides associated with said target peptide, wherein each of said off-target peptides is presented in a complex with the same MHC molecule as in (a) (MHC-off-target peptide complex); c) selecting one or more antigen-recognition molecules based at least in part on the number of MHC-off-target peptide complexes detectably bound by each of the antigen-recognition molecules; and d) optionally, repeating steps (a)-(c) using selected antigen-recognition molecules.

[0206] In some embodiments, the method includes selecting the antigen-recognition molecule(s) that binds to the fewest MHC-off-target peptide complexes. In some embodiments, the selected antigen-recognition molecule(s) detectably bind no more than five (e.g., no more than four, no more than three, no more than two, or no more than one) MHC-off-target peptide complexes, wherein the off-target peptides are expressed in essential, normal tissues.

[0207] In some embodiments, the selected antigen-recognition molecule(s) do not detectably bind to any of the MHC-off-target peptide complexes, wherein the off-target peptides are expressed in essential, normal tissues.

[0208] In some embodiments, if several selected antigen-recognition molecules bind to at least one MHC-off-target peptide complex, the method may also include a step of comparing the level of binding of the antigen-recognition molecule(s) to the MHC-target peptide complex versus to the MHC-off-target peptide complex(es). The antigen-recognition molecule(s) may likely bind to the MHC-target peptide complex stronger than to the MHC-off-target peptide complex(es). ForAttorney Docket No.250298.000720 example, the antigen-recognition molecule(s) may bind to the MHC-target peptide complex about 1000 times, about 500 times, about 200 times, about 100 times, about 90 times, about 80 times, about 70 times, about 60 times, about 50 times, about 40 times, about 30 times, about 20 times, or about 10 times stronger than to the MHC-off-target peptide complex(es). The method may further include selecting the antigen-recognition molecule(s) based at least in part on the MHC-target peptide complex / MHC-off-target peptide complex binding ratio for one or more off-target peptides, wherein a higher MHC-target peptide complex / MHC-off-target peptide complex binding ratio is more desirable.

[0209] In some embodiments, selection of antigen-recognition molecules may take into consideration the level of binding of the plurality of antigen-recognition molecules to the MHC- target peptide complex, wherein stronger binding to the MHC-target peptide complex is more desirable. Accordingly, the method may include selecting the antigen-recognition molecule(s) based at least in part on the level of binding to the MHC-target peptide complex.

[0210] In some embodiments, the selected antigen-recognition molecules(s) bind to at least one potential secondary target peptide in addition to the MHC-target peptide complex.

[0211] In another aspect, the disclosure provides a method for selecting an antigen- recognition molecule that targets DNTT250-258peptide, comprising a) contacting a plurality of antigen-recognition molecules with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258peptide complex) or presented as DNTT250-258peptide or DNTT protein or fragment thereof comprising said DNTT250-258peptide; b) contacting the same plurality of antigen-recognition molecules with one or more off-target peptides, (i) wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with the same kind of MHC molecule, or the fragment or derivative thereof as in (a) (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said off-target peptide; c) selecting one or more antigen-recognition molecules based on their ability to detectably bind to DNTT250-258 peptide complex or DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258peptide and based at least in part on the number of MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereofAttorney Docket No.250298.000720 comprising said off-target peptides detectably bound by each of the antigen-recognition molecules; and d) optionally, repeating steps (a)-(c) using the one or more selected antigen-recognition molecules.

[0212] In another aspect, the disclosure provides a method for selecting an antigen- recognition molecule that specifically binds DNTT250-258 peptide, comprising a) contacting a plurality of antigen-recognition molecules with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC- DNTT250-258 peptide complex) or presented as DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258 peptide; b) contacting the same plurality of antigen- recognition molecules with one or more off-target peptides, (i) wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with the same kind of MHC molecule, or the fragment or derivative thereof as in (a) (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said off-target peptide; c) selecting one or more antigen- recognition molecules based on their ability to detectably bind to DNTT250-258peptide complex or DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258 peptide and based at least in part on the number of MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides detectably bound by each of the antigen-recognition molecules; and d) optionally, repeating steps (a)-(c) using the one or more selected antigen-recognition molecules.

[0213] In some embodiments of the above-described methods for selecting an antigen- recognition molecule that targets DNTT250-258peptide, the DNTT250-258peptide comprises the amino acid sequence KLFTSVFGV (SEQ ID NOs: 75). In some embodiments of the selecting methods, the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NOs: 75).

[0214] In some embodiments of the above-described methods for selecting an antigen- recognition molecule that targets DNTT250-258 peptide, the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the selecting methods, the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.Attorney Docket No.250298.000720

[0215] In some embodiments, the one or more selected antigen-recognition molecules detectably bind no more than five MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

[0216] In some embodiments, the one or more selected antigen-recognition molecules do not detectably bind to any of the tested MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

[0217] In some embodiments, the plurality of antigen-recognition molecules is in a library. The library can be, without limitation, a phage display library or a yeast library.

[0218] In some embodiments, one or more of the DNTT250-258 peptide, DNTT protein or fragment thereof comprising said DNTT250-258peptide, DNTT250-258peptide complexes, MHC-off- target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off- target peptides are immobilized on a solid support.

[0219] In some embodiments, one or more of the DNTT250-258 peptide, DNTT protein or fragment thereof comprising said DNTT250-258peptide, DNTT250-258peptide complexes, MHC-off- target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off- target peptides are present on cells (e.g., on the surface of the cells). As a non-limiting example, off-target peptides can be present on cancer cells (e.g., tumor cells).

[0220] In some embodiments, off-target peptides can be present on cancer cells and / or an antigen-binding molecule targeting DNTT250-258 may be screened for binding and / or reactivity against cancer cells or otherwise immortalized cell lines. In some embodiments, the cancer is a hematologic malignancy, for example, a lymphoma, a leukemia, or a myeloma. In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin’s lymphoma. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), or acute myeloid leukemia (AML). In some embodiments, the cancer is a solid cancer. In one embodiment, the cancer is a head and neck cancer. In one embodiment, the cancer is a renal cell carcinoma. In one embodiment, the cancer is a breast cancer, e.g., triple-negative breast cancer (TNBC). In one embodiment, the cancer is a non-small cell lung cancer (NSCLC), for example, lung adenocarcinoma. In one embodiment, the cancer is liver hepatocellular carcinoma (HCC). In one embodiment, the cancer is lung squamous cell carcinoma (SCC). In one embodiment, the cancer is bladder cancer. In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is uveal melanoma. In oneAttorney Docket No.250298.000720 embodiment, the cancer is nasopharyngeal cancer. In one embodiment, the cancer is synovial sarcoma. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is uterine cancer. In one embodiment, the cancer is endometrial cancer. In one embodiment, the cancer is melanoma. In some embodiments, the cancer cell is derived from a tumor cell line (e.g., an endogenous cell line).

[0221] In some embodiments, the off-target peptides of the present disclosure are present on primary normal cells and / or an antigen-binding molecule targeting DNTT250-258may be screened for binding and / or reactivity against primary normal cells.

[0222] In some embodiments, the off-target peptides of the present disclosure are present on cells and / or an antigen-binding molecule targeting DNTT250-258may be screened for binding and / or reactivity against cells, which may be part of or sampled from tissues of a subject having a disease, disorder, or condition, e.g., a disease, disorder, or condition disclosed herein. In some embodiments, the off-target peptides of the present disclosure are present on cells which may be part of or sampled from a diseased tissue of a subject. In some embodiments, the cells may be part of or sampled from tissues of a subject not having a disease, disorder, or condition. In some embodiments, the cells may be part of sampled from a normal tissue of a subject. In various embodiments, the cells are part of or sampled from, for example, without limitation, adipose tissue, lung tissue, kidney tissue, brain tissue, pituitary tissue, salivary gland tissue, spleen tissue, nerve tissue, colon tissue, adrenal gland tissue, heart tissue, muscle tissue, blood vessel tissue, esophagus tissue, small intestine tissue, bladder tissue, skin tissue, and / or liver tissue. The adipose tissue, lung tissue, kidney tissue, brain tissue, pituitary tissue, salivary gland tissue, spleen tissue, nerve tissue, colon tissue, adrenal gland tissue, heart tissue, muscle tissue, blood vessel tissue, esophagus tissue, small intestine tissue, bladder tissue, skin tissue, and / or liver tissue may be either diseased or normal.

[0223] In some embodiments, one or more of the DNTT250-258, DNTT protein or fragment thereof comprising said DNTT250-258, MHC-DNTT250-258 peptide complexes, MHC-off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off-target peptides are present in a soluble form.

[0224] In some embodiments, the level of binding is determined by detecting the amount of antigen-recognition molecules bound to the DNTT250-258peptide, DNTT protein or fragment thereof comprising said DNTT250-258peptide, MHC-DNTT250-258peptide complexes, MHC-off-Attorney Docket No.250298.000720 target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off- target peptides.

[0225] In some embodiments of any of the above-described methods, the method is performed in a high-throughput format.

[0226] In some embodiments, the method is performed in a multi-well plate (e.g., a 96- well plate).

[0227] In some embodiments of the above-described method of selecting an antigen- recognition molecule that targets DNTT250-258 peptide, the DNTT250-258 peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75). In some embodiments of the selecting methods, the DNTT250-258peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75).

[0228] In some embodiments of the above-described method of selecting an antigen- recognition molecule that specifically binds DNTT250-258 peptide, the DNTT250-258 peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75). In some embodiments of the selecting methods, the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75).

[0229] In some embodiments of the above-described method of selecting an antigen- recognition molecule that targets DNTT250-258 peptide, the off-target peptide(s) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the selecting methods, the off-target peptide(s) consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0230] In some embodiments of the above-described method of selecting an antigen- recognition molecule that specifically binds DNTT250-258 peptide, the off-target peptide(s) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the selecting methods, the off-target peptide(s) consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0231] Methods to determine binding to pMHC complexes include, for example, surface plasmon resonance (e.g., BIACORETM), or any other biosensor technique, enzyme-linked immunosorbent assay (ELISA), ELISpot, luminescence assay, flow cytometry, chromatography, microscopy, or any suitable immunoassay. Alternatively, or in addition, binding may beAttorney Docket No.250298.000720 determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.

[0232] For example, antibodies and TCRs may be obtained from display libraries in which a pMHC complex of the disclosure (e.g., a target pMHCs, such as an MHC-DNTT250-258peptide complex)is used to pan the library. TCRs can be displayed on the surface of phage particles and yeast particles, for example, and such libraries have been used for the isolation of high affinity variants of TCR derived from T cell clones. TCR phage libraries can be used to isolate TCRs with novel antigen specificity. Such libraries can be constructed with α- and β- chain sequences corresponding to those found in a natural repertoire. However, the random combination of these α- and β- chain sequences, which occurs during library creation, can produce a repertoire of TCRs that may not be naturally occurring.

[0233] In some embodiments, a pMHC complex of the disclosure may be used to screen a library of diverse TCRs displayed on the surface of phage particles. The TCRs displayed by said library may not correspond to those contained in a natural repertoire, for example, they may contain α- and β- chain pairing that would not be present in vivo, and / or the TCRs may contain non-natural mutations and / or the TCRs may be in soluble form. Screening may involve panning the phage library with pMHC complexes of the disclosure and subsequently isolating bound phage particles. For this purpose, pMHC complexes may be attached to a solid support, such as a magnetic bead, or column matrix and phage bound pMHC complexes isolated, with a magnet, or by chromatography, respectively. The panning steps may be repeated several times. Isolated phage may be further expanded in E. coli cells. Isolated phage particles may be tested for specific binding to pMHC complexes of the disclosure. Binding can be detected using techniques described herein such as, but not limited to, ELISA, or SPR for example using a BIACORETMinstrument. The DNA sequence of the T cell receptor displayed by pMHC binding phage can be further identified by PCR methods.

