ANTIGEN-BINDING PROTEINS TARGETING SHARED NEOANTIGENS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/930,374, filed on November 4, 2019, the entire contents of which are incorporated by reference herein for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 2, 2020, is named GSO-081WO_SL.txt and is 6,958,930 bytes in size.

BACKGROUND

[0003] Antibody -based immunotherapies targeting tumor-specific antigens hold great promise as a next-generation of personalized cancer immunotherapy. ^1-3 For example, cancers with a high mutational burden, such as non-small cell lung cancer (NSCLC) and melanoma, are particularly attractive targets of such therapy given the relatively greater likelihood of neoantigen generation. ^4,5 However, antibody-based immunotherapy designs face at least two hurdles: (1) discovery of tumor-associated neoantigens and (2) selecting anti-neoantigen leads most likely to generate anti-tumor responses.

[0004] Regarding (1), previous approaches for tumor-associate neoantigen discovery considered only cis-acting mutations, largely neglecting additional sources of neo-ORFs, including mutations in splicing factors, which occur in multiple tumor types and lead to aberrant splicing of many genes ¹³ , and mutations that create or remove protease cleavage sites.

[0005] In addition, approaches to tumor genome and transcriptome analysis can miss somatic mutations that give rise to candidate neoantigens due to suboptimal conditions in library construction, exome and transcriptome capture, sequencing, or data analysis. Likewise, standard tumor analysis approaches can inadvertently promote sequence artifacts or germline polymorphisms as neoantigens, leading to ineffective immunotherapies or increased auto- immunity risk, respectively.

[0006] Regarding (2), one question for neoantigen antibody design is which of the many coding mutations present in subject tumors can generate the “best” therapeutic neoantigen epitopes, e.g., those specifically displayed on the surface of tumor cells, that can robustly serve as addressable targets and cause tumor regression.

[0007] It is recognized that MHCs display intracellularly processed protein fragments on the cell surface. In humans, MHC is referred to as human leukocyte antigen or HLA. In particular, MHC class I molecules are expressed on the surface of virtually all nucleated cells in the body. They are dimeric molecules comprising a transmembrane heavy chain, comprising the peptide antigen binding cleft, and a smaller extracellular chain termed beta2- microglobulin. MHC class I molecules present peptides derived from the degradation of cytosolic proteins by the proteasome, a multi-unit structure in the cytoplasm, (Niedermann G., 2002. Curr Top Microbiol Immunol. 268:91-136; for processing of bacterial antigens, refer to Wick M J, and Ljunggren H G., 1999. Immunol Rev. 172: 153-62). Cleaved peptides are transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) where they are bound to the groove of the assembled class I molecule, and the resultant MHC/peptide complex is transported to the cell membrane to enable antigen presentation to T lymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7; Yewdell J W. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8). Alternatively, cleaved peptides can be loaded onto MHC class I molecules in a TAP- independent manner and can also present extracellularly-derived proteins through a process of cross-presentation.

[0008] MHC genes are highly polymorphic across species populations, comprising multiple common alleles for each individual gene. As such, a given MHC allele/peptide complex comprising a specific HLA subtype and a specific peptide fragment presents a novel protein structure on the cell surface that can be targeted by a novel antigen-binding protein (e.g., antibodies or antigen binding fragments thereof). However, such antibody-based approaches first require the identification of the complex’s structure (peptide sequence and MHC subtype).

[0009] Tumor cells can express neoantigens and may display such antigens on the surface of the tumor cell via MHC presentation. Such tumor-associated neoantigens, comprising the novel protein structure formed by the peptide-MHC subtype complex, can be used for development of novel immunotherapeutic reagents for the specific targeting of tumor cells. For example, tumor-associated antigens can be used to identify therapeutic antigen binding proteins, e.g., TCR-mimetic antibodies, or antigen-binding fragments thereof. However, accurate identification of such neoantigens has been challenging. [0010] Initial methods have been proposed incorporating mutation-based analysis using next- generation sequencing, RNA gene expression, and prediction of MHC binding affinity of candidate neoantigen peptides ⁸ However, these proposed methods can fail to model the entirety of the epitope generation process, which contains many steps (e.g., TAP transport, proteasomal cleavage, and/or TCR recognition) in addition to gene expression and MHC binding ⁹ . Consequently, existing methods are likely to suffer from reduced low positive predictive value (PPV).

[0011] Indeed, analyses of peptides presented by tumor cells performed by multiple groups have shown that <5% of peptides predicted to be presented using gene expression and MHC binding affinity are actually found on the tumor surface MHC ^10,11 . This low correlation between binding predicted and actual MHC presentation was further reinforced by recent observations of the lack of predictive accuracy improvement of binding-restricted neoantigens for checkpoint inhibitor response over the number of mutations alone. ¹² [0012] This low positive predictive value (PPV) of existing methods for predicting presentation presents a problem for neoantigen-based immunotherapy design. If immunotherapies are designed using predictions with a low PPV, many of them will be clinically ineffective.

[0013] Accordingly, there is a need for the discovery and identification of tumor-associated HLA-peptide complexes with high positive predictive value, and a need for the development of antibody-based immunotherapies targeting such complexes.

SUMMARY

[0014] Provided herein is an isolated antigen binding protein (ABP) that specifically binds to an HLA-PEPTIDE antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, wherein the HLA Class I molecule and the HLA-restricted peptide are each selected from an HLA-PEPTIDE antigen as described in any one of SEQ ID NOs: 10,755 to 29,364, and wherein the isolated ABP comprises an antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the HLA-restricted peptide is between about 5-15 amino acids in length. In some embodiments, the HLA-restricted peptide is between about 8-12 amino acids in length, optionally 8, 9, 10, 11, or 12 amino acids in length. [0016] In some embodiments, the HLA-PEPTIDE antigen is selected from Table 5 A. In some embodiments, the HLA-PEPTIDE antigen is selected from Table 5B. In some embodiments, the HLA-PEPTIDE antigen is selected from Table 6. In some embodiments, the HLA-PEPTIDE antigen is selected from Table 7.

[0017] In some embodiments, the HLA-restricted peptide comprises a RAS G12 mutation. In some embodiments, the G12 mutation is a G12C, a G12D, a G12V, or a G12A mutation. In some embodiments, the HLA-PEPTIDE antigen comprises an HLA Class I molecule selected from HLA-A*02:01, HLA-A* 11:01, HLA-A*31:01, HLA-C*01:02, and HLA-A*03:01. In some embodiments, the RAS G12 mutation is any one or more of: a KRAS, NRAS, and HRAS mutation.

[0018] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA-C*01 :02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369); and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[0019] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA-C*01 :02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369); and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[0020] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); and a RAS_G12V MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[0021] In some embodiments, the HLA-PEPTIDE antigen is a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365).

[0022] In some embodiments, the HLA-PEPTIDE antigen is a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366).

[0023] In some embodiments, the HLA-PEPTIDE antigen is a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys. (SEQ ID NO: 29368)

[0024] In some embodiments, the antigen binding protein binds to the HLA-PEPTIDE antigen through at least one contact point with the HLA Class I molecule and through at least one contact point with the HLA-restricted peptide.

[0025] In some embodiments, the antigen binding protein binds to a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365), and wherein the isolated ABP binds to the RAS_G12C MHC Class I antigen at a higher affinity than an HLA-PEPTIDE antigen comprising a different RAS G12 mutation. In some embodiments, the isolated ABP binds to the RAS_G12C MHC Class I antigen at a higher affinity than an HLA-PEPTIDE antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule. In some embodiments, the isolated ABP does not bind to an HLA-PEPTIDE antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule.

[0026] In some embodiments, the antigen binding protein is linked to a scaffold, optionally wherein the scaffold comprises serum albumin or Fc, optionally wherein Fc is human Fc and is an IgG (IgG1, IgG2, IgG3, IgG4), an IgA (IgAl, IgA2), an IgD, an IgE, or an IgM isotype Fc. In some embodiments, the antigen binding protein is linked to a scaffold via a linker, optionally wherein the linker is a peptide linker, optionally wherein the peptide linker is a hinge region of a human antibody.

[0027] In some embodiments, the antigen binding protein comprises an Fv fragment, a Fab fragment, a F(ab’) ₂ fragment, a Fab’ fragment, an scFv fragment, an scFv-Fc fragment, and/or a single-domain antibody or antigen binding fragment thereof. In some embodiments, the antigen binding protein comprises an scFv fragment.

[0028] In some embodiments, the antigen binding protein comprises one or more antibody complementarity determining regions (CDRs), optionally at least six antibody CDRs. In some embodiments, the antigen binding protein comprises an antibody. In some embodiments, the antigen binding protein is a monoclonal antibody. In some embodiments, the antigen binding protein is a humanized, human, or chimeric antibody.

[0029] In some embodiments, the antigen binding protein is multispecific, optionally bispecific. In some embodiments, the antigen binding protein binds greater than one antigen or greater than one epitope on a single antigen.

[0030] In some embodiments, the antigen binding protein comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antigen binding protein comprises a heavy chain constant region of the class human IgG and a subclass selected from IgG1, IgG4, IgG2, and IgG3.

[0031] In some embodiments, the antigen binding protein comprises a modification that extends half-life.

[0032] In some embodiments, the antigen binding protein is a portion of a chimeric antigen receptor (CAR) comprising: an extracellular portion comprising the antigen binding protein; and an intracellular signaling domain. In some embodiments, the antigen binding protein comprises an scFv and the intracellular signaling domain comprises an ITAM. In some embodiments, the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the ABP further comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises a transmembrane portion of CD28. In some embodiments, the ABP further comprises an intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is CD28, 4- IBB, OX-40, ICOS, or any combination thereof.

[0033] Also provided herein is an isolated ABP disclosed herein for use as a medicament. Also provided herein is an isolated ABP disclosed herein for use in treatment of cancer, optionally wherein the cancer expresses or is predicted to express the HLA-PEPTIDE antigen. In some embodiments, the cancer is selected from a solid tumor and a hematological tumor.

[0034] Also provided herein is an antigen binding protein (ABP) that competes for binding with the isolated ABP disclosed herein. Also provided herein is an antigen binding protein (ABP) that binds the same HLA-PEPTIDE antigen epitope bound by the isolated ABP disclosed herein.

[0035] Also provided herein is an engineered cell expressing a receptor comprising the antigen binding protein disclosed herein. In some embodiments, the engineered cells is a T cell, optionally a cytotoxic T cell (CTL). In some embodiments, the antigen binding protein is expressed from a heterologous promoter.

[0036] Also provided herein is an isolated polynucleotide or set of polynucleotides encoding the isolated ABP disclosed herein or an antigen-binding portion thereof. Also provided herein is a vector or set of vectors comprising the polynucleotide or set of polynucleotides disclosed herein. Also provided herein is a virus comprising the isolated polynucleotide or set of polynucleotides disclosed herein. In some embodiments, the virus is a filamentous phage. Also provided herein is a yeast cell comprising the isolated polynucleotide or set of polynucleotides disclosed herein. Also provided herein is a host cell comprising the polynucleotide or set of polynucleotides disclosed herein or the vector or set of vectors disclosed herein, optionally wherein the host cell is CHO or HEK293, or optionally wherein the host cell is a T cell. Also provided herein is a method of producing an antigen binding protein comprising expressing the antigen binding protein with a host cell disclosed herein and isolating the expressed antigen binding protein.

[0037] Also provided herein is a pharmaceutical composition comprising the antigen binding protein disclosed herein and a pharmaceutically acceptable excipient. [0038] Also provided herein is a method of treating cancer in a subject, comprising administering to the subject the ABP disclosed herein or a pharmaceutical composition disclosed herein, optionally wherein the cancer is selected from a solid tumor and a hematological tumor. Also provided herein is a method of stimulating an immune response in a subject, comprising administering to the subject the ABP disclosed herein or a pharmaceutical composition disclosed herein, optionally wherein the subject has cancer, optionally wherein the cancer is selected from a solid tumor and a hematological tumor. Also provided herein is a method of killing a target cell in a subject, comprising administering to the subject the ABP disclosed herein or a pharmaceutical composition of disclosed herein, optionally wherein the subject has cancer and the target cell is a cancer cell, optionally wherein the cancer is selected from a solid tumor and a hematological tumor.

[0039] In some embodiments, the subject is a human subject.

[0040] In some embodiments, the cancer expresses or is predicted to express an HLA- PEPTIDE antigen or HLA Class I molecule as described in any one of SEQ ID NOs: 10,755 to 29,364, and wherein the ABP binds to the HLA-PEPTIDE antigen.

[0041] In some embodiments, the cancer expresses or is predicted to express an HLA- PEPTIDE antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, wherein the HLA Class I molecule and the HLA-restricted peptide are each selected from an HLA-PEPTIDE antigen as described in any one of SEQ ID NOs: 10,755 to 29,364, and wherein the ABP binds to the HLA-PEPTIDE antigen.