[0234] Various methods for generating and / or isolating antigen-recognition molecules, such as antibodies and TCRs, including those that target peptides presented as part of pMHC complexes are well known in the art. Antigen-recognition molecules may be generated by immunizing immunocompetent host animals, such as non-human host animals (e.g., rodents such as mice or rats) with antigens such as peptides or pMHC complexes. In certain implementations, the host animal may be genetically modified such that it generates human or humanized antigenAttorney Docket No.250298.000720 recognition molecules in response to the immunization. For instance, TCRs may be generated by immunizing with antigenic peptide (e.g., a target peptide such as DNTT250-258) a rodent genetically modified to express human or humanized TCRs and, optionally, to further express human or humanized MHC (e.g., MHC I α, MHC II α, and / or MHC II β), human or humanized β2 microglobulin (β2M), and / or human or humanized T Cell co receptor (e.g., CD4, CD8 α, and / or CD8 β). See, e.g., U.S. Pat. No. 11,259,510; U.S. Pub. No. 2022 / 0322648; Moore et al., Sci Immunol.2021 Dec 17; 6(66):eabj4026, which are each herein incorporated by reference in their entirety. Likewise, antibodies, or more specifically TCR-mimetic antibodies, may be generated by immunizing with antigenic pMHC complexes (e.g., a target pMHC complex, such as an MHC- DNTT250-258peptide complex) a rodent genetically modified to express human or humanized immunoglobulins (e.g., IgGs) and, optionally, further express human or humanized MHC (e.g., MHC I α, MHC II α, and / or MHC II β) and / or human or humanized β2 microglobulin (β2M). See, e.g., U.S. Pat No. 8,791,323; U.S. Pat. No. 8,642,835; U.S. Pat. No. 8,754,287; U.S. Pat. No. 10,143,186; U.S. Pat. Pub. No. 2019 / 0292263; Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5153-8, each of which is herein incorporated by reference in its entirety. Methods of screening antigen-recognition molecules (e.g., cells expressing antigen-recognition molecules such as T cells and B cells) and isolating and cloning sequences, such as variable domain coding sequences, are well known in the art.

[0235] Alternatively, antigen binding T cells and TCRs can be isolated from fresh blood obtained from patients or healthy donors. Such a method involves stimulating T cells using autologous dendritic cells (DCs), followed by autologous B cells, and then pulsing with a target / off-target peptide disclosed herein. Several rounds of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for recognition of the target / off- target peptide by measuring cytokine release in the presence of cells (e.g., T2 cells) loaded with the target / off-target peptide of the disclosure (for example using an IFNγ ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNγ), or expression of a cell surface marker (such as CD137). Sorted cells may be expanded and further validated, for example, by ELISpot assay and / or cytotoxicity against target cells and / or staining by peptide-MHC multimer (e.g., tetramer). The TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced. Exemplary methods for isolation andAttorney Docket No.250298.000720 validation of TCRs specific to a target antigen are described in Moore et al., Sci. Immunol. 6, eabj4026 (2021); Deering et al., Sci Rep. 2023 May 25;13(1):8452; and U.S. Pat. Pub. No. 2021 / 0102942, each of which is herein incorporated by reference in its entirety.

[0236] Antigen-recognition molecules may be screened for binding to one or more peptides (e.g., target and / or off-target peptides) according to various methods known in the art. The antigen-recognition molecules (e.g., antibodies, TCRs, CARs) and / or the one or more peptides may be presented in solution, presented on a cell surface (e.g., a reporter cell), or immobilized on a solid support, including the specific cells or solid supports described elsewhere herein. For example, the antigen-recognition molecule may be presented on a cell surface (e.g., on a heterologous host cell) or immobilized on a solid support and the one or more peptides may be presented in solution (e.g., as soluble pMHC complexes), presented on a cell surface (e.g., loaded onto MHC of a heterologous cell), or immobilized on a solid support. Similarly, the one or more peptides may be presented on a cell surface (e.g., loaded onto MHC of a heterologous cell) or immobilized on a solid support (e.g., as a pMHC complex) and the antigen-recognition molecule may be presented in solution (e.g., as soluble pMHC complexes), presented on a cell surface (e.g., on a heterologous host cell), or immobilized on a solid support. In some instances, antigen- recognition molecule binding to a peptide presented on a cell surface may be detected using a genetically modified reporter cell that emits a signal in response to the binding, including in cell- to-cell screening systems in which the antigen-recognition molecule and one or more peptides are presented on different cell surfaces.

[0237] For example, TCR screening can be performed using TCR activation assays. For example, JRT3-T3.5 cells (ATCC TIB-153), a Jurkat subline lacking endogenous TCR surface expression may be utilized as described in Moore et al., Sci. Immunol.6, eabj4026 (2021), which is herein incorporated by reference in its entirety. T cell receptor alpha (TCRA) and T cell receptor beta (TCRB) sequences of interest can be introduced into the cells by lentiviral transduction, and surface TCR+ cells can be sorted. Antigen-presenting cells (e.g., 293T cells) that are pulsed with a target peptide or off-target peptide can be incubated with the TCR-transduced or parental JRT3 cells. A readout (e.g., luciferase activity) is then measured as an indication of TCR-mediated activation.

[0238] In some embodiments, monoclonal antibody screening can be performed with cells isolated from the spleens and lymphoid tissue harvested from mice with optimal titers usingAttorney Docket No.250298.000720 hybridoma and B cell sorting (BST) platforms. Counter-screening approaches using one or more off-target peptides can help to identify and eliminate B-cells and hybridomas that show cross- reactivity with peptides that form pHLA complexes resembling the targeted complex. For example, antigen positive (Ag+) clones that have cross-reactivity to off-target peptides can be identified by examining cell supernatants for antibody binding to cells (e.g., T2 cells) pulsed with the target peptide or off-target peptides using a cell binding assay. As another example, Ag+B- cells can be captured using a biotin-labeled HLA-target peptide complex in the presence of high concentrations of one or more unlabeled, HLA-off-target peptide complexes to enrich for antibodies specific for the HLA-target peptide complex. The antibody variable domains of Ag+B- cells can be subsequently cloned as full-length mAbs and expressed (e.g., in CHO cells) for further screening.

[0239] Antibody binding to target / off-target peptides can be determined using ELISA. For example, MHC-target peptide complexes or MHC-off-target peptide complexes can be coated onto a plate (e.g., 96-well microtiter plate). A sample comprising test antibodies can be added to the plate and the reaction can be incubated under a condition to allow binding to occur. The plate is then washed, and a secondary antibody can be then added to the plate to detect the antibodies bound to an MHC-peptide complex. Typically, the secondary antibody can produce a signal that is indicative of the amount of the antibodies bound to the MHC-peptide complex.

[0240] In various embodiments of the methods described herein, pMHC complexes may be provided in soluble form, or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead (e.g., a magnetic bead), a membrane, sepharose, a plate, a tube, a column. pMHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching pMHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens. In some embodiments, pMHC complexes are labeled with biotin and attached to streptavidin-coated surfaces.

[0241] In various embodiments of the methods described herein, pMHC complexes may be present on a cell (e.g., on a surface of a cell). Such a cell may be a mammalian cell, preferably a cell of the immune system, and a specialized antigen-presenting cell (APC) such as a dendritic cell or a B cell. Other preferred cells include T2 cells. Cells presenting the peptide or pMHCAttorney Docket No.250298.000720 complex of the disclosure may be isolated, preferably in the form of a homogenous population, or provided in a substantially pure form. Such cells may be obtained by pulsing said cells with one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure, or genetically modifying the cells (via DNA or RNA transfer) to express one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure. Pulsing involves incubating the cells with the peptide for several hours using peptide concentrations typically ranging from 10−5to 10−12M. Such cells may additionally be transduced with HLA molecules, such as HLA-A*02 (e.g., HLA-A*02:01) to further induce presentation of the peptide(s). Cells may be produced recombinantly.

[0242] In various embodiments of the methods described herein, the method is performed in a high-throughput format (e.g., a 96-well plate).

[0243] In yet another aspect, provided herein is a method of enriching a sample for antigen- recognition molecules that specifically bind a target peptide, comprising (a) contacting a sample comprising a plurality of antigen-recognition molecules with the target peptide in the presence of one or more off-target peptides associated with said target peptide, wherein each of said target peptide and said one or more off-target peptides is presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-target peptide complex or MHC-off-target peptide complex); and (b) enriching the sample by isolating the antigen-recognition molecules that are bound to the MHC-target peptide complex. The method may further comprise repeating steps (a)-(b) to further enrich the sample.

[0244] In still yet another aspect, provided herein is a method of enriching a sample for antigen-recognition molecules that specifically bind DNTT250-258 peptide, comprising a) contacting a sample comprising a plurality of antigen-recognition molecules with DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258peptide or DNTT250-258peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258 peptide complex) in the presence of one or more off- target peptides, wherein each of said off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID Nos: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, wherein each of said one or more off-target peptides is presented in one of the following forms: (1) as a complex with an MHC molecule, or a fragment or derivative thereof (MHC-off-target peptide complex), (2) as a peptide,Attorney Docket No.250298.000720 or (3) as a protein or fragment thereof comprising said off-target peptide; b) enriching the sample by isolating the antigen-recognition molecules that are detectably bound to said DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258 peptide or to the MHC- DNTT250-258peptide complex; and c) optionally, repeating steps (a)-(b) using the enriched sample.

[0245] In some embodiments of the above-described methods of enriching a sample for antigen-recognition molecules that specifically bind DNTT250-258 peptide, the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the sample enrichment methods, the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0246] In some embodiments, said DNTT250-258peptide or DNTT protein or fragment thereof comprising said DNTT250-258peptide or MHC-DNTT250-258peptide complex is present on a cell (e.g., on a surface of a cell) disclosed herein or solid support disclosed herein and said off- target peptides or proteins or fragments thereof comprising said off-target peptides or MHC-off- target peptide complexes are present in solution.

[0247] In some embodiments, said DNTT250-258 peptide or DNTT protein or fragment thereof comprising said DNTT250-258 peptide or MHC-DNTT250-258 peptide complex is labeled and said off-target peptides or proteins or fragments thereof comprising said off-target peptides or MHC-off-target peptide complexes are not labeled or are labeled differently.

[0248] In some embodiments of the above-described method of enriching a sample for antigen-recognition molecules that specifically bind DNTT250-258peptide, the DNTT250-258peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75). In some embodiments of the enriching methods, the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NO: 75).