[0042] In some embodiments of the method, the HLA-PEPTIDE antigen is selected from Table 5A. In some embodiments of the method, the HLA-PEPTIDE antigen is selected from Table 5B. In some embodiments of the method, the HLA-PEPTIDE antigen is selected from Table 6. In some embodiments of the method, the HLA-PEPTIDE antigen is selected from Table 7.

[0043] In some embodiments, the HLA-PEPTIDE antigen comprises an HLA-restricted peptide which is a peptide fragment of RAS comprising a RAS G12 mutation.

[0044] In some embodiments, the G12 mutation is a G12C, a G12D, a G12V, or a G12A mutation. [0045] In some embodiments, the HLA-PEPTIDE antigen comprises an HLA Class I molecule selected from HLA-A*02:01, HLA-A* 11:01, HLA-A*31:01, HLA-C*01:02, and HLA-A*03:01.

[0046] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA-C*01 :02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369); and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[0047] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA-C*01 :02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369); and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368). [0048] In some embodiments, the HLA-PEPTIDE antigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); or a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368). [0049] In some embodiments, the antigen binding protein binds to a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365), and wherein the isolated ABP binds to the RAS_G12C MHC Class I antigen at a higher affinity than an HLA-PEPTIDE antigen comprising a different RAS G12 mutation.

[0050] In some embodiments, the isolated ABP binds to the RAS_G12C MHC Class I antigen at a higher affinity than an HLA-PEPTIDE antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule.

[0051] In some embodiments, the isolated ABP does not bind to an HLA-PEPTIDE antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule.

[0052] In some embodiments, the method comprises, prior to the administering, determining or having determined the presence of any one or more of the HLA-PEPTIDE antigen, the peptide of the HLA-PEPTIDE antigen, the somatic mutation associated with the HLA- PEPTIDE antigen, and the HLA molecule of the HLA-PEPTIDE antigen in a biological sample obtained from the subject.

[0053] In some embodiments, the biological sample is a blood sample or a tumor sample. In some embodiments, the blood sample is a plasma or serum sample.

[0054] In some embodiments, the determining comprises RNASeq, microarray, PCR, Nanostring, in situ hybridization (ISH), Mass spectrometry, sequencing, or immunohistochemistry (IHC).

[0055] In some embodiments of the method, the method comprises, after having determined the presence of the HLA-PEPTIDE antigen, peptide, or HLA in the biological sample obtained from the subject, administering to the subject an ABP that selectively binds to the HLA-PEPTIDE antigen.

[0056] Also provided herein is a kit comprising the antigen binding protein disclosed herein or a pharmaceutical composition disclosed herein and instructions for use. [0057] Also provided herein is a system, comprising: an isolated HLA-PEPTIDE antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, and wherein the HLA-PEPTIDE antigen is selected from an HLA-PEPTIDE antigen described in any one of SEQ ID NOs: 10,755 to 29,364; and a phage display library.

[0058] In some embodiments, the HLA-PEPTIDE antigen is attached to a solid support. In some embodiments, the solid support comprises a bead, well, membrane, tube, column, plate, sepharose, magnetic bead, cell, or chip. In some embodiments, the HLA-PEPTIDE antigen comprises a first member of an affinity binding pair and the solid support comprises a second member of the affinity binding pair. In some embodiments, the first member is streptavidin and the second member is biotin.

[0059] In some embodiments, the phage display library is a human library. In some embodiments, the phage display library is a humanized library.

[0060] In some embodiments, the system further comprises a negative control HLA- PEPTIDE antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, and wherein the negative control HLA-PEPTIDE antigen comprises a different restricted peptide, a different HLA Class I molecule, or a different restricted peptide and a different HLA Class I molecule. In some embodiments, the negative control HLA-PEPTIDE antigen comprises a different restricted peptide but the same HLA Class I molecule as the HLA-PEPTIDE antigen.

[0061] In some embodiments, the system comprises a reaction mixture, the reaction mixture comprising the HLA-PEPTIDE antigen and a plurality of phages from the phage display library.

[0062] Also provided herein is use of a system disclosed herein for identifying an antigen binding protein that selectively binds the isolated HLA-PEPTIDE antigen.

[0063] Also provided herein is a composition comprising an HLA-PEPTIDE antigen as described by any one of SEQ ID NOs: 10,755 to 29,364, wherein the HLA-PEPTIDE antigen is covalently linked to an affinity tag. In some embodiments, the affinity tag is a biotin tag. [0064] Also provided herein is a composition comprising an HLA-PEPTIDE antigen as described by any one of SEQ ID NOs: 10,755 to 29,364 complexed with a detectable label. In some embodiments, the detectable label comprises a β ₂ -microglobulin binding molecule. In some embodiments, the β ₂ -microglobulin binding molecule is a labeled antibody. In some embodiments, the labeled antibody is a fluorochrome-labeled antibody.

[0065] Also provided herein is a composition comprising an HLA-PEPTIDE antigen as described by any one of SEQ ID NOs: 10,755 to 29,364 attached to a solid support. In some embodiments, the solid support comprises a bead, well, membrane, tube, column, plate, sepharose, magnetic bead, cell, or chip. In some embodiments, the HLA-PEPTIDE antigen comprises a first member of an affinity binding pair and the solid support comprises a second member of the affinity binding pair. In some embodiments, the first member is streptavidin and the second member is biotin.

[0066] Also provided herein is a host cell comprising a heterologous HLA-PEPTIDE antigen as described by any one of SEQ ID NOs: 10,755-29,364. Also provided herein is a host cell which expresses an HLA subtype as defined by any one of the HLA-PEPTIDE antigens described in SEQ ID NOs: 10,755-29,364. Also provided herein is a host cell comprising a polynucleotide encoding an HLA-restricted peptide as defined by any one of the HLA- PEPTIDE antigens in SEQ ID NOs: 10,755-29,364.

[0067] In some embodiments, the host cell does not comprise endogenous MHC. In some embodiments, the host cell comprises an exogenous HLA. In some embodiments, the host cell is a K562 or A375 cell. In some embodiments, the host cell is a cultured cell from a tumor cell line. In some embodiments, the tumor cell line expresses an HLA subtype as defined by the same HLA-PEPTIDE antigen that describes the HLA-restricted peptide. In some embodiments, the tumor cell line is selected from the group consisting of HCC-1599, NCI-H510A, A375, LN229, NCI-H358, ZR-75-1, MS751, OE19, MOR, BV173, MCF-7, NCI-H82, Colo829, SK-MEL-28, KYSE270, 59M, andNCI-H146.

[0068] Also provided herein is a cell culture system comprising a host cell disclosed herein, and a cell culture medium. In some embodiments, the host cell expresses an HLA subtype as defined by any one of the HLA-PEPTIDE antigens in SEQ ID NOs: 10,755-21,015 and SEQ ID NOs: 21,016-29,364, and wherein the cell culture medium comprises a restricted peptide as defined by the same HLA-PEPTIDE antigen as the HLA subtype. In some embodiments, the host cell is a K562 cell which comprises an exogenous HLA, wherein the exogenous HLA is an HLA subtype as defined by any one of the HLA-PEPTIDE antigens in SEQ ID NOs: 10,755-29,364, and the cell culture medium comprises a restricted peptide as defined by the same HLA-PEPTIDE antigen defining the HLA subtype. [0069] Also provided herein is a method of identifying an antigen binding protein disclosed herein, comprising providing at least one HLA-PEPTIDE antigen described in SEQ ID NOs: 10,755-29,364; and binding the at least one target with the antigen binding protein, thereby identifying the antigen binding protein.

[0070] In some embodiments, the antigen binding protein is present in a phage display library comprising a plurality of distinct antigen binding proteins. In some embodiments, the phage display library is substantially free of antigen binding proteins that non-specifically bind the HLA of the HLA-PEPTIDE antigen.

[0071] In some embodiments, the binding step is performed more than once, optionally at least three times.

[0072] In some embodiments, the method further comprises contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE antigen to determine if the antigen binding protein selectively binds the HLA-PEPTIDE antigen, optionally wherein selectivity is determined by measuring binding affinity of the antigen binding protein to soluble target HLA-PEPTIDE complexes versus soluble HLA- PEPTIDE complexes that are distinct from target complexes, optionally wherein selectivity is determined by measuring binding affinity of the antigen binding protein to target HLA- PEPTIDE complexes expressed on the surface of one or more cells versus HLA-PEPTIDE complexes that are distinct from target complexes expressed on the surface of one or more cells.

[0073] Also provided herein is a method of identifying an antigen binding protein disclosed herein, comprising obtaining at least one HLA-PEPTIDE antigen described in SEQ ID NOs: 10,755-29,364; administering the HLA-PEPTIDE antigen to a subject, optionally in combination with an adjuvant; and isolating the antigen binding protein from the subject. [0074] In some embodiments, isolating the antigen binding protein comprises screening the serum of the subject to identify the antigen binding protein.

[0075] In some embodiments, the method further comprises contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE antigen to determine if the antigen binding protein selectively binds to the HLA-PEPTIDE antigen, optionally wherein selectivity is determined by measuring binding affinity of the antigen binding protein to the HLA-PEPTIDE antigen versus soluble HLA-PEPTIDE complexes that are distinct from the HLA-PEPTIDE antigen, optionally wherein selectivity is determined by measuring binding affinity of the antigen binding protein to the HLA- PEPTIDE antigen expressed on the surface of one or more cells versus HLA-PEPTIDE complexes that are distinct from the HLA-PEPTIDE antigen expressed on the surface of one or more cells.

[0076] In some embodiments, the subject is a mouse, a rabbit, or a llama.

[0077] In some embodiments, isolating the antigen binding protein comprises isolating a B cell from the subject that expresses the antigen binding protein and optionally directly cloning sequences encoding the antigen binding protein from the isolated B cell. In some embodiments, the method further comprises creating a hybridoma using the B cell. In some embodiments, the method further comprises cloning CDRs from the B cell. In some embodiments, the method further comprises immortalizing the B cell, optionally via EBV transformation.

[0078] In some embodiments, the method further comprises creating a library that comprises the antigen binding protein of the B cell, optionally wherein the library is phage display or yeast display.

[0079] In some embodiments, the method further comprises humanizing the antigen binding protein.

[0080] Also provided herein is a method of identifying an antigen binding protein disclosed herein, comprising obtaining a cell comprising the antigen binding protein; contacting the cell with an HLA-multimer comprising at least one HLA-PEPTIDE antigen described in SEQ ID NOs: 10,755-29,364; and identifying the antigen binding protein via binding between the HLA-multimer and the antigen binding protein.

[0081] Also provided herein is a method of identifying an antigen binding protein disclosed herein, comprising providing at least one HLA-PEPTIDE antigen described in SEQ ID NOs: 10,755-29,364; and identifying the antigen binding protein using the target.

BRIEF DESCRIPTION OF DRAWINGS

[0082] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0083] FIG. 1 shows the general structure of a Human Leukocyte Antigen (HLA) Class I molecule. By User atropos235 on en. wikipedia - Own work, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=1805424 [0084] FIG. 2 depicts MSD and cell-based binding data of three exemplary scFv clones specific for the neoantigen-based target designated SNA6 (RAS G12V neoantigen HLA-

[0085] FIG. 3 depicts MSD and cell-based binding data of three exemplary scFv clones for the neoantigen-based target designated SNA30 (RAS G12D neoantigen

[0086] FIG. 4 depicts the correlation between EDGE score and the probability of detection of candidate shared neoantigen peptides by targeted Mass Spectrometry.

[0087] FIG. 5A depicts flow cytometry gating strategy for detecting CD8+ T cells.

[0088] FIG. 5B depicts flow cytometry results demonstrating that a large proportion of CD8+ T cells exhibit binding to the RAS G12V:HLA*1101 pHLA.

[0089] FIG. 6 depicts sensorgrams from binding affinity assessment of selected “SNA6” scFv PPEs using a Carterra biosensor.

[0090] FIG. 7 shows titration of DOX administration in regulating expression of a representative neoantigen under a Tet-On system in multiple K562-HLA cell-lines.

[0091] FIG. 8 shows a representative KRAS G12V peptide VVGAVGVGK (SEQ ID NO: 29362) observed by mass-spectrometry in a HLA-A* 11:01 expressing K562 cell line. Top panels shows detection was DOX dependent (left column no DOX; right panel DOX added), and bottom panels show detection of the heavy peptide control standard was equivalent.

DETAILED DESCRIPTION

[0092] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted. [0093] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated. [0094] As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise. For example, a multispecific ABP “comprising a diabody” includes a multispecific ABP “consisting of a diabody” and a multispecific ABP “consisting essentially of a diabody.”