[0249] In some embodiments of the above-described method of enriching a sample for antigen-recognition molecules that specifically bind DNTT250-258 peptide, the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments of the enriching methods, the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

[0250] There are various ways that can allow isolation of the antigen-recognition molecules that are bound to the MHC-target peptide complex. For example, the MHC-target peptide complex may be present on antigen-presenting cells while the MHC-off-target peptideAttorney Docket No.250298.000720 complexes are not present on antigen-presenting cells (e.g., in a soluble form). Alternatively, the MHC-target peptide complex may be immobilized on a solid support while the MHC-off-target peptide complexes may be soluble or immobilized to a different solid support. The MHC-target peptide complex and the MHC-off-target peptide complexes may also be differentially labeled such that specific detection and isolation of the MHC-target peptide complex and the antigen- recognition molecules bound thereon can be achieved. The antigen-recognition molecules may be eluted from the MHC-target peptide complex after isolation.

[0251] Certain embodiments and implementations of the disclosed technology are described above with reference to block and flow diagrams of systems and methods and / or computer program products according to example embodiments or implementations of the disclosed technology. It will be understood that one or more blocks of the block diagrams and flow diagrams, and combinations of blocks in the block diagrams and flow diagrams, respectively, can be implemented by computer-executable program instructions. Likewise, some blocks of the block diagrams and flow diagrams may not necessarily need to be performed in the order presented, may be repeated, or may not necessarily need to be performed at all, according to some embodiments or implementations of the disclosed technology.

[0252] These computer-executable program instructions may be loaded onto a general- purpose computer, a special-purpose computer, a processor, or other programmable data processing apparatus to produce a particular machine, such that the instructions that execute on the computer, processor, or other programmable data processing apparatus create means for implementing one or more functions specified in the flow diagram block or blocks. These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means that implement one or more functions specified in the flow diagram block or blocks.

[0253] As an example, embodiments or implementations of the disclosed technology may provide for a computer program product, including a computer-usable medium having a computer- readable program code or program instructions embodied therein, said computer-readable program code adapted to be executed to implement one or more functions specified in the flow diagram block or blocks. Likewise, the computer program instructions may be loaded onto a computer orAttorney Docket No.250298.000720 other programmable data processing apparatus to cause a series of operational elements or steps to be performed on the computer or other programmable apparatus to produce a computer- implemented process such that the instructions that execute on the computer or other programmable apparatus provide elements or steps for implementing the functions specified in the flow diagram block or blocks.

[0254] Accordingly, blocks of the block diagrams and flow diagrams support combinations of means for performing the specified functions, combinations of elements or steps for performing the specified functions, and program instruction means for performing the specified functions. It will also be understood that each block of the block diagrams and flow diagrams, and combinations of blocks in the block diagrams and flow diagrams, can be implemented by special- purpose, hardware-based computer systems that perform the specified functions, elements or steps, or combinations of special-purpose hardware and computer instructions.

[0255] Certain implementations of the disclosed technology are described above with reference to customer devices that may include mobile computing devices. Those skilled in the art will recognize that there are several categories of mobile devices, generally known as portable computing devices that can run on batteries but are not usually classified as laptops. For example, mobile devices can include, but are not limited to portable computers, tablet PCs, internet tablets, PDAs, ultra-mobile PCs (UMPCs), wearable devices, and smart phones. Additionally, implementations of the disclosed technology can be utilized with internet of things (IoT) devices, smart televisions and media devices, appliances, automobiles, toys, and voice command devices, along with peripherals that interface with these devices.

[0256] While certain embodiments of this disclosure have been described in connection with what is presently considered to be the most practical and various embodiments, it is to be understood that this disclosure is not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0257] This written description uses examples to disclose certain embodiments of the technology and also to enable any person skilled in the art to practice certain embodiments of this technology, including making and using any apparatuses or systems and performing any incorporated methods. The patentable scope of certain embodiments of the technology is definedAttorney Docket No.250298.000720 in the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims. Exemplary Compositions

[0258] In one aspect, the disclosure provides isolated peptides which can be identified, e.g., using any of various methods described herein. In some embodiments, the isolated peptide may be identified for a DNTT target peptide (e.g., for DNTT250-258 target KLFTSVFGV (SEQ ID NO: 75)) as described herein.

[0259] In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments, the isolated peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0260] In some embodiments, one or more isolated peptides of the present disclosure may have a Degree of Similarity (DoS) score, as described elsewhere herein, with the target peptide, e.g., SEQ ID NO: 75, of at least 7 residues. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76-78, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 76-78. In some embodiments, one or moreAttorney Docket No.250298.000720 isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76-78. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of any one of SEQ ID NOs: 76-78. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 76-78, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0261] In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76 and 78, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 76 and 78. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76 and 78. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of any one of SEQ ID NOs: 76 and 78. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 76 and 78, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0262] In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76-77, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 76-77. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76-77. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of any one of SEQ ID NOs: 76-77. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptidesAttorney Docket No.250298.000720 comprise two or more sequences selected from any one or SEQ ID NOs: 76-77, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0263] In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 76, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of SEQ ID NO: 76. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of SEQ ID NO: 76. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of SEQ ID NO: 76.

[0264] In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 77, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of SEQ ID NO: 77. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of SEQ ID NO: 77. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of SEQ ID NO: 77.

[0265] In some embodiments, one or more isolated peptides of the present disclosure may have a Degree of Similarity (DoS) score, as described elsewhere herein, with the target peptide, e.g., SEQ ID NO: 75, of at least 6 residues. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 79-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 79-82. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 79-82. In some embodiments, the one or more isolated peptides consist of anAttorney Docket No.250298.000720 amino acid sequence of any one of SEQ ID NOs: 79-82. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 79-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0266] In some embodiments, one or more isolated peptides of the present disclosure may have been detected by mass spectrometry as being presented by an HLA molecule in a human tissue sample. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76 and 78- 82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 76 and 78-82. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76 and 78-82. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of any one of SEQ ID NOs: 76 and 78-82. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 76 and 78-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0267] In some embodiments, one or more isolated peptides of the present disclosure may comprise a sequence that is not expressed within the proteome of a non-human host animal (e.g., a rodent such as a mouse or a rat) such that the non-human host animal is unlikely to be tolerized to the peptide (e.g., as part of a pMHC complex). Immunization of such non-human host animals for the purposes of generating antigen-recognition molecules (e.g., antibodies or TCRs) may be more prone to generating antigen-recognition molecules that cross-react with the peptide (e.g., as part of a pMHC complex). In some embodiments, one or more isolated peptides of the present disclosure comprise an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76-77 and 81, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, one or more isolated peptides of the present disclosure comprise an aminoAttorney Docket No.250298.000720 acid sequence of any one of SEQ ID NOs: 76-77 and 81. In some embodiments, one or more isolated peptides of the present disclosure consist essentially of an amino acid sequence of any one of SEQ ID NOs: 76-77 and 81. In some embodiments, the one or more isolated peptides consist of an amino acid sequence of any one of SEQ ID NOs: 76-77 and 81. In some embodiments, the one or more isolated peptides comprise a plurality of isolated peptides. In some embodiments, the one or more isolated peptides comprise two or more sequences selected from any one or SEQ ID NOs: 76-77 and 81, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0268] In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76-78, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76 and 78, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76-77, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of SEQ ID NO: 76, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of SEQ ID NO: 77, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 79-82, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76 and 78-82, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76-77 and 81, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure provides one or more isolated peptides comprising or consisting of an amino acid sequence selected from any subset of sequences formed by identifying the common sequences within one or more sets of sequences disclosed herein.Attorney Docket No.250298.000720

[0269] In some embodiments, the isolated peptide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more, amino acids in length. In certain embodiments, the isolated peptide is 8-12 amino acids in length. In certain embodiments, the isolated peptide is 8-11 amino acids in length. In certain embodiments, the isolated peptide is 8-10 amino acids in length. In certain embodiments, the isolated peptide is 9-12 amino acids in length. In certain embodiments, the isolated peptide is 9- 11 amino acids in length. In certain embodiments, the isolated peptide is 9-10 amino acids in length. In certain embodiments, the isolated peptide is 9 amino acids in length.

[0270] Amino acids sequences derived disclosed herein may include naturally occurring proteogenic amino acids as well as non-proteogenic amino acids and non-naturally occurring amino acids such as amino acid analogs. In some embodiments, the amino acids that may be used in the practice of the present disclosure may include, for example, without limitation, naturally occurring proteogenic (L)-amino acids, their optical (D)-isomers, chemically modified amino acids, including, e.g., amino acid analogs such as, e.g., selenocysteine (Sec), penicillamine (3- mercapto-D-valine), pyroglutamic acid (5-oxoproline), etc., naturally occurring non-proteogenic amino acids such as norleucine, and chemically synthesized amino acids that have properties known in the art to be characteristic of an amino acid, and amino acid equivalents.

[0271] Inserted amino acids and substituted amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain, and / or be linked together via non-native peptide bonds. Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007; 50(7):1598-609 and Hoppes et al., J. Immunol 2014; 193(10):4803-13 and references therein. If more than one amino acid residue is substituted and / or inserted, the replacement / inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.

[0272] D-amino acids may be substituted for the L-amino acids in the peptides of the disclosure. In addition, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids such as β-γ-δ-amino acids, as well as many derivatives of L-α-amino acids) may also be used for substitutions or additions to produce peptides of the present disclosure.

[0273] One can also replace the naturally occurring side chains of the 20 genetically encoded amino acids (or the stereoisomeric D-amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lowerAttorney Docket No.250298.000720 alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. For example, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0274] Other examples of amino acid replacements include stereoisomers (e.g., D-amino acids) and unnatural amino acids such as, for example, L-ornithine, L-homocysteine, L- homoserine, L-citrulline, 3-sulfino-L-alanine, N-(L-arginino)succinate, 3,4-dihydroxy-L- phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, triiodothyronine, L-thyroxine, L- selenocysteine, N-(L-arginino)taurine, 4-aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a- disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, 3-pyridylalanine, 4- hydroxyproline, O-phosphoserine, N-methylglycine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, nor-leucine, and other similar amino acids and imino acids.

[0275] In some embodiments, an isolated peptide described herein, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, can comprise one or more reverse peptide bonds, one or more non-peptide bonds, one or more D-isomers of amino acids, one or more chemical modifications, or any combination thereof.

[0276] In some embodiments, an isolated peptide described herein, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, is produced by expression in a heterologous host cell. In some embodiments an isolated peptide described herein, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, is produced synthetically.Attorney Docket No.250298.000720

[0277] In some embodiments, the peptide disclosed herein forms a complex with one or more MHC class I α heavy chains. In some embodiments, the MHC class I α heavy chain is fully human. In some embodiments, the MHC class I α heavy chain is humanized. Humanized MHC class I α heavy chains are described, e.g., in U.S. Pat. Pub. Nos.2013 / 0111617, 2013 / 0185819 and 2014 / 0245467. In some embodiments, the MHC class I α heavy chain comprises a human extracellular domain (human α1, α2, and / or α3 domains) and a cytoplasmic domain of another species. In some embodiments, the class I α heavy chain polypeptide is HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In various aspects, the peptide-MHC can include all the domains of an MHC class I heavy chain. In some embodiments, the MHC molecule is a class I MHC molecule. In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule. In some embodiments, the class I HLA molecule is an HLA- A molecule. In some embodiments, the HLA-A molecule is an HLA-A*02 molecule. In some specific embodiments, the HLA-A molecule is an HLA-A*02:01 molecule.

[0278] In some embodiments, the MHC molecule comprises a β2-microglobulin. In some embodiments, the β2-microglobulin is fully human. In some embodiments, the β2-microglobulin is humanized.