[0095] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).

[0096] The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g.,

Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (V _H ) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, CH2, and CH3. Each light chain typically comprises a light chain variable region (V _L ) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0097] The term “antigen binding protein” or “ABP” is used herein in its broadest sense and includes certain types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.

[0098] In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. An ABP specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, ABP fragments, and multi-specific antibodies. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, a CAR comprises an ABP.

[0099] An “HLA-PEPTIDE ABP,” “anti -HLA-PEPTIDE ABP,” or “HLA-PEPTIDE-specific ABP” is an ABP, as provided herein, which specifically binds to the antigen HLA-PEPTIDE. An ABP includes proteins comprising one or more antigen-binding domains that specifically bind to an antigen or epitope via a variable region, such as a variable region derived from a B cell (e.g., antibody).

[00100] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri- scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[00101] As used herein, “variable region” refers to a variable nucleotide sequence that arises from a recombination event, for example, it can include a V, J, and/or D region of an immunoglobulin or T cell receptor (TCR) sequence from a B cell or T cell, such as an activated T cell or an activated B cell.

[00102] The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope. One example of an antigen-binding domain is an antigen-binding domain formed by an antibody V _H -V _L dimer of an ABP. Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin. An antigen-binding domain can include antibody CDRs 1, 2, and 3 from a heavy chain in that order; and antibody CDRs 1, 2, and 3 from a light chain in that order. An antigen-binding domain can include TCR CDRs, e.g., αCDRl, αCDR2, αCDR3, βCDRI, βCDR2, and βCDR3. TCR CDRs are described herein.

[00103] The antibody V _H and V _L regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each V _H and V _L generally comprises three antibody CDRs and four FRs, arranged in the following order (from N-terminus to C- terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The antibody CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the ABP. See Rabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[00104] The light chain from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of its constant domain.

[00105] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.

[00106] The amino acid sequence boundaries of an antibody CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Rabat et al., supra (“Rabat” numbering scheme); Al-Lazikani et al., 1997, ./. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, ./. Mol.

Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol .,

2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[00107] Table 1 provides the positions of antibody CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Rabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Rabat and Chothia numbering schemes.

[00108] Antibody CDRs may be assigned, for example, using ABP numbering software, such as Abnum, available at www.bioinf.org.uk/abs/abnum/, and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.

* The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR.

[00109] The “EU numbering scheme” is generally used when referring to a residue in an ABP heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in ABP heavy chain constant regions described herein.

[00110] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region. For example, when used to refer to an IgG molecule, a “full length antibody” is an antibody that comprises two heavy chains and two light chains.

[00111] The amino acid sequence boundaries of a TCR CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including but not limited to the IMGT unique numbering, as described by LeFranc, M.-P, Immunol Today. 1997 Nov;18(l 1):509; Lefranc, M.-P., "IMGT Locus on Focus: A new section of Experimental and Clinical Immunogenetics", Exp. Clin. Immunogenet., 15, 1-7 (1998); Lefranc and Lefranc, The T Cell Receptor FactsBook; and M.-P. Lefranc/ Developmental and Comparative Immunology 27 (2003) 55-77, all of which are incorporated by reference.

[00112] An “ABP fragment” comprises a portion of an intact ABP, such as the antigen- binding or variable region of an intact ABP. ABP fragments include, for example, Fv fragments, Fab fragments, F(ab’) ₂ fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments. ABP fragments include antibody fragments. Antibody fragments can include Fv fragments, Fab fragments, F(ab’) ₂ fragments, Fab’ fragments, scFv (sFv) fragments, scFv-Fc fragments, and TCR fragments. [00113] “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[00114] “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (C _H1 ) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length ABP.

[00115] “F(ab’) ₂ ” fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds. F(ab’) ₂ fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact ABP. The F(ab’) fragments can be dissociated, for example, by treatment with ß-mercaptoethanol.

[00116] “Single-chain Fv” or “sFv” or “scFv” fragments comprise a V _H domain and a V _L domain in a single polypeptide chain. The V _H and V _L are generally linked by a peptide linker. See Pliickthun A. (1994). Any suitable linker may be used. In some embodiments, the linker is a (GGGGS)n (SEQ ID NO: 29371). In some embodiments, n = 1, 2, 3, 4, 5, or 6. See ABPs from Escherichia coli. In Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal ABPs vol. 113 (pp. 269-315). Springer- Verlag, New York, incorporated by reference in its entirety.

[00117] “scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the V _H or V _L , depending on the orientation of the variable domains in the scFv (i.e., V _H -V _L or V _L - V _H ). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[00118] The term “single domain antibody” refers to a molecule in which one variable domain of an ABP specifically binds to an antigen without the presence of the other variable domain. Single domain ABPs, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain ABPs are also known as sdAbs or nanobodies.

[00119] The term “Fc region” or “Fc” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol ., 2010, 125:S41-52, incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region, or an Fc region modified as described in the art or elsewhere in this disclosure.

[00120] The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an ABP. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the b-sandwich (e.g., iMab), lipocalin (e.g, Anticalins ^® ), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g, Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody ^® ), ankyrin repeats (e.g, DARPins), gamma-B-crystallin/ubiquitin (e.g, Affilins), CTLD3 (e.g. Tetranectins), Fynomers, and (LDLR-A module) (e.g, Avimers). Additional information on alternative scaffolds is provided in Binz et al, Nat. Biotechnol. , 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et al, J. Biol. Chem , 2014, 289:14392- 14398; each of which is incorporated by reference in its entirety. An alternative scaffold is one type of ABP.

[00121] A “multispecific ABP” is an ABP that comprises two or more different antigen- binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g, a single HLA-PEPTIDE molecule expressed by a cell) or on different antigens (e.g, different HLA-PEPTIDE molecules expressed by the same cell, or a HLA-PEPTIDE molecule and a non-HLA- PEPTIDE molecule). In some aspects, a multi-specific ABP binds two different epitopes (i.e, a “bispecific ABP”). In some aspects, a multi-specific ABP binds three different epitopes (i.e, a “trispecific ABP”).

[00122] A “monospecific ABP” is an ABP that comprises one or more binding sites that specifically bind to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule which, while divalent (i.e, having two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[00123] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject. [00124] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[00125] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature , 1986, 321:522-525; Riechmann et al., Nature , 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[00126] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody -encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[00127] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., ABP and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (K _D ). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below.

Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE ^® ) or biolayer interferometry (e.g., FORTEBIO ^® ).

[00128] With regard to the binding of an ABP to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less than about 50% of the affinity for HLA-PEPTIDE. In some aspects, the affinity of a HLA- PEPTIDE ABP for a non-target molecule is less than about 40% of the affinity for HLA- PEPTIDE. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less than about 30% of the affinity for HLA-PEPTIDE. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less than about 20% of the affinity for HLA-PEPTIDE. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less than about 10% of the affinity for HLA-PEPTIDE. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less than about 1% of the affinity for HLA-PEPTIDE. In some aspects, the affinity of a HLA-PEPTIDE ABP for a non- target molecule is less than about 0.1% of the affinity for HLA-PEPTIDE.

[00129] The term “kd” (sec ^-1 ), as used herein, refers to the dissociation rate constant of a particular ABP - antigen interaction. This value is also referred to as the k _off value.

[00130] The term “k _a ” (M ^-1 sec ^-1 ), as used herein, refers to the association rate constant of a particular ABP -antigen interaction. This value is also referred to as the k _on value.

[00131] The term “K _D ” (M), as used herein, refers to the dissociation equilibrium constant of a particular ABP -antigen interaction. K _D = k _d /k _a. In some embodiments, the affinity of an ABP is described in terms of the K _D for an interaction between such ABP and its antigen.

For clarity, as known in the art, a smaller K _D value indicates a higher affinity interaction, while a larger K _D value indicates a lower affinity interaction. [00132] The term “K _A ” (M ^-1 ), as used herein, refers to the association equilibrium constant of a particular ABP-antigen interaction. K _A = k _a /k _d .

[001.331 An “immunoconjugate” is an ABP conjugated to one or more heterologous molecule(s), such as a therapeutic (cytokine, for example) or diagnostic agent.

[00134] “Fc effector functions” refer to those biological activities mediated by the Fc region of an ABP having an Fc region, which activities may vary depending on isotype. Examples of ABP effector functions include Clq binding to activate complement dependent cytotoxicity (CDC), Fc receptor binding to activate ABP-dependent cellular cytotoxicity (ADCC), and ABP dependent cellular phagocytosis (ADCP).

[00135] When used herein in the context of two or more ABPs, the term “competes with” or “cross-competes with” indicates that the two or more ABPs compete for binding to an antigen (e g., HLA-PEPTIDE). In one exemplary assay, HLA-PEPTIDE is coated on a surface and contacted with a first HLA-PEPTIDE ABP, after which a second HLA-PEPTIDE ABP is added. In another exemplary assay, a first HLA-PEPTIDE ABP is coated on a surface and contacted with HLA-PEPTIDE, and then a second HLA-PEPTIDE ABP is added. If the presence of the first HLA-PEPTIDE ABP reduces binding of the second HLA-PEPTIDE ABP, in either assay, then the ABPs compete with each other. The term “competes with” also includes combinations of ABPs where one ABP reduces binding of another ABP, but where no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one ABP reduces binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. A skilled artisan can select the concentrations of the ABPs used in the competition assays based on the affinities of the ABPs for HLA-PEPTIDE and the valency of the ABPs. The assays described in this definition are illustrative, and a skilled artisan can utilize any suitable assay to determine if ABPs compete with each other. Suitable assays are described, for example, in Cox et al., “Immunoassay Methods,” in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov/books/NBK92434/; accessed September 29, 2015); Silman et al., Cytometry , 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated by reference in its entirety.

[00136] The term “epitope” means a portion of an antigen that specifically binds to an ABP. Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and may have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination such as, for example, testing for ABP binding to HLA-PEPTIDE variants with different point-mutations, or to chimeric HLA-PEPTIDE variants.

[00137] As used herein, the term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[00138] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alternatively, sequence similarity or dissimilarity can be established by the combined presence or absence of particular nucleotides, or, for translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).

[00139] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). [00140] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[00141] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. By way of example, the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for one another.

[00142] Table 2. Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.

[00143] Table 3. Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.

[00144] Table 4. Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.

[00145] Additional conservative substitutions may be found, for example, in Creighton,

Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. An ABP generated by making one or more conservative substitutions of amino acid residues in a parent ABP is referred to as a “conservatively modified variant.”

[00146] The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;

S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[00147] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[00148] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom. Such progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations.

[00149] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[00150] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[00151] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with an ABP provided herein. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[00152] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.

[00153] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematologic malignancy.

[00154] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition. [00155] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[00156] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[00157] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%,

50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[00158] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor. [00159] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.

[00160] The terms “nucleic acids” and “polynucleotides” may be used interchangeably herein to refer to polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can include, but are not limited to coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA, isolated RNA, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Exemplary modified nucleotides include, e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-( carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthioN6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6- diaminopurine.

[00161 ] As used herein the term “antigen” is a substance that induces an immune response. An antigen can be a neoantigen. An antigen can be a “shared antigen” that is an antigen found among a specific population, e.g., a specific population of cancer patients. Antigens can include HLA-PEPTIDE antigens.

[00162] As used herein the term “neoantigen” is an antigen that has at least one alteration that makes it distinct from the corresponding wild-type antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. In some embodiments, the alteration occurs in tumor or cancer cells. In some embodiments, the alteration does not occur in a non-tumor or a non-cancer cell. In some embodiments, the alteration is absent from normal tissue. A neoantigen can include a polypeptide sequence or a nucleotide sequence. A mutation can include a frameshift or nonframeshift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF. A mutation can also include a splice variant. Post-translational modifications specific to a tumor cell can include aberrant phosphorylation. Post-translational modifications specific to a tumor cell can also include a proteasome- generated spliced antigen. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced peptides; Science. 2016 Oct 21;354(6310):354-358. A neoantigen can be a shared neoantigen if it can be found among multiple patients in a specific population (e.g., a specific population of cancer patients). Neoantigens can include HLA- PEPTIDE neoantigens.

[00163] As used herein, the terms “HLA-PEPTIDE,” “pHLA,” “peptide-HLA,” and “peptide-HLA complex,” are used interchangeably herein to refer to an antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA- restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule. Such antigens are defined by a specific HLA-restricted peptide having a defined amino acid sequence complexed with a specific HLA Class I subtype. [00164] In some embodiments, an “HLA-PEPTIDE neoantigen,” a “pHLA neoantigen,” and a “peptide-HLA neoantigen” are used interchangeably herein to refer to an HLA- PEPTIDE that comprises at least one alteration that makes it distinct from the corresponding wild-type HLA-PEPTIDE antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. In some embodiments, the at least one alteration is in the restricted peptide sequence, such that the restricted peptide of the HLA-PEPTIDE neoantigen is distinguished from a corresponding restricted peptide sequence without the alteration, e.g., a restricted peptide containing the wild-type sequence.

[00165] Exemplary HLA-PEPTIDE neoantigens and shared HLA-PEPTIDE neoantigens are shown in Table A (SEQ ID NO: 10,755-21,015), in the AACR GENIE Results (SEQ ID NO:21, 016-29, 357), and in SEQ ID NOs 29358-29364; corresponding genes and somatic alterations associated with each antigen are also shown. Such pHLA neoantigens and shared pHLA neoantigens are useful for inducing an immune response in a subject via administration. The subject can be identified for administration through the use of various diagnostic methods, e.g., patient selection methods described herein.

[00166] As used herein the term “tumor antigen” is a antigen present in a subject’s tumor cell or tissue but not in the subject’s corresponding normal cell or tissue, or derived from a polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.

[00167] As used herein the term “candidate antigen” is a mutation or other aberration giving rise to a sequence that may represent an antigen.

[00168] As used herein the term “coding region” is the portion(s) of a gene that encode protein.

[00169] As used herein the term “coding mutation” is a mutation occurring in a coding region.

[00170] As used herein the term “ORF” means open reading frame.

[00171] As used herein the term “NEO-ORF” is a tumor-specific ORF arising from a mutation or other aberration such as splicing.

[00172] As used herein the term “missense mutation” is a mutation causing a substitution from one amino acid to another.

[00173] As used herein the term “nonsense mutation” is a mutation causing a substitution from an amino acid to a stop codon or causing removal of a canonical start codon.

[00174] As used herein the term “frameshift mutation” is a mutation causing a change in the frame of the protein.

[00175] As used herein the term “indel” is an insertion or deletion of one or more nucleic acids.

[00176] As used herein the term “non-stop or read-through” is a mutation causing the removal of the natural stop codon.

HLA-PEPTIDE ANTIGENS

[00177] The major histocompatibility complex (MHC) is a complex encoded by a group of linked loci, which are collectively termed H-2 in the mouse and HLA in humans. The two principal classes of the MHC antigens, class I and class II, each comprise a set of cell surface glycoproteins which play a role in determining tissue type and transplant compatibility. In transplantation reactions, cytotoxic T-cells (CTLs) respond mainly against class I glycoproteins, while helper T-cells respond mainly against class II glycoproteins.

[00178] Human major histocompatibility complex (MHC) class I molecules, referred to interchangeably herein as HLA Class I molecules, are expressed on the surface of nearly all cells. These molecules function in presenting peptides which are mainly derived from endogenously synthesized proteins to, e.g., CD8+ T cells via an interaction with the alpha- beta T-cell receptor. The class I MHC molecule comprises a heterodimer composed of a 46- kDa a chain which is non-covalently associated with the 12-kDa light chain beta-2 microglobulin. The a chain generally comprises al and α2 domains which form a groove for presenting an HLA-restricted peptide, and an a3 plasma membrane-spanning domain which interacts with the CD8 co-receptor of T-cells. FIG. 1 depicts the general structure of a Class I HLA molecule. Some TCRs can bind MHC class I independently of CD8 coreceptor (see, e.g., Kerry SE, Buslepp J, Cramer LA, et al. Interplay between TCR Affinity and Necessity of Coreceptor Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack of CD8 Engagement. Journal of immunology (Baltimore, Md : 1950). 2003;171(9):4493-4503.)

[00179] Class I MHC-restricted peptides (also referred to interchangeably herein as HLA- restricted antigens, HLA-restricted peptides, antigenic peptides, MHC-restricted antigens, restricted peptides, or peptides) generally bind to the heavy chain alphal-alpha2 groove via about two or three anchor residues that interact with corresponding binding pockets in the MHC molecule. The beta-2 microglobulin chain plays an important role in MHC class I intracellular transport, peptide binding, and conformational stability. For most class I molecules, the formation of a heterotrimeric complex of the MHC class I heavy chain, peptide (self, non-self, and/or antigenic) and beta-2 microglobulin leads to protein maturation and export to the cell-surface.

[00180] Binding of a given HLA subtype to an HLA-restricted peptide forms a complex with a unique and novel surface that can be specifically recognized by an ABP such as, e.g., a TCR on a T cell or an antibody or antigen-binding fragment thereof.

[00181] Accordingly, provided herein are HLA-PEPTIDE antigens comprising a specific HLA-restricted peptide having a defined amino acid sequence complexed with a specific HLA subtype.

[00182] HLA-PEPTIDE antigens identified herein may be useful for cancer immunotherapy. In some embodiments, the HLA-PEPTIDE antigens identified herein are presented on the surface of a tumor cell. The HLA-PEPTIDE antigens identified herein may be expressed by tumor cells in a human subject. The HLA-PEPTIDE antigens identified herein may be expressed by tumor cells in a population of human subjects. For example, the HLA-PEPTIDE antigens identified herein may be shared HLA-PEPTIDE antigens which are commonly expressed in a population of human subjects with cancer.

[00183] The HLA-PEPTIDE antigens identified herein may have a prevalence with an individual tumor type The prevalence with an individual tumor type may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,

57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The prevalence with an individual tumor type may be about 0.1%-100%, 0.2-50%, 0.5-25%, or 1-10%.

Exemplary HLA Class I subtypes of the pHLA neoantisens

[00184] In humans, there are many MHC haplotypes (referred to interchangeably herein as MHC subtypes, HLA subtypes, MHC types, and HLA types). Exemplary HLA subtypes include, by way of example only, 2 digit, 4 digit, 6 digit, and 8 digit subtypes. A full list of HLA Class Alleles can be found on http://hla.alleles.org/alleles/. For example, a full list of HLA Class I Alleles can be found on http://hla.alleles.org/alleles/classl.html. Exemplary HLA Class I subtypes include any of the HLA subtypes disclosed in in Table A (see SEQ ID NO: 10,755-21,015) in the AACR GENIE results (see SEQ ID NO: 21,016-29,357), and in SEQ ID NOs: 29358-29364 disclosed herein. Table A neoantigens and the AACR GENIE Results are disclosed in PCT/US2019/033830, filed on May 23, 2019, which application is hereby incorporated by reference in its entirety.

Exemplary HLA-restricted peptides

[00185] The HLA-restricted peptides (referred to interchangeably herein) as “restricted peptides” can be peptide fragments of tumor-associated neoantigens, e.g., shared neoantigens. The peptide fragments can include any of the amino acid sequences disclosed in Table A (see SEQ ID NO: 10,755-21,015), in the AACR GENIE results (see SEQ ID NO: 21,016-29,357), and in SEQ ID NOs: 29358-29364 disclosed herein. Table A neoantigens and the AACR GENIE Results are disclosed in PCT/US2019/033830, filed on May 23, 2019, which application is hereby incorporated by reference in its entirety.

[00186] Accordingly, disclosed herein are isolated peptides that comprise tumor specific mutations identified by the methods disclosed herein, peptides that comprise known tumor specific mutations, and mutant polypeptides or fragments thereof identified by methods disclosed herein. Neoantigen peptides can be described in the context of their coding sequence where a neoantigen includes the nucleotide sequence (e.g., DNA or RNA) that codes for the related polypeptide sequence.

[00187] Also disclosed herein are peptides, e.g., restricted peptides derived from any polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue, for example any polypeptide known to or have been found to be aberrantly expressed in a tumor cell or cancerous tissue in comparison to a normal cell or tissue. Suitable polypeptides from which the restricted peptides can be derived can be found for example in the COSMIC database. COSMIC curates comprehensive information on somatic mutations in human cancer. In some embodiments, the restricted peptide contains the tumor specific mutation.

[00188] One or more restricted peptides can comprise at least one of: a binding affinity with MHC with an IC50 value of less than 1000nM, for MHC Class I peptides a length of 8- 15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, presence of sequence motifs within or near the peptide promoting proteasome cleavage, and presence or sequence motifs promoting TAP transport.

[00189] The restricted peptides may have a size of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acid residues, and any range derivable therein. In particular embodiments, the restricted peptide has a size of about 8, about 9, about 10, about 11, or about 12 amino molecule residues. The restricted peptide may be about 5-15 amino acids in length, preferably may be about 7-13 amino acids in length, or more preferably may be about 8-12 amino acids in length.

Exemplary shared HLA-PEPTIDE neoantisens

[00190] Exemplary shared HLA-PEPTIDE neoantigens are shown in Table A (see SEQ ID NO: 10,755-21,015), in the AACR GENIE results (see SEQ ID NO: 21,016-29,357), and in SEQ ID NOs: 29358-29364 disclosed herein. Table A neoantigens and the AACR GENIE Results are disclosed in PCT/US2019/033830, filed on May 23, 2019, which application is hereby incorporated by reference in its entirety.

[00191] One or more HLA-PEPTIDE neoantigens can be presented on the surface of a tumor.

[00192] One or more HLA-PEPTIDE neoantigens can be immunogenic in a subject having a tumor, e.g., capable of eliciting a T cell response or a B cell response in the subject. [00193] If desirable, a longer peptide can be designed in several ways. In one case, when presentation likelihoods of peptides on HLA alleles are predicted or known, a longer peptide could consist of either: (1) individual presented peptides with an extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the presented peptides with extended sequences for each. In another case, when sequencing reveals a long (>10 residues) neoepitope sequence present in the tumor (e.g. due to a frameshift, read-through or intron inclusion that leads to a novel peptide sequence), a longer peptide would consist of: (3) the entire stretch of novel tumor-specific amino acids— thus bypassing the need for computational or in vitro test-based selection of the strongest HLA-presented shorter peptide. In both cases, use of a longer peptide allows endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses.

[00194] Antigenic peptides and polypeptides can be presented on an HLA protein. In some aspects antigenic peptides and polypeptides are presented on an HLA protein with greater affinity than a wild-type peptide. In some aspects, a antigenic peptide or polypeptide can have an IC50 of at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.

[00195] In some aspects, antigenic peptides and polypeptides do not induce an autoimmune response and/or invoke immunological tolerance when administered to a subject.

[00196] Also provided are compositions comprising at least two or more antigenic peptides. In some embodiments the composition contains at least two distinct peptides. At least two distinct peptides can be derived from the same polypeptide. By distinct polypeptides is meant that the peptide vary by length, amino acid sequence, or both. The peptides are derived from any polypeptide known to or have been found to contain a tumor specific mutation or peptides derived from any polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue, for example any polypeptide known to or have been found to be aberrantly expressed in a tumor cell or cancerous tissue in comparison to a normal cell or tissue. Suitable polypeptides from which the antigenic peptides can be derived can be found for example in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC curates comprehensive information on somatic mutations in human cancer. AACR GENIE aggregates and links clinical-grade cancer genomic data with clinical outcomes from tens of thousands of cancer patients. The peptide contains the tumor specific mutation. In some aspects the tumor specific mutation is a driver mutation for a particular cancer type.

[00197] Antigenic peptides and polypeptides having a desired activity or property can be modified to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.

For instance, antigenic peptide and polypeptides can be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding, stability or presentation. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as Gly, Ala; Val, lie, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications can be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341- 347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

[00198] Modifications of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in increasing the stability of the peptide and polypeptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Half- life of the peptides can be conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4 degrees C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions. [00199] The peptides and polypeptides can be modified to provide desired attributes other than improved serum half-life. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Immunogenic peptides/T helper conjugates can be linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus can be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[00200] A antigenic peptide can be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

[00201] Proteins or peptides can be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. The nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and can be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases located at the National Institutes of Health website. The coding regions for known genes can be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.

[00202] In some embodiments, an antigen can include a nucleic acid (e.g. polynucleotide) that encodes a antigenic peptide or portion thereof. The polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as, e.g., polynucleotides with a phosphorothiate backbone, or combinations thereof and it may or may not contain introns. [00203] A still further aspect provides an expression vector capable of expressing a polypeptide or portion thereof. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found e.g. in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

[00204] HLA Class I molecules which do not associate with a restricted peptide ligand are generally unstable. Accordingly, the association of the restricted peptide with the al/α2 groove of the HLA molecule may stabilize the non-covalent association of the b2- microglobulin subunit of the HLA subtype with the a-subunit of the HLA subtype.

[00205] Stability of the non-covalent association of the β2-microglobulin subunit of the HLA subtype with the a-subunit of the HLA subtype can be determined using any suitable means. For example, such stability may be assessed by dissolving insoluble aggregates of HLA molecules in high concentrations of urea (e.g., about 8M urea), and determining the ability of the HLA molecule to refold in the presence of the restricted peptide during urea removal, e.g., urea removal by dialysis. Such refolding approaches are described in, e.g., Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3429-3433, April 1992, hereby incorporated by reference.