[0279] In some embodiments, the MHC class I molecule comprises a mutation in a β2- microglobulin (β2m or Β2M) polypeptide and in the Heavy Chain sequence to affect a disulfide bond between the Β2M and the Heavy Chain. In some cases, the Heavy Chain is an HLA and wherein the disulfide bond links one of the following pairs of residues: Β2M residue 12, HLA residue 236; Β2M residue 12, HLA residue 237; Β2M residue 8, HLA residue 234; Β2M residue 10, HLA residue 235; Β2M residue 24, HLA residue 236; Β2M residue 28, HLA residue 232; Β2M residue 98, HLA residue 192; Β2M residue 99, HLA residue 234; Β2M residue 3, HLA residue 120; Β2M residue 31, HLA residue 96; Β2M residue 53, HLA residue 35; Β2M residue 60, HLA residue 96; Β2M residue 60, HLA residue 122; Β2M residue 63, HLA residue 27; Β2M residue Arg3, HLA residue Gly120; Β2M residue His31, HLA residue Gln96; Β2M residue Asp53, HLA residue Arg35; Β2M residue Trp60, HLA residue Gln96; Β2M residue Trp60, HLA residue Asp122; Β2M residue Tyr63, HLA residue Tyr27; Β2M residue Lys6, HLA residue Glu232; Β2M residue Gln8, HLA residue Arg234; Β2M residue Tyr10, HLA residue Pro235; Β2M residue Ser11, HLA residue Gln242; Β2M residue Asn24, HLA residue Ala236; Β2M residue Ser28, HLA residue Glu232; Β2M residue Asp98, HLA residue His192; and Β2M residue Met99, HLA residueAttorney Docket No.250298.000720 Arg234, first linker position Gly2, Heavy Chain (HLA) position Tyr84; Light Chain (Β2M) position Arg12, HLA Ala236; and / or Β2M residue Arg12, HLA residue Gly237.

[0280] In some embodiments, at least one chain of the MHC and the peptide are comprised within a fusion protein. In one specific embodiment, the MHC and the peptide are separated by a linker sequence. For example, the single chain molecule can comprise, from amino to carboxy terminal, an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence. Alternatively, the single chain molecule can comprise, from amino to carboxy terminal, an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence. The single-chain molecule can further comprise a signal peptide sequence at the amino terminal. In certain embodiments, there can be a linker sequence between the peptide sequence and the β2- microglobulin sequence. In certain embodiments, there can be a linker sequence between the β2- microglobulin sequence and the class I α (heavy) chain sequence. A single-chain molecule can further comprise a signal peptide sequence at the amino terminal, as well as first linker sequence extending between the peptide sequence and the β2-microglobulin sequence, and / or a second linker sequence extending between the β2-microglobulin sequence and the class I heavy chain sequence. In certain embodiments, the β2-microglobulin and the class I α (heavy) chain sequences can be human, murine, or porcine.

[0281] In some embodiments, a single-chain molecule can comprise a first flexible linker between the peptide ligand segment and the β2-microglobulin segment. For example, linkers can extend from and connect the carboxy terminal of the peptide ligand segment to the amino terminal of the β2-microglobulin segment. Preferably, the linkers are structured to allow the linked peptide ligand to fold into the binding groove resulting in a functional MHC-antigen peptide. In some embodiments, this linker can comprise at least about 10 amino acids, up to about 15 amino acids. In some embodiments, a single-chain molecule can comprise a second flexible linker inserted between the β2-microglobulin and heavy chain segments. For example, linkers can extend from and connect the carboxy terminal of the β2-microglobulin segment to the amino terminal of the heavy chain segment. In certain embodiments, the β2-microglobulin and the heavy chain can fold into the binding groove resulting in a molecule which can function in promoting T cell expansion.

[0282] Suitable linkers used in the MHCs can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acidsAttorney Docket No.250298.000720 to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Non-limiting examples of linkers include, e.g., glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 131) and (GGGS)n (SEQ ID NO: 132), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains). Exemplary linkers can comprise amino acid sequences including, but not limited to, those listed in Table 1. In some embodiments, a linker peptide includes a cysteine residue that can form a disulfide bond with a cysteine residue present in a second polypeptide. Table 1. Examples of Linker Sequences Linker amino acid SEQ Linker codon-optimized SEQ Linker codon-optimized SEQ sequence ID nucleotide sequence ID nucleotide sequence ID :Attorney Docket No.250298.000720 GGGASGGGGSGG 94GGGGGGGGCGCTTCA110GGAGGCGGCGCTTCTG126 GGS GGCGGAGGTGGAAGT GGGGCGGGGGTAGTG [0eptide covalently attached to an MHC class I α (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cystines). In certain embodiments, the disulfide bond comprises a first cysteine, comprising a linker extending from the carboxy terminal of an antigen peptide, and a second cysteine comprising an MHC class I heavy chain (e.g., an MHC class I α (heavy) chain which has a non-covalent binding site for the antigen peptide). In certain embodiments, the second cysteine can be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In certain embodiments, the single-chain molecule can comprise one contiguous polypeptide chain as well as a disulfide bridge. In certain embodiments, the single-chain molecule can comprise two contiguous polypeptide chains which are attached via the disulfide bridge as the only covalent linkage. In some embodiments, the linking sequences can comprise at least one amino acid in addition to the Cys residues, including one or more Gly residues, one or more Ala residues, and / or one or more Ser residues.

[0284] In certain embodiments, the disulfide bridge can link an antigen peptide described herein in the class I groove of the pMHC complex if the pMHC complex comprises a first cysteine in a Gly-Ser linker extending between the C-terminus of the peptide and the β2-microglobulin, and a second cysteine in a proximal heavy chain position.Attorney Docket No.250298.000720

[0285] Attaching the peptide to the MHC class I or MHC class II molecule via a flexible linker has the can help ensure that the peptide will occupy and stay associated with the MHC molecule during biosynthesis, transport, and display. However, there may be situations in which this linker can interfere with peptide binding to the MHC molecule or with recognition of the complex by an antigen-recognition molecule. As an alternate approach, in some embodiments, the MHC molecule and the peptide are expressed separately.

[0286] In some embodiments, the β2-microglobulin sequence can comprise a full-length β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks the leader peptide sequence. As such, in some configurations, the β2-microglobulin sequence can comprise about 99 amino acids, and can be a mouse ^2-microglobulin sequence (e.g., GenBank Accession No. X01838). In some other configurations, the β2-microglobulin sequence can comprise about 99 amino acids, and can be a human ^2-microglobulin sequence (e.g., GenBank Accession No. AF072097.1).

[0287] In some embodiments, a peptide of the present disclosure may be covalently linked to an MHC molecule, such as by a disulfide bond. In some embodiments, the pMHC complex can contain MHC sequences as disclosed in U.S. Patent Nos. 4,478,823; 6,011,146; 8,518,697; 8,895,020; 8,992,937; WO 96 / 04314; Mottez et al. J. Exp. Med.181: 493-502, 1995; Madden et al. Cell 70: 1035-1048, 1992; Matsumura et al., Science 257: 927-934, 1992; Mage et al., Proc. Natl. Acad. Sci. USA 89: 10658-10662, 1992; Toshitani et al., Proc. Nat’l Acad. Sci.93: 236-240, 1996; Chung et al, J. Immunol.163:3699-3708, 1999; Uger and Barber, J. Immunol.160: 1598- 1605, 1998; Uger et al., J. Immunol. 162, pp. 6024-6028, 1999; White et al., J. Immunol. 162: 2671-2676, 1999; Yu et al., J. Immunol.168:3145-3149, 2002; Truscott et al., J. Immunol. 178: 6280–6289, 2007; Mitaksov et al., Chem Biol.2007 August; 14(8):909–922; Hansen et al., Trends Immunol.2010 Oct; 31(10):363-9, all of which are incorporated by reference in their entireties.

[0288] In some embodiments, the MHC comprises a class II MHC polypeptide or a fragment, mutant or derivative thereof. In one specific embodiment, the MHC comprises α and β polypeptides of a class II MHC complex or a fragment, mutant or derivative thereof. In one specific embodiment, the α and β polypeptides are linked by a peptide linker. In one specific embodiment, the MHC comprises α and β polypeptides of a human class II MHC complex selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM and HLA-DO. In anotherAttorney Docket No.250298.000720 specific embodiment, the MHC comprises α and β polypeptides of a murine H-2A or H-2E class II MHC complex.

[0289] Naturally occurring MHC class II molecules can contain two polypeptide chains, α and β. The chains may come from the DP, DQ, or DR gene groups. There are about 40 known different human MHC class II molecules. All have the same basic structure but can vary subtly in their molecular structure. MHC class II molecules can bind peptides of 13-18 amino acids in length.

[0290] In some embodiments, the MHC class II α chain is fully human. In some embodiments, the MHC class II α chain is humanized. Humanized MHC class II α chains are described, e.g., in U.S. Pat. Nos. 8,847,005, 9,043,996, and 10,154,658, which are incorporated herein by reference in their entireties. In some embodiments, the humanized MHC class II α chain polypeptide comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA. In some embodiments, the class II α chain polypeptide is humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA and / or HLA-DRA.

[0291] In some embodiments, the peptide of the present disclosure forms a complex with one or more MHC class II β chains. In some embodiments, the MHC class II β chain is fully human. In some embodiments, the MHC class II β chain polypeptide is humanized. Humanized MHC class II β chain polypeptides are described, e.g., in U.S. Pat. Nos.8,847,005, 9,043,996, and 10,154,658, which are incorporated herein by reference in their entireties. In some embodiments, the humanized MHC class II β chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB. In some embodiments, the class II β chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB and / or HLA-DRB. In some embodiments, a peptide of the present disclosure may be covalently linked to an MHC class II molecule, such as by a disulfide bond. In some embodiments, the α chain and β chain of an MHC class II molecule may be linked, such as by a Jun-Fos zipper, electrostatic engineering, knobs-into-holes, an immunoglobulin scaffold, an immunoglobulin Fc region, or a linker. See, e.g., U.S. Pat. Pub. No. 2022 / 0409732, which is incorporated herein by reference in its entirety.

[0292] Peptides or pMHC complexes disclosed herein may be fused or conjugated to one or more heterologous molecules. Peptides or pMHC complexes of the disclosed herein may alsoAttorney Docket No.250298.000720 be in multimeric form. Accordingly, the present disclosure also provides fusion proteins, conjugates, and oligomeric complexes comprising a peptide or a pMHC complex of the disclosure.

[0293] In some embodiments, peptides are fused or conjugated to one or more heterologous molecules which includes an MHC molecule (or fragments or derivatives thereof). In some embodiments, the MHC molecule is a class I MHC molecule. In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule. In some embodiments, the class I HLA molecule is an HLA-A molecule. In some embodiments, the HLA- A molecule is an HLA-A*02 molecule. In some specific embodiments, the HLA-A molecule is an HLA-A*02:01 molecule.

[0294] Heterologous molecules suitable for genetical fusion and / or chemical conjugation with the peptides or the pMHC complexes of the disclosure include, but are not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc. The heterologous molecule(s) may be fused at the N- and / or C-terminus of the peptide and / or another polypeptide chain in the pMHC complex.