[00206] For other example, such stability may be assessed using conditional HLA Class I ligands. Conditional HLA Class I ligands are generally designed as short restricted peptides which stabilize the association of the b2 and a subunits of the HLA Class I molecule by binding to the al/α2 groove of the HLA molecule, and which contain one or more amino acid modifications allowing cleavage of the restricted peptide upon exposure to a conditional stimulus. Upon cleavage of the conditional ligand, the b2 and a-subunits of the HLA molecule dissociate, unless such conditional ligand is exchanged for a restricted peptide which binds to the al/α2 groove and stabilizes the HLA molecule. Conditional ligands can be designed by introducing amino acid modifications in either known HLA peptide ligands or in predicted high-affinity HLA peptide ligands. For HLA alleles for which structural information is available, water-accessibility of side chains may also be used to select positions for introduction of the amino acid modifications. Use of conditional HLA ligands may be advantageous by allowing the batch preparation of stable HLA-peptide complexes which may be used to interrogate test restricted peptides in a high throughput manner. Conditional HLA Class I ligands, and methods of production, are described in, e.g., Proc Natl Acad Sci U S A. 2008 Mar 11; 105(10): 3831-3836; Proc Natl Acad Sci U S A. 2008 Mar 11; 105(10): 3825-3830; J Exp Med. 2018 May 7; 215(5): 1493-1504; Choo, J. A. L. et al. Bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides. Angew Chem Int Ed Engl 53, 13390-13394 (2014); Am ore, A. et al. Development of a Hypersensitive Periodate-Cleavable Amino Acid that is Methionine- and Disulfide-Compatible and its Application in MHC Exchange Reagents for T Cell Characterisation. ChemBioChem 14, 123-131 (2012); Rodenko, B. et al. Class I Major Histocompatibility Complexes Loaded by a Periodate Trigger. J Am Chem Soc 131, 12305-12313 (2009); and Chang, C. X. L. et al. Conditional ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses in acute and chronic viral diseases. Eur J Immunol 43, 1109-1120 (2013). These references are incorporated by reference in their entirety.

[00207] Accordingly, in some embodiments, the ability of an HLA-restricted peptide described herein, e.g., described in Table A (SEQ ID NO: 10,755-21,015), AACR GENIE results (SEQ ID NOs: 21,016-29,357), or in SEQ ID NOs: 29358-29364, to stabilize the association of the b2- and a-subunits of the HLA molecule, is assessed by performing a conditional ligand mediated-exchange reaction and assay for HLA stability. HLA stability can be assayed using any suitable method, including, e.g., mass spectrometry analysis, immunoassays (e.g., ELISA), size exclusion chromatography, and HLA multimer staining followed by flow cytometry assessment of T cells.

[00208] Other exemplary methods for assessing stability of the non- covalent association of the β2-microglobulin subunit of the HLA subtype with the a-subunit of the HLA subtype include peptide exchange using dipeptides. Peptide exchange using dipeptides has been described in, e.g., Proc Natl Acad Sci U S A. 2013 Sep 17, 110(38): 15383-8; Proc Natl Acad Sci U S A. 2015 Jan 6, 112(l):202-7, which is hereby incorporated by reference.

[00209] The HLA-PEPTIDE antigen may be isolated and/or in substantially pure form. For example, the HLA-PEPTIDE antigens may be isolated from their natural environment, or may be produced by means of a technical process. In some cases, the HLA-PEPTIDE antigen is provided in a form which is substantially free of other peptides or proteins. [00210] The HLA-PEPTIDE antigens may be presented in soluble form, and optionally may be a recombinant HLA-PEPTIDE antigen complex. The skilled artisan may use any suitable method for producing and purifying recombinant HLA-PEPTIDE antigens. Suitable methods include, e.g., use of E. coli expression systems, insect cells, and the like. Other methods include synthetic production, e.g., using cell free systems. An exemplary suitable cell free system is described in WO2017089756, which is hereby incorporated by reference in its entirety.

[00211] Also provided herein are compositions comprising an HLA-PEPTIDE antigen. [00212] In some cases, the composition comprises an HLA-PEPTIDE antigen attached to a solid support. Exemplary solid supports include, but are not limited to, beads, wells, membranes, tubes, columns, plates, sepharose, magnetic beads, and chips. Exemplary solid supports are described in, e.g., Catalysts 2018, 8, 92; doi:10.3390/catal8020092, which is hereby incorporated by reference in its entirety.

[00213] The HLA-PEPTIDE antigen may be attached to the solid support by any suitable methods known in the art. In some cases, the HLA-PEPTIDE antigen is covalently attached to the solid support.

[00214] In some cases, the HLA-PEPTIDE antigen is attached to the solid support by way of an affinity binding pair. Affinity binding pairs generally involved specific interactions between two molecules. A ligand having an affinity for its binding partner molecule can be covalently attached to the solid support, and thus used as bait for immobilizing. Common affinity binding pairs include, e.g., streptavidin and biotin, avidin and biotin; polyhistidine tags with metal ions such as copper, nickel, zinc, and cobalt; and the like. Accordingly, provided herein are compositions comprising an HLA-PEPTIDE antigen disclosed herein, wherein the HLA-PEPTIDE antigen is covalently linked to an affinity tag.

[00215] The HLA-PEPTIDE antigen may comprise a detectable label. In some embodiments, the HLA-PEPTIDE antigen is complexed with the detectable label. In some embodiments, the detectable label comprises a β ₂ -microglobulin binding molecule, e.g., a labeled antibody, e.g., a fluorochrome labeled antibody.

[00216] Also provided herein are pharmaceutical compositions comprising HLA-PEPTIDE antigens.

[00217] The composition comprising an HLA-PEPTIDE antigen may be a pharmaceutical composition. Such a composition may comprise multiple HLA-PEPTIDE antigens. Exemplary pharmaceutical compositions are described herein. The composition may be capable of eliciting an immune response. The composition may comprise an adjuvant. Suitable adjuvants include, but are not limited to 1018 ISS, alum, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand. 1998; 92:3-11). Also cytokines can be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T- lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418). HLA surface expression and processing of intracellular proteins into peptides to present on HLA can also be enhanced by interferon-gamma (IFN-g). See, e.g., York IA, Goldberg AL, Mo XY, Rock KL. Proteolysis and class I major histocompatibility complex antigen presentation. Immunol Rev. 1999;172:49-66; and Rock KL, Goldberg AL. Degradation of cell proteins and the generation of MHC class I-presented peptides. Ann Rev Immunol. 1999;17: 12. 739-779, which are incorporated herein by reference in their entirety.

[00218] Also provided herein are host cells comprising an HLA-PEPTIDE antigen disclosed herein. In some embodiments, the host cell comprises a polynucleotide encoding an HLA-restricted peptide as defined by the HLA-PEPTIDE antigen. In some embodiments, the polynucleotide is heterologous to the host cell. In some embodiments, the host cell does not comprise endogenous MHC. In some embodiments, the host cell comprises an exogenous HLA Class I molecule. In some embodiments, the host cell is a K562 or A375 cell. In some embodiments, the host cell is a cultured cell from a tumor cell line. In some embodiments, the tumor cell line expresses an HLA subtype as defined by the HLA- PEPTIDE antigen.

[00219] Also provided herein are cell culture systems comprising a host cell disclosed herein and a cell culture medium. In some embodiments, the host cell expresses the HLA Class I subtype as defined by the HLA-PEPTIDE antigen and the cell culture medium comprises the restricted peptide as defined by the HLA-PEPTIDE antigen.

ABPs

[00220] Also provided herein are ABPs that specifically bind to an HLA-PEPTIDE antigen disclosed herein. In some embodiments, an ABP disclosed herein specifically binds to an HLA-PEPTIDE neoantigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, wherein the HLA Class I molecule and the HLA-restricted peptide are each selected from an HLA-PEPTIDE neoantigen as described in any one of SEQ ID NOs: 10,755 to 29,364, and wherein the isolated ABP comprises an antibody or antigen-binding fragment thereof. For example, for an ABP disclosed herein, the target of the ABP is an HLA Class I molecule and the associated HLA-restricted peptide that are each selected from a single HLA-PEPTIDE neoantigen described in any one of the aforementioned SEQ ID NOs, i.e., the HLA Class I molecule and the HLA-restricted peptide are each selected from the same SEQ ID NO. For example, the target of an ABP against SEQ ID NO: 19865 would bind to HLA- A* 11:01 in complex with a restricted peptide of the sequence: VVVGADGVGK (SEQ ID NO: 19865).

[00221] The HLA-PEPTIDE neoantigen may be expressed on the surface of any suitable target cell including a tumor cell.

[00222] In some embodiments, the ABP specifically binds a complex comprising HLA and an HLA-restricted peptide (HLA-PEPTIDE), e.g., derived from a tumor. In some embodiments, the ABP does not bind to the HLA in the absence of the HLA-restricted peptide. In some embodiments, the ABP does not bind HLA-restricted peptide in the absence of the HLA. In some embodiments, the ABP binds tumor cells presenting human MHC complexed with the HLA - restricted peptide, optionally wherein the HLA restricted peptide is a tumor antigen characterizing the cancer. In some aspects, the ABP binds a complex comprising HLA and HLA-restricted peptide when naturally presented on a cell such as a tumor cell. [00223] An ABP can bind to each portion of an HLA-PEPTIDE complex (i.e., HLA and peptide representing each portion of the complex), which when bound together form a novel target and protein surface for interaction with and binding by the ABP, distinct from a surface presented by the peptide alone or HLA subtype alone. Generally the novel target and protein surface formed by binding of HLA to peptide does not exist in the absence of each portion of the HLA-PEPTIDE complex. In some embodiments, the ABP binds to the HLA-PEPTIDE neoantigen through at least one contact point with the HLA Class I molecule and through at least one contact point with the HLA-restricted peptide.

[00224] In some embodiments, an ABP provided herein modulates binding of HLA- PEPTIDE to one or more ligands of HLA-PEPTIDE.

[00225] In more particular embodiments, the ABP specifically binds to a neoantigen described in Table 5A. In more particular embodiments, the ABP specifically binds to a neoantigen described in Table 5B. In more particular embodiments, the ABP specifically binds to a neoantigen described in Table 6. In more particular embodiments, the ABP specifically binds to a neoantigen described in Table 7.

[00226] In some embodiments of the ABP, the HLA-restricted peptide comprises a RAS mutation. In some embodiments of the ABP, the RAS mutation is a RAS G12 mutation. The RAS may be KRAS, NRAS, or HRAS. In some embodiments of the ABP, the HLA- restricted peptide comprises a RAS G12 mutation. In some embodiments of the ABP, the HLA-restricted peptide comprises a NRAS G12 mutation. In some embodiments of the ABP, the HLA-restricted peptide comprises a HRAS G12 mutation. Because amino acid positions 1-50 of HRAS, KRAS, and NRAS are identical, a skilled artisan understands that an HLA- Class I restricted peptide comprising a RAS G12 mutation corresponds to the KRAS G12, NRAS G12, and HRAS G12 mutation. By way of example only, SEQ ID NO: 14954, described as a KRAS G12C neoantigen, and SEQ ID NO: 14955, described as an NRAS G12C neoantigen, both have identical HLA-PEPTIDE pairings (HLA-A*02:01_ KLVVVGACGV (SEQ ID NO: 14954)). Accordingly, SEQ ID NOs 14954 and 14955 describe identical KRAS/NRAS/HRAS G12C HLA-PEPTIDE neoantigens.

[00227] In some embodiments, the G12 mutation is a G12C, a G12D, a G12V, or a G12A mutation. In some embodiments wherein the HLA-restricted peptide comprises the RAS G12 mutation, the HLA Class I molecule is selected from HLA-A*02:01, HLA-A*11:01, HLA- A*31:01, HLA-C*01:02, and HLA-A*03:01. [00228] In particular embodiments of the ABP, the HLA-PEPTIDE neoantigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367) ; aRAS_G12VMHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA- C*01 :02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369) ; and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[00229] In some embodiments of the ABP, the HLA-PEPTIDE neoantigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365); a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366); a RAS_G12D MHC Class I antigen comprising HLA- A* 11 :01 and the restricted peptide Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29367) ; a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A*31:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368); a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29362); a RAS_G12V MHC Class I antigen comprising HLA-C*01:02 and the restricted peptide Ala Val Gly Val Gly Lys Ser Ala Leu (SEQ ID NO: 29369) ; and a RAS_G12V MHC Class I antigen comprising HLA-A*03:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[00230] In some embodiments of the ABP, the HLA-PEPTIDE neoantigen is selected from: a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) ; a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366) ; and a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368). In some embodiments of the ABP, the antigen comprises HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365). In some embodiments of the ABP, the antigen comprises HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (SEQ ID NO: 29366). In some embodiments of the ABP, the antigen comprises HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[00231] In some embodiments of an ABP that binds to a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val, (SEQ ID NO: 29365) the ABP binds to such RAS_G12 MHC Class I antigen at a higher affinity than a RAS_G12C MHC Class I antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) and a different HLA subtype. In some embodiments, the ABP binds to such RAS_G12 MHC Class I antigen at a higher affinity than a RAS_G12C MHC Class I antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) and a different HLA-A2 subtype. In some embodiments, the ABP does not bind to a RAS_G12C MHC Class I antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) and a different HLA-A2 subtype.

[00232] In some embodiments of an ABP that binds to an antigen comprising a particular RAS G12 mutation, the ABP does not binds to the particular antigen at a lower affinity than an antigen comprising a different RAS G12 mutation. For example, an ABP that binds to a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) does not bind to that RAS_G12C MHC Class I antigen at a lower affinity than an antigen comprising a different RAS G12 mutation. In some embodiments of an ABP that binds to an antigen comprising a particular RAS G12 mutation, the ABP binds to the particular antigen at a higher affinity than an antigen comprising a different RAS G12 mutation. For example, an ABP that binds to a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) may bind to that RAS_G12C MHC Class I antigen at a higher affinity than an antigen comprising a different RAS G12 mutation. In some embodiments, the ABP binds a RAS_G12C MHC Class I antigen comprising HLA-A*02:01 and the restricted peptide Lys Leu Val Val Val Gly Ala Cys Gly Val (SEQ ID NO: 29365) at a higher affinity than an antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule. In particular embodiments, such ABP does not bind to an antigen comprising the restricted peptide Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 29370) and an HLA-A2 molecule.

[00233] In some embodiments, the higher affinity is at least 2-fold, at least 5-fold, or at least 10-fold.

[00234] Affinity differences can be determined by any means known in the art. In some embodiments, such affinity differences are assessed by MSD-ECL, SPR, BLI, or flow cytometry.

[00235] In some embodiments, an ABP is an ABP that competes with an illustrative ABP provided herein. In some aspects, the ABP that competes with the illustrative ABP provided herein binds the same epitope as an illustrative ABP provided herein.

[00236] In some embodiments, the ABPs described herein are referred to herein as “variants.” In some embodiments, a variant is derived from any of the sequences provided herein, wherein one or more conservative amino acid substitutions are made. Conservative amino acid substitutions are described herein. In preferred embodiments, the non- conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. In yet more preferred embodiments, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent ABP.

ABPs comprising an antibody or antigen-binding fragment thereof [00237] An ABP may comprise an antibody or antigen-binding fragment thereof.

[00238] In some embodiments, the ABPs provided herein comprise a light chain. In some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain.

[00239] In some embodiments, the ABPs provided herein comprise a heavy chain. In some aspects, the heavy chain is an IgA. In some aspects, the heavy chain is an IgD. In some aspects, the heavy chain is an IgE. In some aspects, the heavy chain is an IgG. In some aspects, the heavy chain is an IgM. In some aspects, the heavy chain is an IgG1. In some aspects, the heavy chain is an IgG2. In some aspects, the heavy chain is an IgG3. In some aspects, the heavy chain is an IgG4. In some aspects, the heavy chain is an IgAl. In some aspects, the heavy chain is an IgA2.

[00240] In some embodiments, the ABPs provided herein comprise an antibody fragment.

In some embodiments, the ABPs provided herein consist of an antibody fragment. In some embodiments, the ABPs provided herein consist essentially of an antibody fragment. In some aspects, the ABP fragment is an Fv fragment. In some aspects, the ABP fragment is a Fab fragment. In some aspects, the ABP fragment is a F(ab’) ₂ fragment. In some aspects, the ABP fragment is a Fab’ fragment. In some aspects, the ABP fragment is an scFv (sFv) fragment. In some aspects, the ABP fragment is an scFv-Fc fragment. In some aspects, the ABP fragment is a fragment of a single domain ABP.

[00241] In some embodiments, an ABP fragment provided herein is derived from an illustrative ABP provided herein. In some embodiments, an ABP fragments provided herein is not derived from an illustrative ABP provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining ABP fragments.

[00242] In some embodiments, an ABP fragment provided herein retains the ability to bind the antigen, as measured by one or more assays or biological effects described herein. In some embodiments, an ABP fragment provided herein retains the ability to prevent HLA- PEPTIDE from interacting with one or more of its ligands, as described herein.

[00243] In some embodiments, the ABPs provided herein are monoclonal ABPs. In some embodiments, the ABPs provided herein are polyclonal ABPs.

[00244] In some embodiments, the ABPs provided herein comprise a chimeric ABP. In some embodiments, the ABPs provided herein consist of a chimeric ABP. In some embodiments, the ABPs provided herein consist essentially of a chimeric ABP. In some embodiments, the ABPs provided herein comprise a humanized ABP. In some embodiments, the ABPs provided herein consist of a humanized ABP. In some embodiments, the ABPs provided herein consist essentially of a humanized ABP. In some embodiments, the ABPs provided herein comprise a human ABP. In some embodiments, the ABPs provided herein consist of a human ABP. In some embodiments, the ABPs provided herein consist essentially of a human ABP.

[00245] In some embodiments, the ABPs provided herein comprise an alternative scaffold. In some embodiments, the ABPs provided herein consist of an alternative scaffold. In some embodiments, the ABPs provided herein consist essentially of an alternative scaffold. Any suitable alternative scaffold may be used. In some aspects, the alternative scaffold is selected from an Adnectin™, an iMab, an Anticalin ^® , an EETI-II/AGRP, a Kunitz domain, a thioredoxin peptide aptamer, an Affibody ^® , a DARPin, an Affilin, a Tetranectin, a Fynomer, and an Avimer.

[00246] Also disclosed herein is an isolated humanized, human, or chimeric ABP that competes for binding to an HLA-PEPTIDE with an ABP disclosed herein.

[00247] Also disclosed herein is an isolated humanized, human, or chimeric ABP that binds an HLA-PEPTIDE epitope bound by an ABP disclosed herein.

[00248] In certain aspects, an ABP comprises a human Fc region comprising at least one modification that reduces binding to a human Fc receptor.

[00249] It is known that when an ABP is expressed in cells, the ABP is modified after translation. Examples of the posttranslational modification include cleavage of lysine at the C terminus of the heavy chain by a carboxypeptidase; modification of glutamine or glutamic acid at the N terminus of the heavy chain and the light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation, and it is known that such posttranslational modifications occur in various ABPs ( See Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447, incorporated by reference in its entirety). In some embodiments, an ABP is an ABP or antigen-binding fragment thereof which has undergone posttranslational modification. Examples of an ABP or antigen-binding fragment thereof which have undergone posttranslational modification include an ABP or antigen-binding fragments thereof which have undergone pyroglutamylation at the N terminus of the heavy chain variable region and/or deletion of lysine at the C terminus of the heavy chain. It is known in the art that such posttranslational modification due to pyroglutamylation at the N terminus and deletion of lysine at the C terminus does not have any influence on the activity of the ABP or fragment thereof (Analytical Biochemistry, 2006, Vol. 348, p. 24-39, incorporated by reference in its entirety).

Monospecific and Multispecific HLA-PEPTIDE ABPs

[00250] In some embodiments, the ABPs provided herein are monospecific ABPs.

[00251] In some embodiments, the ABPs provided herein are multispecific ABPs.

[00252] In some embodiments, a multispecific ABP provided herein binds more than one antigen. In some embodiments, a multispecific ABP binds 2 antigens. In some embodiments, a multispecific ABP binds 3 antigens. In some embodiments, a multispecific ABP binds 4 antigens. In some embodiments, a multispecific ABP binds 5 antigens. [00253] In some embodiments, a multispecific ABP provided herein binds more than one epitope on a HLA-PEPTIDE antigen. In some embodiments, a multispecific ABP binds 2 epitopes on a HLA-PEPTIDE antigen. In some embodiments, a multispecific ABP binds 3 epitopes on a HLA-PEPTIDE antigen.

[00254] Many multispecific ABP constructs are known in the art, and the ABPs provided herein may be provided in the form of any suitable multispecific suitable construct.

[00255] In some embodiments, the multispecific ABP comprises an immunoglobulin comprising at least two different heavy chain variable regions each paired with a common light chain variable region (i.e., a “common light chain ABP”). The common light chain variable region forms a distinct antigen-binding domain with each of the two different heavy chain variable regions. See Merchant et al., Nature Biotechnol. , 1998, 16:677-681, incorporated by reference in its entirety.

[00256] In some embodiments, the multispecific ABP comprises an immunoglobulin comprising an ABP or fragment thereof attached to one or more of the N- or C-termini of the heavy or light chains of such immunoglobulin. See Coloma and Morrison, Nature Biotechnol. , 1997, 15:159-163, incorporated by reference in its entirety. In some aspects, such ABP comprises a tetravalent bispecific ABP.

[00257] In some embodiments, the multispecific ABP comprises a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello, Nature , 1983, 305:537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA , 1986, 83:1453-1457; each of which is incorporated by reference in its entirety.

[00258] In some embodiments, the multispecific ABP comprises immunoglobulin chains with alterations to reduce the formation of side products that do not have multispecificity. In some aspects, the ABPs comprise one or more “knobs-into-holes” modifications as described in U.S. Pat. No. 5,731,168, incorporated by reference in its entirety.

[00259] In some embodiments, the multispecific ABP comprises immunoglobulin chains with one or more electrostatic modifications to promote the assembly of Fc hetero-mul timers. See WO 2009/089004, incorporated by reference in its entirety.

[00260] In some embodiments, the multispecific ABP comprises a bispecific single chain molecule. See Traunecker et al., EMBO J. , 1991, 10:3655-3659; and Gruber et al., J.

Immunol ., 1994, 152:5368-5374; each of which is incorporated by reference in its entirety. [00261] In some embodiments, the multispecific ABP comprises a heavy chain variable domain and a light chain variable domain connected by a polypeptide linker, where the length of the linker is selected to promote assembly of multispecific ABP with the desired multispecificity. For example, monospecific scFvs generally form when a heavy chain variable domain and light chain variable domain are connected by a polypeptide linker of more than 12 amino acid residues. See U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety. In some embodiments, reduction of the polypeptide linker length to less than 12 amino acid residues prevents pairing of heavy and light chain variable domains on the same polypeptide chain, thereby allowing pairing of heavy and light chain variable domains from one chain with the complementary domains on another chain. The resulting ABP therefore has multispecificity, with the specificity of each binding site contributed by more than one polypeptide chain. Polypeptide chains comprising heavy and light chain variable domains that are joined by linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabodies) and tetramers (termed tetrabodies) are favored. However, the exact type of oligomerization appears to depend on the amino acid residue composition and the order of the variable domain in each polypeptide chain (e.g., V _H - linker-V _L vs. V _L -linker-V _H ), in addition to the linker length. A skilled person can select the appropriate linker length based on the desired multispecificity.

Fc Region

[00262] In certain embodiments, an ABP provided herein comprises an Fc region. In some embodiments, the Fc region is a wild-type Fc region.

[00263] The term “Fc-region-comprising ABP” refers to an ABP that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the ABP or by recombinant engineering the nucleic acid encoding the ABP. Accordingly, an ABP having an Fc region can comprise an ABP with or without K447.

ABPs specific for the RAS_G12D neoantigen HTA-AH1:01 VVVGADGVGK

(“SNA30”) (SEO ID NO: 19865)

[00264] In some aspects, provided herein are ABPs comprising antibodies or antigen- binding fragments thereof that specifically bind an antigen, wherein the antigen is a RAS_G12D MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Asp Gly Val Gly Lys (“SNA30”) (SEQ ID NO: 19865).

[002651 The ABP specific for the RAS_G12D antigen HLA-A* 11 :01_ VVVGADGVGK (SEQ ID NO: 29366) may comprise one or more antibody complementarity determining region (CDR) sequences, e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3). The ABP specific for the RAS_G12D antigen HLA-A* 11 :01_ VVVGADGVGK (SEQ ID NO: 29366) may comprise a VH sequence. The ABP specific for the RAS_G12D antigen HLA-A* 11 :01_ VVVGADGVGK (SEQ ID NO: 29366) may comprise a VL sequence. The ABP specific for the RAS_G12D antigen HLA-A* 11 :01_ VVVGADGVGK (SEQ ID NO: 29366) may comprise a VH sequence and a VL sequence.