[0295] Heterologous peptides and polypeptides include, but are not limited to, an epitope (e.g., FLAG) or a tag sequence (e.g., His6 (SEQ ID NO: 133), and the like) to allow for the detection and / or isolation of a fusion protein; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region (e.g., an Fc domain); a half-life-extending sequence comprising a combination of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non-naturally occurring charged and / or uncharged amino acids (e.g., Ser, Gly, Glu or Asp) designed to form a predominantly hydrophilic or predominantly hydrophobic fusion partner for a fusion protein; a functional or non-functional antibody (e.g., an antibody that is specific for dendritic cells), or a heavy or light chain thereof; and a polypeptide which has an activity, such as a therapeutic activity, different from fusion proteins of the present disclosure. In some embodiments, the one or more heterologous molecules enhances a peptide-specific immune response in a subject. In some embodiments, the one or more heterologous molecules mediates peptide delivery to a specific site within a subject.Attorney Docket No.250298.000720

[0296] In some embodiments, fusion proteins of the disclosure may comprise one or more affinity tags, e.g., to allow for affinity purification or coupling to another molecule. Examples of affinity tags include, but are not limited to, a His6 tag (SEQ ID NO: 133), an Avi-tag, a biotin, a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, a GST tag, a MBP tag, a chitin binding protein tag, a calmodulin tag, a V5 tag, a streptavidin binding tag, a green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, SUMO tag, and Ubiquitin tag.

[0297] Peptides or pMHC complexes of the disclosure may be conjugated to additional moieties such as carrier molecules or adjuvants for use as vaccines. Examples of adjuvants used in vaccines include microbes, such as the bacterium Bacillus Calmette-Guérin (BCG), and / or substances produced by bacteria, such as Detox B (an oil droplet emulsion of monophosphoryl lipid A and mycobacterial cell wall skeleton). KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), the E2 core protein of the pyruvate dehydrogenase complex are examples of suitable carrier proteins used in vaccine compositions. Additional examples of carrier proteins suitable for use in the compositions of the present disclosure include, but are not limited to, ovalbumin (OVA), blue carrier protein (BCP), thyroglobulin (THY), a soybean trypsin inhibitor (STI), and multiple attachment peptide (MAP), albumin, serum albumin, c-reactive protein, conalbumin, lactalbumin, ion carrier protein, acyl carrier protein, signal transduction adapter protein, androgen binding protein, calcium binding protein, calmodulin binding protein, ceruloplasmin, cholesterol Ester transfer protein, f box protein, fatty acid binding protein, follistatin, follistatin related protein, GTP binding protein, insulin-like growth factor binding protein, iron binding protein, latent TGF beta binding protein, light-harvesting protein complex, lymph Sphere antigen, membrane transport protein, neurophysin, periplasmic binding protein, phosphate binding protein, phosphatidylethanolamine binding protein, phospholipid transport protein, retinol binding protein, RNA binding protein, s-phase kinase related protein, sex hormone binding globulin, Thyroxine binding protein, transcobalamin, transcortin, transferrin binding protein, and / or vitamin D binding protein.

[0298] Peptides or pMHC complexes of the present disclosure may also be attached, covalently (e.g., via a linker) or non-covalently, to a moiety capable of eliciting a therapeutic effect, such as antibodies, or cytokines. Alternatively or additionally, the peptides or pMHC complexes may be encapsulated into liposomes.Attorney Docket No.250298.000720

[0299] In certain aspects the, present disclosure contemplates a non-covalent complex comprising (i) an isolated peptide described herein or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, and (ii) an MHC molecule, or a fragment or derivative thereof. In some embodiments, the MHC molecule is a class I MHC molecule. In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule. In some embodiments, the class I HLA molecule is an HLA-A molecule. In some embodiments, the HLA- A molecule is an HLA-A*02 molecule. In some specific embodiments, the HLA-A molecule is an HLA-A*02:01 molecule.

[0300] Other suitable heterologous molecules include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes, and contrast reagents, antibodies, or enzymes that produce a detectable product. Methods for detecting heterologous molecules may include flow cytometry, microscopy, electrophoresis, or scintillation counting.

[0301] In some embodiments, peptides or pMHC complexes of the disclosure may be conjugated with fluorocarbon to increase cellular immunogenicity. Where the peptide or another polypeptide chain of the pMHC complex is linked to a fluorocarbon, the terminus of the peptide or polypeptide chain, such as the terminus that is not conjugated to the fluorocarbon, or other attachment, can be altered, for example to promote solubility of the fluorocarbon- peptide / polypeptide construct via the formation of micelles. To facilitate large-scale synthesis of the construct, the N- or C-terminal amino acid residues of the peptide or another polypeptide chain of the pMHC complex can be modified. When the desired peptide or another polypeptide chain of the pMHC complex is particularly sensitive to cleavage by peptidases, the normal peptide bond can be replaced by a non-cleavable peptide mimetic. Such bonds and methods of synthesis are well known in the art.

[0302] Peptides or pMHC complexes of the disclosure may be provided in soluble form, or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead (e.g., a magnetic bead), a membrane, sepharose, a plate, a tube, and a column. pMHC complexes may be attached, for instance, to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching peptides or pMHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens. In some embodiments, peptides or pMHC complexes are labeled with biotin and attached to streptavidin-coated surfaces.Attorney Docket No.250298.000720 In some embodiments, the peptides or pMHC complexes of the disclosure may be conjugated to a particle or a solid support described herein.

[0303] Peptides or pMHC complexes of the disclosure may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octameric, or greater. Accordingly, in some aspects, the present disclosure provides oligomeric complexes comprising the peptides or pMHC complexes of the present disclosure. In certain embodiments, an oligomeric complex of the present disclosure may comprise, for example, two or more isolated peptides described herein, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. As used herein, the terms “oligomer”, “oligomeric”, “oligomerize” and “oligomerization” or the like encompass a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher species of polymerized monomers that comprise the peptide or pMHC complex. Having multiple copies of the peptides or pMHC complexes in a large complex may enhance their biological activity, e.g., immunogenic activity.

[0304] For example, the peptides of the disclosure may be oligomerized using the biotin / streptavidin system. Biotinylated analogs of peptide monomers may be synthesized by standard techniques. For example, the peptide may be C-terminally biotinylated. These biotinylated peptide monomers are then oligomerized by incubation with streptavidin [e.g., at a 4:1 molar ratio at room temperature in phosphate buffered saline (PBS) or HEPES-buffered RPMI medium for 1 hour]. In a variation of this embodiment, biotinylated peptide monomers may be oligomerized by incubation with anti-biotin antibodies [e.g., goat anti-biotin IgG].

[0305] In general, oligomeric pMHC complexes may be produced using pMHC tagged with a biotin residue and complexed through fluorescently labeled streptavidin. A biotinylation site may be introduced to the pMHC complex to which biotin can be added, for example, using the BirA enzyme. Alternatively, oligomeric pMHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, the extracellular domains of MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker. Oligomeric pMHC complexes have also been produced using carrier molecules such as dextran. Oligomeric pMHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.Attorney Docket No.250298.000720

[0306] In other embodiments, the peptides or pMHC complexes of the disclosure can be oligomerized by covalent attachment to at least one linker. The linker moiety can be a peptide linker, such as those described herein. In some embodiments, polyethylene glycol (PEG) may serve as the linker that oligomerizes the peptide monomers. For example, a single PEG moiety may be simultaneously attached to the N-termini of both peptide chains of a peptide dimer.

[0307] Alternatively, oligomeric peptide or pMHC complexes may also contain one or more intramolecular disulfide bonds between cysteine residues of the peptide or pMHC monomers. Preferably, the two monomers contain at least one intramolecular disulfide bond. Most preferably, both monomers contain an intramolecular disulfide bond, such that each monomer contains a cyclic group. Such disulfide bonds may be formed by oxidation of the cysteine residues of the peptide core sequence. In one embodiment the control of cysteine bond formation is exercised by choosing an oxidizing agent of the type and concentration effective to optimize formation of the desired isomer. For example, oxidation of a peptide dimer to form two intramolecular disulfide bonds (one on each peptide chain) is preferentially achieved (over formation of intermolecular disulfide bonds) when the oxidizing agent is DMSO. The formation of cysteine bonds can be controlled by the selective use of thiol-protecting groups during peptide synthesis.

[0308] In some embodiments, peptides or pMHC complexes described herein may be fused or conjugated to a dimerization moiety. The dimerization moiety may contain, for example, an immunoglobulin domain, such as from an IgG antibody (e.g., human IgG), which connects two monomers generating a homodimer or heterodimer molecule. As a non-limiting example, the dimerization motif in the proteins according to the present disclosure may be constructed to include a hinge region and an immunoglobulin domain (e.g. Cγ3 domain), e.g., carboxyterminal C domain (CH3 domain), or a sequence that is substantially identical to the C domain. The hinge region may be Ig derived and contributes to the dimerization through the formation of an interchain covalent bond(s), e.g. disulfide bridge(s). In addition, such homodimer or heterodimer molecules may further comprise one or more targeting moieties that bind to target molecules present on, for example, antigen-presenting cells (APCs) such as dendritic cells or B cells. In such instances, the hinge region may function as a flexible spacer between the domains allowing the two targeting units to bind simultaneously to two target molecules on the APC expressed with variable distances. The immunoglobulin domains contribute to dimerization through non-covalent interactions, e.g. hydrophobic interactions. In a preferred embodiment the CH3 domain is derived from IgG. TheseAttorney Docket No.250298.000720 dimerization moieties may be exchanged with other multimerization moieties from e.g., other Ig isotypes / subclasses. Preferably the dimerization motif is derived from native human proteins, such as human IgG. Examples of such homodimer protein construct are described in US 10,590,195, which is incorporated herein by reference in its entirety.

[0309] In one aspect, the present disclosure provides a composition comprising (i) one or more isolated peptides described herein, one or more fusion proteins described herein, one or more conjugates described herein, one or more oligomeric complexes of claim described herein, or one or more non-covalent complexes described herein, or any combination thereof; and (ii) a carrier or excipient. In some embodiments, such composition described herein may be formulated as a pharmaceutical composition.

[0310] A pharmaceutical composition of the disclosure may be in any suitable form (depending upon the desired method of administering to a patient). Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood.2009; 114(3):535-46.

[0311] The pharmaceutical composition may comprise the peptides or peptide-based molecules of the disclosure either in the free form or in the form of a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” as used herein 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 —NH2group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, benzoic acid, citric acid, propionic acid, glycolic acid, trifluoroacetic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the 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 which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.

[0312] The pharmaceutical composition may be adapted for administration by any appropriate route such as, e.g., parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes.Attorney Docket No.250298.000720

[0313] Such compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0314] In another aspect, the present disclosure provides an isolated cell comprising one or more fusion proteins described herein, one or more conjugates described herein, one or more oligomeric complexes described herein, or one or more non-covalent complexes described herein, or any combination thereof. In some embodiments, the isolated cell is an immune cell. In some embodiments, the isolated cell is an antigen-presenting cell (APC).

[0315] In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding one or more peptide(s) and / or peptide-based molecules (such as complexes (e.g., pMHC complexes), fusion proteins, or conjugates comprising the described peptides) of the disclosure.

[0316] In some embodiments, the polynucleotide described herein encodes a peptide comprising an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 76- 82, or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 76-82, or a fragment or derivative thereof.

[0317] In some embodiments, the polynucleotide described herein encodes more than one peptide selected from any one of SEQ ID NOs: 76-82, or fragments thereof. For example, the polynucleotide described herein may encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 peptides as described herein (e.g., SEQ ID NOs: 76-82), or fragments thereof. The peptides may be arranged in any order, and may be identical or different.

[0318] In some embodiments, the polynucleotide described herein may comprise a nucleotide sequence which is operably linked to a promoter. A promoter of the present disclosure can be selected from a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide is under the control of a human promoter. In some embodiments, the nucleotide is under the control of a non-human promoter.Attorney Docket No.250298.000720

[0319] Non-limiting examples of promoters include cytomegalovirus (CMV) promoter (U.S. Pat. Nos.5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (VIIIa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0320] In some embodiment, the polynucleotide described herein is a DNA molecule.

[0321] In some embodiments, the polynucleotide described herein is an RNA molecule. For example, the RNA molecule may be mRNA or a self-replicating RNA.

[0322] A nucleic acid molecule described herein may be generated synthetically. One method is the phosphoramidite method. Without wishing to be bound by theory, in this chemistry, a phosphoramidite (a nucleoside with side protecting groups that preserve the integrity of the sugar, the phosphodiester linkage, and the base during chain extension steps) is coupled through its reactive 3' phosphorous group to the 5' hydroxyl group of a nucleoside immobilized on a solid support column. The steps of oligonucleotide synthesis can include the following: (1) Detritylation, in which the dimethoxytrityl (DMT or trityl) group on the 5' hydroxyl of the support nucleoside is removed by treatment with trichloroacetic acid (TCA). (2) In the coupling step, a phosphoramidite, made reactive by tetrazole (a weak acid), is chemically coupled to the last base added to the column support material. (3) In the capping step, any free 5' hydroxyl groups of unreacted column nucleotides are acetylated by treatment with acetic anhydride and N- methylimidazole. (4) In the oxidation step, the unstable internucleotide phosphate linkage between the previously coupled base and the most recently added base is oxidized by treatment with iodine and water to a more stable phosphotriester linkage. Following coupling of all bases in the oligonucleotide's sequence, the completed nucleic acid chain may be cleaved from the column byAttorney Docket No.250298.000720 treatment with ammonium hydroxide, and the base protecting groups are removed by heating in the ammonium hydroxide solution.

[0323] By way of a non-limiting example, a synthesis cycle may comprise growth of the nucleotide chain from an initial protected nucleoside derivatized via its terminal 3′ hydroxyl to a solid support. Reagents and solvents can be pumped through the support to induce the consecutive removal and addition of sugar protecting groups in order to isolate the reactivity of a specific chemical moiety on the monomer and effect its stepwise addition to the growing oligonucleotide chain. Assembly of the protected oligonucleotide chain can be carried out in chemical steps, for example, without limitation, deblocking, activation / coupling, oxidation, and capping. Cleavage and deprotection then reveal the single-stranded nucleic acid.

[0324] Nucleic acid synthesis methods disclosed herein can comprise, for example, oligonucleotide synthesis, column-based oligonucleotide synthesis, microarray-based oligonucleotide synthesis, gene synthesis from oligonucleotides, gene synthesis from array- derived oligonucleotide pools, and any of various error correction and sequence validation steps, or any combination thereof.

[0325] RNA chemical synthesis may be similar to that used for DNA. In some embodiments, RNA chemical synthesis methods may comprise an additional protecting group at the 2′ hydroxyl of ribose. The 2′ hydroxyl of ribose position may be protected with tert- butyldimethyl silyl groups, which can be stable throughout the synthesis, and can be removed at the final deprotection step by addition of a basic fluoride ion such as tetrabutylammonium fluoride (TBAF). The remaining positions on both the sugar and the bases can be protected in the same fashion as for DNA. By adjusting several parameters in the DNA synthesis protocol such as, but not limited to the coupling times, monomer delivery rate, frequency of washing steps, and types of capping reagents, stepwise coupling efficiencies of up to 99% can be obtained.

[0326] In a further aspect, the disclosure provides a vector comprising a nucleic acid sequence described herein. The vector utilized in the context of the present disclosure desirably comprises sequences appropriate for introduction into cells. In some embodiments, the vector is an expression vector.

[0327] In the context of the present disclosure, the term “vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acidAttorney Docket No.250298.000720 molecule of the disclosure, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.

[0328] The following vectors are provided by way of example: bacteriophages such as lambda (X) bacteriophage, EMBL bacteriophage; bacterial vectors such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5; eukaryotic vectors such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG and pSVL; and transposons such as Sleeping Beauty transposon and PiggyBac transposon.

[0329] In some embodiments, the vector is a viral vector. Viral vectors can be derived from naturally occurring virus genomes, which typically are modified to be replication incompetent, e.g. non-replicating. Non-replicating viruses require the provision of proteins in trans for replication. Typically, those proteins are stably or transiently expressed in a viral producer cell line, thereby allowing replication of the virus. The viral vectors are, thus, typically infectious and non-replicating. Viral vectors may be adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g., vectors derived from cytomegaloviruses, like rhesus cytomegalovirus (RhCMV)), arena virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors (canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retrovirus vectors, lentivirus vectors, simian virus 40 (SV40), bovine papilloma viruses, Epstein-Barr viruses, Moloney murine leukemia viruses, Harvey murine sarcoma viruses, murine mammary tumor viruses, Rous sarcoma viruses, poxvirus viral like particles, baculoviral vectors and bacterial spores.

[0330] As further examples, adenovirus vectors may be derived from human adenovirus (Ad) but also from adenoviruses that infect other species, such as bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or great apes, such as Chimpanzee (Pan), Gorilla (Gorilla), Orangutan (Pongo), Bonobo (Pan paniscus) and common chimpanzee (Pan troglodytes). Poxvirus (Poxviridae) vectors may beAttorney Docket No.250298.000720 derived from smallpox virus (variola), vaccinia virus, cowpox virus or monkeypox virus. Exemplary vaccinia viruses are the Copenhagen vaccinia virus (W), New York Attenuated Vaccinia Virus (NYVAC), ALVAC, TROVAC and Modified Vaccinia Ankara (MVA).

[0331] In certain embodiments, the present disclosure provides a composition comprising (i) an isolated polynucleotide described herein or a vector described herein; and (ii) a carrier or excipient. In some embodiments, such composition described herein may be formulated as a pharmaceutical composition described herein.

[0332] A non-limiting example of a carrier is a lipid nanoparticle carrier. A carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0333] The present disclosure further encompasses compositions comprising (i) one or more isolated peptides described herein, one or more fusion proteins described herein, one or more conjugates described herein, one or more oligomeric complexes described herein, one or more non-covalent complexes described herein, or one or more cells described herein, or any combination thereof, conjugated to a solid support. Examples of solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column. pMHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching peptides, fusion proteins, conjugates, oligomeric complexes, non-covalent, and cells described herein to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens. In some embodiments, peptides or pMHC complexes are labeled with biotin and attached to streptavidin-coated surfaces. In a preferred embodiment, a suitable solid can be a multi-well plate (e.g., a 96-well plate).Attorney Docket No.250298.000720

[0334] In yet another aspect, the disclosure provides a host cell comprising the vector of the disclosure. The host cell can be either a prokaryotic cell or a eukaryotic (e.g., an immune cell such as, but not limited to, an antigen-presenting cell (APC)). 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 and RR1. Non-limiting examples of eukaryotic host cells include yeast, insect, and mammalian cells (e.g., from a mouse, rat, monkey, or human cell lines). Non- limiting examples of yeast host cells include, e.g., YPH499, YPH500, and YPH501. Non-limiting examples of mammalian host cells include Chinese hamster ovary (CHO) cells, NIH Swiss mouse embryo cells NIH / 3T3, monkey kidney-derived COS-1 cells, and 293 cells which are human embryonic kidney cells. Examples of insect cells include Sf9 cells, which can be transfected with baculovirus expression vectors.

[0335] Transformation of appropriate cell hosts with a DNA construct of the present disclosure is accomplished by well-known methods that typically depend on the type of vector used. Successfully transformed cells, i.e. cells that contain a DNA construct of the present disclosure, can be identified by, for example, PCR. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0336] It will be appreciated that certain host cells of the disclosure are useful in the preparation of the peptides or peptide-based molecules of the disclosure, for example bacterial, yeast, and insect cells. However, other host cells may be useful in certain methods. For example, antigen-presenting cells (APCs), such as dendritic cells or B cells, may be used to express the peptides of the disclosure such that the peptides may be loaded into appropriate MHC molecules.

[0337] A further aspect of the disclosure provides a method of producing peptides or peptide-based molecules of the disclosure, the method comprising culturing a host cell and isolating the peptide or peptide-based molecule from the host cell or its culture medium.

[0338] The present disclosure further comprises a kit which may comprise any of various compositions of the present disclosure, including the peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties of the disclosure.

[0339] In one aspect, the present disclosure may include a kit comprising, for example: (i) a) one or more isolated peptides described herein; b) one or more fusion proteins of described herein; c) one or more conjugates described herein; d) one or more oligomeric complexes describedAttorney Docket No.250298.000720 herein; e) one or more non-covalent complexes described herein; f) one or more compositions described herein; g) one or more cells described herein; h) one or more polynucleotides described herein; and / or i) one or more vectors described herein; and (ii) optionally, packaging and / or instructions for use for the same. In various embodiments the kit or any composition described herein which comprises one or more off-target peptides and / or one or more polynucleotides or vectors encoding one or more off-target peptides may further comprise one or more antigen- recognition molecules, such as an antibody or TCR (e.g., as expressed on a cell), that targets the relevant target peptide (e.g., that targets DNTT250-258 peptide target KLFTSVFGV (SEQ ID NOs: 75)). The one or more antigen-recognition molecules may, for example, be in solution with a composition comprising one or more off-target peptides disclosed herein (e.g., in a container) and / or may be bound to one or more off-target peptides disclosed herein (e.g., bound to one or more pMHC complexes comprising off-target peptides). EXAMPLES

[0340] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. Development of Peptide in Groove Similarity Prediction (PIGSPRED) to Predict Off-targets Associated with Target Peptide-HLA (pHLA) Complexes

[0341] Peptide-HLA (pHLA) complexes specifically expressed on cancer cells present a unique set of targets to destroy cancer cells using antibody-based or cell-based therapeutics approaches. However, it is important to take into account the potential off-targets associated with these pHLA complexes to avoid off-target toxicity. While expression of these targets should be specific to cancer cells, it has become increasingly clear that there exists another set of off-targets: pHLA complexes that are highly similar to the target pHLA [Cameron BJ et al., Sci Transl Med. 2013; Linette GP et al., Blood. 2013]. The three-dimensional nature of the interactions between pHLA and its cognate therapeutic molecules has made it challenging to ascertain the similarity of pHLA complexes. However, most of the off-target toxicity that has been reported has resulted from off-targets that share the same HLA as the target pHLA, and also contain a peptide that is similar / homologous to the peptide sequence in the target pHLA. The present Example describes the development of a method called PIGSPRED (Peptide in Groove Similarity Prediction) usefulAttorney Docket No.250298.000720 in the prediction of such off-targets. After a target pHLA of interest was identified, PIGSPRED predicted off-targets associated with the target, which helped in evaluation of the risk associated with the target at the target selection step, as well as in screening highly specific T-cell Receptor (TCR) or antibodies (Abs). Following input of a target pHLA, the PIGSPRED method operated in the following multi-step fashion: 1. Identification of peptide positions important for TCR (T-cell Receptor) or antibody binding