ABPs specific for the RAS G12V neoantigen HLA-A*11:01 VVVGAVGVGK

(“SNA6”) (SEQ ID NO: 19976)

[00266] In some aspects, provided herein are ABPs comprising antibodies or antigen- binding fragments thereof that specifically bind an antigen, wherein the antigen is a RAS_G12V MHC Class I antigen comprising HLA-A* 11:01 and the restricted peptide Val Val Val Gly Ala Val Gly Val Gly Lys (SEQ ID NO: 29368).

[002671 The ABP specific for the RAS G12V antigen HLA-A* 11 :01_ VVVGAVGVGK (SEQ ID NO: 29368) may comprise one or more antibody complementarity determining region (CDR) sequences, e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3). The ABP specific for the RAS G12V antigen HLA-A* 11 :01_ VVVGAVGVGK (SEQ ID NO: 29368) may comprise a VH sequence. The ABP specific for the RAS G12V antigen HLA-A* 11 :01_ VVVGAVGVGK (SEQ ID NO: 29368) may comprise a VL sequence. The ABP specific for the RAS G12V antigen HLA-A* 11 :01_ VVVGAVGVGK (SEQ ID NO: 29368) may comprise a VH sequence and a VL sequence.

Nucleotides, Vectors, Host Cells, and Related Methods [00268] Also provided are isolated nucleic acids encoding the ABPs or antigens disclosed herein, vectors comprising the nucleic acids, and host cells comprising the vectors and nucleic acids, as well as recombinant techniques for the production of the ABPs.

[00269] The nucleic acids may be recombinant. The recombinant nucleic acids may be constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or replication products thereof. For purposes herein, the replication can be in vitro replication or in vivo replication.

[00270] For recombinant production of an ABP, the nucleic acid(s) encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression. In some aspects, the nucleic acid may be produced by homologous recombination, for example as described in U.S. Patent No. 5,204,244, incorporated by reference in its entirety.

[00271] Many different vectors are known in the art. The vector components generally include one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Patent No. 5,534,615, incorporated by reference in its entirety. [00272] Exemplary vectors or constructs suitable for expressing an ABP, e.g., a CAR, antibody, or antigen binding fragment thereof, include, e.g., the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as AGTIO, AGT1 1, AZapII (Stratagene), AEMBL4, and ANMl 149, are also suitable for expressing an ABP disclosed herein.

[00273] Illustrative examples of suitable host cells are provided below. These host cells are not meant to be limiting, and any suitable host cell may be used to produce the ABPs provided herein.

[00274] Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia ( E . coif), Enterohacter , Erwinia , Klebsiella , Proteus , Salmonella ( S. typhimurium ), Serratia (S. marcescans ), Shigella , Bacilli ( B . subtilis and B. licheniformis ), Pseudomonas (P. aeruginosa ), and Streptomyces. One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are also suitable.

[00275] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for ABP-encoding vectors. Saccharomyces cerevisiae , or common baker’s yeast, is a commonly used lower eukaryotic host microorganism. However, a number of other genera, species, and strains are available and useful, such as Schizosaccharomyces pombe , Kluyveromyces ( K . lactis , K. fragilis, K. bulgaricus K. wickeramii , K. waltii , K. drosophilarum , K. thermotolerans , and if. marxianus ), Yarrowia, Pichia pastoris , Candida (C. albicans ), Trichoderma reesia , Neurospora crassa, Schwanniomyces (S. occidentalis ), and filamentous fungi such as, for example Penicillium, Tolypocladium , and Aspergillus (A. nidulans and A niger).

[00276] Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertoli cells; African green monkey kidney cells (VERO-76), and the like.

[00277] The host cells used to produce the HLA-PEPTIDE ABP may be cultured in a variety of media. Commercially available media such as, for example, Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco’s Modified Eagle’s Medium (DMEM) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz., 1979, 58:44; Barnes et al., Anal. Biochem., 1980, 102:255; and U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469; or WO 90/03430 and WO 87/00195 may be used. Each of the foregoing references is incorporated by reference in its entirety.

[00278] Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[00279] The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[00280] When using recombinant techniques, the ABP can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the ABP is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. For example, Carter et al.

( Bio/Technology , 1992, 10:163-167, incorporated by reference in its entirety) describes a procedure for isolating ABPs which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation.

[00281] In some embodiments, the ABP is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs , 2012, 4:217-225, incorporated by reference in its entirety. In some aspects, the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell.

In some aspects, the prokaryotic cell is E. coli. Cell-free expression of the ABP may be useful, for example, where the ABP accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.

[00282] Where the ABP is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon ^® or Millipore ^® Pellcon ^® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[00283] The ABP composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the ABP. Protein A can be used to purify ABPs that comprise human gΐ, g2, or g4 heavy chains (Lindmark et al., J. Immunol. Meth ., 1983, 62: 1-13, incorporated by reference in its entirety). Protein G is useful for all mouse isotypes and for human g3 (Guss et al., EMBO J., 1986, 5:1567-1575, incorporated by reference in its entirety).

[00284] The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the ABP comprises a C _H3 domain, the BakerBond ABX ^® resin is useful for purification.

[00285] Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin Sepharose ^® , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, and can be applied by one of skill in the art. [00286] Following any preliminary purification step(s), the mixture comprising the ABP of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 to about 4.5, generally performed at low salt concentrations (e.g., from about 0 to about 0.25 M salt).

Methods of Makinp HLA-PEPTIDE ABPs HLA-PEPTIDE Antisen Preparation

[00287] The HLA-PEPTIDE antigen used for isolation or creation of the ABPs provided herein may be intact HLA-PEPTIDE or a fragment of HLA-PEPTIDE. The HLA-PEPTIDE antigen may be, for example, in the form of isolated protein or a protein expressed on the surface of a cell.

[00288] In some embodiments, the HLA-PEPTIDE antigen is a non-naturally occurring variant of HLA-PEPTIDE, such as a HLA-PEPTIDE protein having an amino acid sequence or post-translational modification that does not occur in nature.

[00289] In some embodiments, the HLA-PEPTIDE antigen is truncated by removal of, for example, intracellular or membrane-spanning sequences, or signal sequences. In some embodiments, the HLA-PEPTIDE antigen is fused at its C-terminus to a human IgG1 Fc domain or a polyhistidine tag.

Methods and Systems for Identifying ABPs

[00290] ABPs that bind HLA-PEPTIDE can be identified using any method known in the art, e.g., phage display or immunization of a subject.

[00291] One method of identifying an antigen binding protein includes providing at least one HLA-PEPTIDE target; and binding the at least one target with an antigen binding protein, thereby identifying the antigen binding protein. The antigen binding protein can be present in a library comprising a plurality of distinct antigen binding proteins.

[00292] In some embodiments, the library is a phage display library. The phage display library can be developed so that it is substantially free of antigen binding proteins that non- specifically bind the HLA of the HLA-PEPTIDE target. The antigen binding protein can be present in a yeast display library comprising a plurality of distinct antigen binding proteins. The yeast display library can be developed so that it is substantially free of antigen binding proteins that non-specifically bind the HLA of the HLA-PEPTIDE target.

[00293] In some embodiments, the library is a yeast display library. [00294] In some aspects, the binding step is performed more than once, optionally at least three times, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or lOx.

[00295] In addition, the method can also include contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE target to determine if the antigen binding protein selectively binds the HLA-PEPTIDE target.

[00296] Accordingly, provided herein are systems for identifying an ABP that selectively binds one or more antigens described herein. In some embodiments, the system comprises (a) an isolated antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, and wherein the antigen is selected from an antigen described in any one of SEQ ID NOs: 10,755 to 29,364; and (b) a library comprising a plurality of distinct antigen binding proteins. In some embodiments, the library is a phage display library.

[00297] In some embodiments of the system, the antigen is attached to a solid support. The solid support can cormpise, e.g., ahead, well, membrane, tube, column, plate, sepharose, magnetic bead, cell, or chip. In some embodiments, the antigen comprises a first member of an affinity binding pair and the solid support comprises a second member of the affinity binding pair. In some embodiments, the first member is streptavidin and the second member is biotin.

[00298] In some embodiments of the system, the library (e.g., the phage display library) is a human library. In some embodiments of the system, the library (e.g., the phage display library) is a humanized library.

[00299] In some embodiments, the system further comprises a negative control antigen comprising an HLA-restricted peptide complexed with an HLA Class I molecule, wherein the HLA-restricted peptide is located in the peptide binding groove of an al/α2 heterodimer portion of the HLA Class I molecule, and wherein the negative control antigen comprises a different restricted peptide, a different HLA Class I molecule, or a different restricted peptide and a different HLA Class I molecule. In some embodiments, the negative control antigen comprises a different restricted peptide but the same HLA Class I molecule as the antigen. [00300] In some embodiments, the system comprises a reaction mixture, the reaction mixture comprising the antigen and a plurality of phages from the phage display library. [00301] Another method of identifying an antigen binding protein can include obtaining at least one HLA-PEPTIDE target; administering the HLA-PEPTIDE target to a subject (e.g., a mouse, rabbit or a llama), optionally in combination with an adjuvant; and isolating the antigen binding protein from the subject. Isolating the antigen binding protein can include screening the serum of the subject to identify the antigen binding protein. The method can also include contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE target, e.g., to determine if the antigen binding protein selectively binds to the HLA-PEPTIDE target. An antigen binding protein that is identified can be humanized.

[00302] In some aspects, isolating the antigen binding protein comprises isolating a B cell from the subject that expresses the antigen binding protein. The B cell can be used to create a hybridoma. The B cell can also be used for cloning one or more of its CDRs. The B cell can also be immortalized, for example, by using EBV transformation. Sequences encoding an antigen binding protein can be cloned from immortalized B cells or can be cloned directly from B cells isolated from an immunized subject. A library that comprises the antigen binding protein of the B cell can also be created, optionally wherein the library is phage display or yeast display.

[00303] Another method of identifying an antigen binding protein can include obtaining a cell comprising the antigen binding protein; contacting the cell with an HLA-multimer (e.g., a tetramer) comprising at least one HLA-PEPTIDE target; and identifying the antigen binding protein via binding between the HLA-multimer and the antigen binding protein.

[00304] The cell can be, e.g., a T cell, optionally a cytotoxic T lymphocyte (CTL), or a natural killer (NK) cell, for example. The method can further include isolating the cell, optionally using flow cytometry, magnetic separation, or single cell separation. The method can further include sequencing the antigen binding protein.

[00305] Another method of identifying an antigen binding protein can include obtaining one or more cells comprising the antigen binding protein; activating the one or more cells with at least one HLA-PEPTIDE target presented on at least one antigen presenting cell (APC); and identifying the antigen binding protein via selection of one or more cells activated by interaction with at least one HLA-PEPTIDE target.

[00306] The cell can be, e.g., a T cell, optionally a CTL, or an NK cell, for example. The method can further include isolating the cell, optionally using flow cytometry, magnetic separation, or single cell separation. The method can further include sequencing the antigen binding protein. Methods of Making Monoclonal ABPs

[00307] Monoclonal ABPs may be obtained, for example, using the hybridoma method first described by Kohler et al., Nature , 1975, 256:495-497 (incorporated by reference in its entirety), and/or by recombinant DNA methods (see e.g., U.S. Patent No. 4,816,567, incorporated by reference in its entirety). Monoclonal ABPs may also be obtained, for example, using phage or yeast-based libraries. See e.g, U.S. Patent Nos. 8,258,082 and 8,691,730, each of which is incorporated by reference in its entirety.

[00308] In the hybridoma method, a mouse or other appropriate host animal is immunized to elicit lymphocytes that produce or are capable of producing ABPs that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See Goding J.W., Monoclonal ABPs: Principles and Practice 3 ^rd ed. (1986) Academic Press, San Diego, CA, incorporated by reference in its entirety.

[00309] The hybridoma cells are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[00310] Useful myeloma cells are those that fuse efficiently, support stable high-level production of ABP by the selected ABP-producing cells, and are sensitive media conditions, such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA), and SP-2 or X63- Ag8-653 cells (available from the American Type Culture Collection, Rockville, MD). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal ABPs. See e.g., Kozbor, J. Immunol., 1984, 133:3001, incorporated by reference in its entirety.

[00311] After the identification of hybridoma cells that produce ABPs of the desired specificity, affinity, and/or biological activity, selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[00312] DNA encoding the monoclonal ABPs may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal ABPs). Thus, the hybridoma cells can serve as a useful source of DNA encoding ABPs with the desired properties. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as bacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia sp .), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce ABP, to produce the monoclonal ABPs.

Methods of Makins Chimeric ABPs

[00313] Illustrative methods of making chimeric ABPs are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81:6851-6855; each of which is incorporated by reference in its entirety. In some embodiments, a chimeric ABP is made by using recombinant techniques to combine a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) with a human constant region.