[0342] When evaluating similarity / homology of a peptide to the target peptide, it is necessary to evaluate similarity at peptide positions that may be involved in binding interactions with the TCR / antibody. These important positions can be ascertained by analyzing the experimental structures of pHLA in complex with the TCR / antibody typically derived using crystallography or cryo-electron microscopy (cryoEM) techniques. Not only are such structures difficult to obtain, but at the initial target selection stage, where the potential risks associated with the target are evaluated, a TCR / antibody against the target is unavailable. To address this challenge, a computational mutagenesis algorithm using a commercial tool called NetMHCpan, which can predict the binding affinity of the peptide to an HLA using a machine learning model (Jurtz V et al., J Immunol.2017), was implemented. First, the binding affinity of the peptide to the HLA was predicted. Then the amino acid at each position in the peptide was iteratively mutated to a glycine amino acid, and the affinity of the mutated peptide subsequently predicted. Positions that did not result in the loss of binding affinity to the HLA were flagged for additional consideration, as such positions were not likely to be involved in HLA binding, and therefore were free to interact with the TCR / antibody. Out of these free positions, the ones containing a non-glycine amino acid were identified as the positions important for TCR / antibody binding. As described above, if the structure of a pHLA in complex with the TCR / antibody was available, then the binding motifs in the peptide sequence involved in TCR / antibody interactions could be obtained. 2. Identification of similar peptides

[0343] Canonical human protein sequences were obtained from UniprotKB and similar peptides were identified that were the same length as the target peptide and identical to the target peptide at three or more important amino acid positions. Similar peptides were additionally identified based on experimentally-derived binding motifs in the target peptide sequence. For these similar peptides to be potential off-targets, they would have to be capable of binding the HLA in the target pHLA. Therefore, NetMHCpan was used to predict the binding affinity of each similarAttorney Docket No.250298.000720 peptide to the HLA, and the peptides that were not predicted to bind were discarded from the dataset. A similar peptide may be identical to the target peptide, although from a different protein source as the target peptide. 3. Expression analysis

[0344] Once similar peptides were identified, it was necessary to establish that the peptides could potentially cause off-target effects to avoid overestimation of the risk associated with a given target. Therefore, the expression of the peptides in essential, normal tissues or essential cell types was ascertained. For this purpose, the Gene Tissue Expression database or GTEx (gtexportal.org / home / ) was queried. The GTEx database version 6, based on genome build GRCh38, contains gene expression data across 51 tissue types from 549 healthy donors. Gene expression values were queried only in those tissue types considered essential, in other words, tissues that cannot be sacrificed for the purpose of saving a patient’s life, unlike non-essential tissues such as, e.g., breast, ovary, testes, etc. For each gene, the 95-percentile expression value was calculated in each essential tissue type, followed by calculation of the maximum of that value across all essential tissue types. If the maximum expression value of a gene was greater than 0.5 transcripts per million (TPM), then it was assumed that any similar peptide that was derived from the gene would be a potential off-target. 4. Final output

[0345] Once all potential off-targets were identified, a Degree of Similarity (DoS) score was calculated for each off-target peptide to quantify the homology between the off-target and the target peptide. DoS represents the number of identical amino acids at identical positions, or hamming distance, between peptides. Once the DoS scores were determined, the number of off- targets at different threshold values was computed to compare the likelihood of off-target toxicity associated with different targets. Using immunopeptidomics data generated in the present Example, off-target peptides observed in mass-spectrometry experiments were annotated, thereby providing additional evidence for the potential off-targets. Identification of potential off-targets associated with an HLA-A2 peptide Identification of potential off-targets associated with a first MAGEA4 target peptide: GVYDGREHTV (SEQ ID NO: 1)

[0346] Tables 2A-4C illustrate results of applying the PIGSPRED method inputting MAGEA4230-239target GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at step 102.Attorney Docket No.250298.000720

[0347] Table 2A includes a number of potential high risk off-target peptides associated with the MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at different DoS threshold values. The number of potential high risk off-target peptides identified computationally are provided. Important positions were predicted computationally at step 104 to be positions 4 and 6-9. The table also provides the number of computationally predicted potential high risk off- target peptides that were also identified in immunopeptidomics mass-spectrometry data. Table 2A: Identified DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>=1 3 4 5 6 7 8 9 0get GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at a DoS greater than or equal to 6. The predicted binding affinity of each off-target is represented by the half maximal inhibitory concentration (IC50) value and a binding affinity percentile rank value (%Rank_BA in NetMHCpan). The mRNA level (TMP from GTEx) in the normal tissue with the highest expression for each off-target gene is provided, as well as the top three highly expressing normal tissues. Peptides found in mice may be useful for conducting antigen-recognition molecule binding studies (e.g., screenings) in mice. Potential off-target (1) GLADGRTHTV (SEQ ID NO: 2) was detected in mass-spectrometry experiments, while other potential off-targets in the table were not. Potential off-target peptides GLYDGREHSV (SEQ ID NO: 73, DoS = 8) from the MAGEA8 gene (TPM = 0.4, not expressed in mouse) and GLYDGMEHLI (SEQ ID NO: 74, DoS = 6) from the MAGEA10 gene (TPM = 0.1, not expressed in mouse) were filtered from the list of potential off-target peptides in Table 2B due to their low expression levels in essential normal tissue. However, as both MAGEA8 and MAGEA10 are cancer / testis (CT) antigens that may be useful for targeting in cancer immunotherapy applications, another working list comprising such potential secondary targets may be prepared, and additional testing (e.g., antigen-recognition molecule screening) may optionally be performed on one or more such potential secondary targets to assess whether cross-reactivity with those peptides could provide a beneficial therapeutic effect. Table 2B:Attorney Docket No.250298.000720 No Off-target DoS IC50 percentile Off target TPM Normal In Mass rank gene tissue mouse Spec l m l

[0349] Table 3A includes the number of potential off-targets associated with the MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at different DoS threshold values. An experimentally derived binding motif, XXXDXREXXX, was used where X represents any amino acid. Table 3A: Identified DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>=1 4 7

[0350] Table 3B includes potential off-targets associated with the MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at DoS>=3. An experimentally derived binding motif, XXXDXREXXX, was used where X represents any amino acid. Potential off-target (9) ALVDQRELYL (SEQ ID NO: 9) was detected in mass spectrometry experiments, while other potential off-targets in the table were not.Attorney Docket No.250298.000720 Table 3B: No Off-target DoS IC50percentile Off TPM Normal In Mass rank value target tissue mouse Spec l

[0351] Table 4A includes the number of potential off-targets associated with the MAGEA4230-239 target GVYDGREHTV (SEQ ID NO: 1)-HLA-A*02:01 at different DoS threshold values. An experimentally derived binding motif,Attorney Docket No.250298.000720 XX[YFWM][DENQ][GA][R][DENQ]XXX (SEQ ID NO: 10), was used where X represents any amino acid. Table 4A: Identified DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>= DoS>=1 3 4 5 6 7 8 9 0get GVYDGREHTV(SEQ ID NO: 1)-HLA-A*02:01 at DoS>=2. An experimentally derived binding motif, XX[YFWM][DENQ][GA][R][DENQ]XXX (SEQ ID NO: 10), was used where X represents any amino acid. Potential off-target (5) ALVDQRELYL (SEQ ID NO: 9) was detected in mass spectrometry experiments, while other potential off-targets in the table were not. Table 4B No Off-target DoS IC50 percentile Off target TPM Normal In Mass rank gene tissue mouse SpecAttorney Docket No.250298.000720 No Off-target DoS IC50 percentile Off target TPM Normal In Mass rank gene tissue mouse Spec l m l: KVLEHVVRV (SEQ ID NO: 48)

[0353] Tables 5A-5B illustrates results of applying the PIGSPRED method when inputting an alternative MAGEA4 peptide-derived pMHC target, MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48)-HLA-A*02:01, at step 102.

[0354] Table 5A includes a number of potential high risk off-target peptides associated with the MAGEA4286-294 target KVLEHVVRV (SEQ ID NO: 48)-HLA-A*02:01 at different DoS threshold values. The number of potential high risk off-target peptides identified computationally are provided. Important positions were predicted computationally at step 104. The table also provides the number of computationally predicted potential high risk off-target peptides that were also identified in immunopeptidomics mass-spectrometry data. Table 5A: Identified DoS>=3 DoS>=4 DoS>=5 DoS>=6 DoS>=7 DoS>=8 DoS>=9

[0355] ab e 5 nc udes potent a o -targets assoc ated w t t e target at a oS greater than or equal to 6. Eleven different potential off-targets were detected in mass-spectrometry experiments, while other potential off-targets in the table were not. Off-target peptide KVLEHVVRV (SEQ ID NO: 49, DoS = 9) from the MAG...

Claims

Attorney Docket No.250298.000720 CLAIMS What is claimed is:

1. An isolated peptide comprising an amino acid sequence of any one of SEQ ID NOs: 76- 82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is 8-12 amino acids in length.

2. The peptide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82.

3. The peptide of claim 2, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

4. The peptide of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide comprises one or more non- proteogenic amino acids, one or more non-naturally occurring amino acids, one or more non-native peptide bonds, or any combination thereof, optionally wherein the isolated peptide comprises one or more reverse peptide bonds, one or more D-isomers of amino acids, one or more chemical modifications, or any combination thereof.

5. The peptide of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is produced by expression in a heterologous host cell.

6. The peptide of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is produced synthetically.

7. A fusion protein comprising one or more peptides of any one of claims 1-6, or fragments or derivatives thereof, fused to one or more heterologous molecules.Attorney Docket No.250298.000720 8. The fusion protein of claim 7, wherein the one or more heterologous molecules comprise an MHC molecule, or a fragment or derivative thereof.

9. The fusion protein of claim 8, wherein the MHC molecule is a class I MHC molecule.

10. The fusion protein of claim 9, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

11. The fusion protein of claim 10, wherein the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

12. The fusion protein of claim 11, wherein the HLA-A molecule is an HLA-A*02:01 molecule.

13. A conjugate comprising one or more peptides of any one of claims 1-6, or fragments or derivatives thereof, conjugated to one or more heterologous molecules.

14. The conjugate of claim 13, wherein the one or more heterologous molecules comprise an MHC molecule, or a fragment or derivative thereof.

15. The conjugate of claim 14, wherein the MHC molecule is a class I MHC molecule.

16. The conjugate of claim 15, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

17. The conjugate of claim 16, wherein the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

18. The conjugate of claim 17, wherein the HLA-A molecule is an HLA-A*02:01 molecule.Attorney Docket No.250298.000720 19. The conjugate of claim 13, wherein the one or more peptides, or fragments or derivatives thereof, are conjugated to a particle or a solid support.

20. An oligomeric complex comprising two or more isolated peptides of any one of claims 1- 6 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

21. A non-covalent complex comprising (i) the isolated peptide of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, and (ii) an MHC molecule, or a fragment or derivative thereof.

22. The non-covalent complex of claim 21, wherein the MHC molecule is a class I MHC molecule.

23. The non-covalent complex of claim 22, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

24. The non-covalent complex of claim 23, wherein the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

25. The non-covalent complex of claim 24, wherein the HLA-A molecule is HLA-A*02:01 molecule.

26. A composition comprising (i) one or more isolated peptides of any one of claims 1-6, one or more fusion proteins of any one of claims 7-12, one or more conjugates of any one of claims 13-19, one or more oligomeric complexes of claim 20, or one or more non-covalent complexes of any one of claims 21-25, or any combination thereof; and (ii) a carrier or excipient.

27. An isolated cell comprising the one or more fusion proteins of any one of claims 7-12, one or more conjugates of any one of claims 13-19, one or more oligomeric complexes of claimAttorney Docket No.250298.000720 20, or one or more non-covalent complexes of any one of claims 21-25, or any combination thereof.

28. The isolated cell of claim 27, wherein the isolated cell is an immune cell.

29. The isolated cell of claim 27 or claim 28, wherein the isolated cell is an antigen-presenting cell (APC).

30. An isolated polynucleotide comprising a nucleotide sequence encoding one or more isolated peptides of any one of claims 1-6 or one or more fusion proteins of any one of claims 7-12.

31. The isolated polynucleotide of claim 30, wherein the nucleotide sequence is operably linked to a promoter.

32. The isolated polynucleotide of claim 30 or claim 31, wherein the isolated polynucleotide comprises DNA.

33. The isolated polynucleotide of claim 30 or claim 31, wherein the isolated polynucleotide comprises RNA.

34. The isolated polynucleotide of claim 33, wherein the RNA is mRNA.

35. The isolated polynucleotide of claim 33, wherein the RNA is self-replicating RNA.

36. A vector comprising the isolated polynucleotide of any one of claims 30-35.

37. The vector of claim 36, wherein the vector is an expression vector.

38. The vector of claim 36 or claim 37, wherein the vector is a viral vector.Attorney Docket No.250298.000720 39. A host cell comprising the isolated polynucleotide of any one of claims 30-35 or the vector of any one of claims 36-38.

40. The host cell of claim 39, wherein the host cell is a prokaryotic cell.

41. The host cell of claim 39, wherein the host cell is a eukaryotic cell.

42. The host cell of claim 41, wherein the host cell is an immune cell.

43. The host cell of claim 41 or claim 42, wherein the host cell is an antigen-presenting cell (APC).

44. A composition comprising (i) the isolated polynucleotide of any one of claims 30-35 or the vector of any one of claims 36-38; and (ii) a carrier or excipient.

45. The composition of claim 44, wherein the carrier is a lipid nanoparticle carrier.

46. A composition comprising: one or more isolated peptides of any one of claims 1-6, one or more fusion proteins of any one of claims 7-12, one or more conjugates of any one of claims 13-19, one or more oligomeric complexes of claim 20, one or more non-covalent complexes of any one of claims 21-25, or one or more cells of any one of claims 27-29 and 39-43, or any combination thereof, conjugated to a solid support.

47. The composition of claim 46, wherein the solid support is a multi-well plate.

48. A kit comprising: (i) a) one or more isolated peptides of any one of claims 1-6; b) one or more fusion proteins of any one of claims 7-12; c) one or more conjugates of any one of claims 13-19; d) one or more oligomeric complexes of claim 20; e) one or more non- covalent complexes of any one of claims 21-25; f) one or more compositions of any one of claims 26 and 44-47; g) one or more cells of any one of claims 27-29 andAttorney Docket No.250298.000720 39-43; h) one or more polynucleotides of any one of claims 30-35; and / or i) one or more vectors of any one of claims 36-38; (ii) optionally, an antigen-recognition molecule that targets DNTT250-258 peptide; and / or (iii) optionally, packaging and / or instructions for use for the same.

49. The kit of claim 48, wherein the antigen-recognition molecule(s) comprises an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

50. An in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258peptide, comprising: a) contacting the antigen-recognition molecule with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258peptide complex); b) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) is presented in a complex with the same kind of MHC molecule, or the fragment or derivative thereof, as in (a) (MHC-off-target peptide complex); and c) determining the level of binding of the antigen-recognition molecule to the MHC- DNTT250-258 peptide complex and each of the MHC-off-target peptide complexes.

51. An in vitro method of assessing off-target effects of an antigen-recognition molecule that targets DNTT250-258 peptide, comprising: a) contacting the antigen-recognition molecule with one or more off-target peptides, wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with a major histocompatibility complex (MHC) molecule,Attorney Docket No.250298.000720 or a fragment or derivative thereof (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said peptide; and b) determining the level of binding of the antigen-recognition molecule to said one or more MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

52. The method of claim 50 or claim 51, further comprising determining that the antigen- recognition molecule is likely to have off-target effects if it detectably binds to at least one MHC-off-target peptide complex, off-target peptide or protein or fragment thereof comprising said off-target peptide.

53. A method for selecting an antigen-recognition molecule that targets DNTT250-258 peptide, comprising: a) contacting a plurality of antigen-recognition molecules with DNTT250-258peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258 peptide complex); b) contacting the same plurality of antigen-recognition molecules with one or more off-target peptides, (i) wherein each of said off-target peptides comprises an amino acid sequence of any one of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, and (ii) wherein each of said off-target peptides is presented in one of the following forms: (1) as a complex with the same kind of MHC molecule, or the fragment or derivative thereof as in (a) (MHC-off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said off-target peptide; c) selecting one or more antigen-recognition molecules based on their level of binding to MHC-DNTT250-258peptide complex and based at least in part on the number of different MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides detectably bound by each of the antigen-recognition molecules; andAttorney Docket No.250298.000720 d) optionally, repeating steps (a)-(c) using the one or more selected antigen- recognition molecules.

54. The method of claim 53, wherein the one or more selected antigen-recognition molecules detectably bind no more than five MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

55. The method of claim 53, wherein the one or more selected antigen-recognition molecules do not detectably bind to any of the tested MHC-off-target peptide complexes, off-target peptides or proteins or fragments thereof comprising said off-target peptides.

56. The method of any one of claims 53-55, wherein the plurality of antigen-recognition molecules is in a library.

57. The method of claim 56, wherein the library is a phage display library or a yeast library.

58. The method of any one of claims 50-57, wherein one or more of the MHC-DNTT250-258peptide complexes, MHC-off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off-target peptides are immobilized on a solid support.

59. The method of any one of claims 50-57, wherein one or more of the MHC-DNTT250-258peptide complexes, MHC-off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off-target peptides are present on cells.

60. The method of any one of claims 50-57, wherein one or more of the MHC-DNTT250-258 peptide complexes, MHC-off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off-target peptides are present in a soluble form.

61. The method of any one of claims 50-60, wherein the level of binding is determined by detecting the amount of antigen-recognition molecules bound to the MHC-DNTT250-258Attorney Docket No.250298.000720 peptide complexes, MHC-off-target peptide complexes, off-target peptides, or proteins or fragments thereof comprising said off-target peptides.

62. The method of any one of claims 50-61, wherein the method is performed in a high- throughput format.

63. The method of claim 62, wherein the method is performed in a multi-well plate.

64. A method of enriching a sample for antigen-recognition molecules that target DNTT250-258 peptide, comprising: a) contacting a sample comprising a plurality of antigen-recognition molecules with DNTT250-258 peptide presented in a complex with a major histocompatibility complex (MHC) molecule, or a fragment or derivative thereof (MHC-DNTT250-258 peptide complex) in the presence of one or more off-target peptides, wherein each of said off-target peptides (i) comprises an amino acid sequence of any one of SEQ ID Nos: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the peptide is 8-12 amino acids in length, wherein each of said one or more off-target peptides is presented in one of the following forms: (1) as a complex with an MHC molecule, or a fragment or derivative thereof (MHC- off-target peptide complex), (2) as a peptide, or (3) as a protein or fragment thereof comprising said off-target peptide; b) enriching the sample by isolating the antigen-recognition molecules that are detectably bound to said MHC-DNTT250-258 peptide complex; and c) optionally, repeating steps (a)-(b) using the enriched sample.

65. The method of claim 64, wherein said MHC-DNTT250-258 peptide complex is present on a cell or solid support and said off-target peptides or proteins or fragments thereof comprising said off-target peptides or MHC-off-target peptide complexes are present in solution.Attorney Docket No.250298.000720 66. The method of claim 64 or claim 65, wherein said MHC-DNTT250-258peptide complex is labeled and said off-target peptides or proteins or fragments thereof comprising said off- target peptides or MHC-off-target peptide complexes are not labeled or are labeled differently.

67. The method of any one of claims 50-66, wherein the DNTT250-258 peptide comprises the amino acid sequence of KLFTSVFGV (SEQ ID NO: 75).

68. The method of claim 67, wherein the DNTT250-258 peptide consists of the amino acid sequence KLFTSVFGV (SEQ ID NOs: 75).

69. The method of any one of claims 50-68, wherein the off-target peptide comprises an amino acid sequence of any one of SEQ ID NOs: 76-82.

70. The method of claim 69, wherein the off-target peptide consists of an amino acid sequence of any one of SEQ ID NOs: 76-82.

71. The method of any one of claims 50-70, wherein each of said off-target peptides is presented as an MHC-off-target peptide complex.

72. The method of any one of claims 50-71, wherein the MHC molecule is a class I MHC molecule.

73. The method of claim 72, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

74. The method of claim 73, wherein the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

75. The method of claim 74, wherein the HLA-A molecule is an HLA-A*02:01 molecule.Attorney Docket No.250298.000720 76. The method of any one of claims 50-75, wherein the peptide within the MHC-DNTT250-258peptide complex and / or MHC-off-target peptide complex is covalently bound to the MHC.

77. The method of any one of claims 50-75, wherein the peptide within the MHC-DNTT250-258peptide complex and / or MHC-off-target peptide complex is non-covalently bound to the MHC.

78. The method of any one of claims 50-77, wherein the antigen-recognition molecule(s) comprises an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

79. A library of off-target peptides, said library comprising two or more peptides each selected from the amino acid sequences of SEQ ID NOs: 76-82, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is 8-12 amino acids in length.

80. The library of claim 79, wherein the at least two off-target peptides are each present in a complex with a major histocompatibility complex (MHC) molecule.

81. The library of claim 79 or claim 80, wherein the MHC molecule is a class I MHC molecule.

82. The library of claim 81, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

83. The library of claim 82, wherein the class I HLA molecule is an HLA-A molecule, optionally an HLA-A*02 molecule.

84. The library of claim 83, wherein the HLA-A molecule is an HLA-A*02:01 molecule.

85. The library of any one of claims 79-84, wherein the peptide within the MHC-off-target peptide complex is covalently bound to the MHC.Attorney Docket No.250298.000720 86. The library of any one of claims 79-84, wherein the peptide within the MHC-off-target peptide complex is non-covalently bound to the MHC.

87. A library comprising two or more proteins or fragments thereof each comprising one or more off-target peptides, each off-target peptide being selected from the amino acid sequences of SEQ ID NOs: 76-82.

88. A library comprising: a) two or more isolated peptides of any one of claims 1-6; b) two or more fusion proteins of any one of claims 7-12; c) two or more conjugates of any one of claims 13-19; d) two or more oligomeric complexes of claim 20; e) two or more non- covalent complexes of any one of claims 21-25; f) two or more compositions of any one of claims 26 and 44-47; g) two or more cells of any one of claims 27-29 and 39-43; h) two or more polynucleotides of any one of claims 30-35; and / or i) two or more vectors of any one of claims 36-38, optionally wherein each element of the library exists in a separate container.

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