Methods of Makins Humanized ABPs

[00314] Humanized ABPs may be generated by replacing most, or all, of the structural portions of a non-human monoclonal ABP with corresponding human ABP sequences. Consequently, a hybrid molecule is generated in which only the antigen-specific variable, or CDR, is composed of non-human sequence. Methods to obtain humanized ABPs include those described in, for example, Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. U.S. A., 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86:10029-10033; and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; each of which is incorporated by reference in its entirety.

Methods of Makins Human ABPs

[00315] Human ABPs can be generated by a variety of techniques known in the art, for example by using transgenic animals (e.g., humanized mice). See, e.g., Jakobovits et al.,

Proc. Natl. Acad. Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807; each of which is incorporated by reference in its entirety. Human ABPs can also be derived from phage-display libraries ( see e.g., Hoogenboom et al., ./. Mol. Biol ., 1991, 227:381-388; Marks et al., J. Mol. Biol., 1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905; each of which is incorporated by reference in its entirety). Human ABPs may also be generated by in vitro activated B cells (see e.g, U.S. Patent. Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety). Human ABPs may also be derived from yeast-based libraries (see e.g, U.S. Patent No. 8,691,730, incorporated by reference in its entirety).

Methods of Makins ABP Fragments

[00316] The ABP fragments provided herein may be made by any suitable method, including the illustrative methods described herein or those known in the art. Suitable methods include recombinant techniques and proteolytic digestion of whole ABPs.

Illustrative methods of making ABP fragments are described, for example, in Hudson et al., Nat. Med., 2003, 9:129-134, incorporated by reference in its entirety. Methods of making scFv ABPs are described, for example, in Pluckthun, in The Pharmacology of Monoclonal ABPs, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458; each of which is incorporated by reference in its entirety.

Methods of Makins Multispecific ABPs

[00317] The multispecific ABPs provided herein may be made by any suitable method, including the illustrative methods described herein or those known in the art.

Methods for Engineering Cells with ABPs

[00318] Also provided are methods, nucleic acids, compositions, and kits, for expressing the ABPs, including receptors comprising antibodies, CARs, and the like, and for producing genetically engineered cells expressing such ABPs. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.

[00319] In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.

[00320] In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[00321] In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[00322] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso- Camino et al. (2013) Mol TherNucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557.

[00323] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102- 109.

[00324] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[00325] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298; Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437; and Roth et al. (2018) Nature 559:405-409). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[00326] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.

[00327] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

Preparation of Engineered Cells

[00328] In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the HLA-PEPTIDE-ABP, e.g., antibodies, antigen binding fragments thereof, CARs, and the like, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. [00329] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[00330] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources. [00331] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.

[00332] In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

[00333] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.

[00334] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[00335] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[00336] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffmity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[00337] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[00338] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[00339] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[00340] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques. [00341] For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS.RTM. M-450 CD3/CD28 T Cell Expander).

[00342] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker ¹ ^ ¹¹ ) on the positively or negatively selected cells, respectively.

[00343] In some embodiments, T cells are separated from a peripheral blood mononuclear cell (PBMC) sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.

[003441 In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[00345] In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. Peripheral blood mononuclear cell (PBMC) can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti- CD8 and anti-CD62L antibodies.

[00346] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[00347] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[00348] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[00349] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immune-magnetic (or affinity-magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher Humana Press Inc., Totowa, N.J.). [00350] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[00351] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S.

Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[00352] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[00353] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[00354] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell- type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[00355] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable. [00356] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[00357] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.

[00358] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps. [00359] In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[00360] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labeled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[00361] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[00362] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale fluorescence activated cell sorting (FACS). In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[00363] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[00364] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This can then be diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. Other examples include Cryostor®, CTL-Cryo™ ABC freezing media, and the like. The cells are then frozen to -80 degrees C at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[00365] In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.

[00366] Thus, in some embodiments, the cell populations are incubated in a culture- initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.

[00367] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[00368] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. [00369] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.

[00370] In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al.

(2012) J Immunother. 35(9):689-701.

[00371] In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some embodiments, the PBMC feeder cells are inactivated with Mytomicin C. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[00372] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1. [00373] In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

Assays

[00374] A variety of assays known in the art may be used to identify and characterize an HLA-PEPTIDE ABP provided herein.

Binding , Competition , and Epitope Mapping Assays

[00375] Specific antigen-binding activity of an ABP provided herein may be evaluated by any suitable method, including using SPR, BLI, RIA, Carterra biosensor, and MSD-SET, as described elsewhere in this disclosure. Additionally, antigen-binding activity may be evaluated by ELISA assays, using flow cytometry, and/or Western blot assays. [00376] Assays for measuring competition between two ABPs, or an ABP and another molecule (e.g., one or more ligands of HLA-PEPTIDE such as a TCR) are described elsewhere in this disclosure and, for example, in Harlow and Lane, ABPs: A Laboratory Manual ch.14, 1988, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y, incorporated by reference in its entirety.

[00377] Assays for mapping the epitopes to which an ABP provided herein bind are described, for example, in Morris “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66, 1996, Humana Press, Totowa, N.J., incorporated by reference in its entirety. In some embodiments, the epitope is determined by peptide competition. In some embodiments, the epitope is determined by mass spectrometry. In some embodiments, the epitope is determined by mutagenesis. In some embodiments, the epitope is determined by crystallography.

Assays for Effector Functions

[00378] Effector function following treatment with an ABP and/or cell provided herein may be evaluated using a variety of in vitro and in vivo assays known in the art, including those described in Ravetch and Kinet, Annu. Rev. Immunol ., 1991, 9:457-492; U.S. Pat. Nos. 5,500,362, 5,821,337; Hellstrom et al., Proc. Nat 7 Acad. Sci. USA , 1986, 83:7059-7063; Hellstrom et al., Proc. Nat Ί Acad. Sci. USA , 1985, 82:1499-1502; Bruggemann et al., J. Exp. Med., 1987, 166:1351-1361; Clynes et al., Proc. Nat’lAcad. Sci. USA, 1998, 95:652-656;

WO 2006/029879; WO 2005/100402; Gazzano- Santoro et al., J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003, 101:1045-1052; Cragg et al. Blood, 2004, 103:2738- 2743; and Petkova et al., Int’l. Immunol., 2006, 18:1759-1769; each of which is incorporated by reference in its entirety.

Pharmaceutical Compositions

[00379] An ABP, cell, or HLA-PEPTIDE target provided herein can be formulated in any appropriate pharmaceutical composition and administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, the intra- arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

[00380] The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Sheskey et al. (Eds.) 8th Ed. (2017), incorporated by reference in its entirety.

[00381] In some embodiments, the pharmaceutical composition comprises a carrier.

Methods of Treatment

[00382] For therapeutic applications, ABPs and/or cells are administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above. For example, ABPs and/or cells may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, or intratumoral routes. The ABPs also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.

[00383] The ABPs and/or cells provided herein can be useful for the treatment of any disease or condition involving HLA-PEPTIDE antigen. In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with an anti-HLA- PEPTIDE ABP and/or cell. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer.

[00384] In some embodiments, the ABPs and/or cells provided herein are provided for use as a medicament. In some embodiments, the ABPs and/or cells provided herein are provided for use in the manufacture or preparation of a medicament. In some embodiments, the medicament is for the treatment of a disease or condition that can benefit from an anti-HLA- PEPTIDE ABP and/or cell. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer.

[00385] Provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of an ABP and/or cell provided herein to the subject. In some aspects, the disease or condition is a cancer. [00386] Also provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of an ABP and/or cell provided herein to the subject, wherein the disease or condition is a cancer, and the cancer is selected from a solid tumor and a hematological tumor.

[00387] Also provided herein is a method of modulating an immune response in a subject in need thereof, comprising administering to the subject an effective amount of an ABP and/or cell or a pharmaceutical composition disclosed herein.

[00388] A modulated immune response in the subject may be evaluated by any means known in the art.

[00389] In some embodiments, a modulated immune response in the subject comprises an increase in or induction of antibody-dependent cellular toxicity (ADCC), e.g., of a target cell with surface expression of the neoantigen target of the ABP. ADCC can be evaluated by any means known in the art.

[00390] In some embodiments, a modulated immune response in the subject comprises an increase in or induction of complement dependent cytotoxicity (CDC), e.g., of a target cell with surface expression of the neoantigen target of the ABP. CDC can be evaluated by any means known in the art.

[00391] By way of example only, immune response in the subject can be evaluated by evaluating lymphocytes obtained from the subject or the subject’s tumor for binding to the HLA-PEPTIDE antigen. In some embodiments, tumor-infiltrating lymphocytes from the subject or evaluated for binding to the HLA-PEPTIDE antigen. By way of other example only, modulated immune response in the subject can include an expansion of pre-existing neoantigen-specific T cell population, a broader repertoire of new T-cell specificities in the subject, or both. Methods for evaluating such modulated immune response are described in Ott et al., An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 547, 217-221 (13 July 2017), which is hereby incorporated be reference in its interity. Methods for evaluating immune response are also described in Sahin et al., Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222-226 (13 July 2017), which is hereby incorporated by reference in its entirety. [00392] To perform immune monitoring, PBMCs are commonly used. PBMCs can be isolated before prime vaccination, and after prime vaccination (e.g. 4 weeks and 8 weeks). PBMCs can be harvested just prior to boost vaccinations and after each boost vaccination (e.g. 4 weeks and 8 weeks). [00393] In some embodiments, a modulated immune response in the subject comprises a modulated T cell response. T cell responses can be measured using one or more methods known in the art such as ELISpot, intracellular cytokine staining, cytokine secretion and cell surface capture, T cell proliferation, MHC multimer staining, or by cytotoxicity assay. T cell responses to HLA-PEPTIDE antigens disclosed herein can be monitored from PBMCs by measuring induction of cytokines, such as IFN-gamma, using an ELISpot assay. Specific CD4 or CD8 T cell responses to HLA-PEPTIDE antigens disclosed herein can be monitored from PBMCs by measuring induction of cytokines captured intracellularly or extracellularly, such as IFN-gamma, using flow cytometry. Specific CD4 or CD8 T cell responses to HLA- PEPTIDE antigens disclosed herein can be monitored from PBMCs by measuring T cell populations expressing T cell receptors specific for epitope/MHC class I complexes using MHC multimer staining. Specific CD4 or CD8 T cell responses to HLA-PEPTIDE antigens disclosed herein can be monitored from PBMCs by measuring the ex vivo expansion of T cell populations following 3H-thymidine, bromodeoxyuridine and carboxyfluoresceine-diacetate- succinimidylester (CFSE) incorporation. The antigen recognition capacity and lytic activity of PBMC-derived T cells that are specific HLA-PEPTIDE antigens disclosed herein can be assessed functionally by chromium release assay or alternative colorimetric cytotoxicity assays.

[00394] In particular embodiments, immune response in the subject is evaluated by enzyme linked immunospot (ELISPOT) analysis.

[00395] Also provided herein is a method of killing a target cell in a subject in need thereof, comprising administering to the subject an effective amount of an ABP and/or cell or a pharmaceutical composition disclosed herein. In some embodiments, the subject has cancer. In some embodiments, the target cell is a cancer cell.

[00396] In some embodiments, the cancer or cancer cell expresses or is predicted to express an HLA-PEPTIDE antigen or HLA Class I molecule as described in any one of SEQ ID NOs: 10,755 to 29,364. In some embodiments, the cancer or cancer cell is determined or predicted to comprise the somatic mutation in the gene that is associated with the HLA- PEPTIDE antigen. In some embodiments, the ABP selectively binds to the HLA-PEPTIDE antigen. In some embodiments, the ABP selectively binds to the HLA Class I subtype comprised in the HLA-PEPTIDE antigen.

[00397] In some embodiments, prior to administering the ABP to the subject, the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to express the HLA-PEPTIDE antigen . In some embodiments, prior to administering the ABP to the subject, the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to comprise the HLA Class I subtype of the HLA-PEPTIDE antigen. By way of example only, prior to administering an ABP that selectively binds to RAS G12D neoantigen HLA- A* 11:01 _VVV GADGV GK (SEQ ID NO: 29366) (“SNA30”), the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to comprise HLA- A* 11 :01. In some embodiments, prior to administering the ABP to the subject, the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to comprise the somatic mutation in the gene that is associated with the HLA-PEPTIDE antigen. By way of example only, prior to administering an ABP that selectively binds to RAS G12D neoantigen HLA-A* 11 :01_VVVGADGVGK (SEQ ID NO: 29366) (“SNA30”), the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to comprise a RAS G12D mutation. In some embodiments, prior to administering the ABP to the subject, the cancer or cancer cell of the subject, or a biological sample from the subject, is determined to comprise the HLA Class I subtype of the HLA-PEPTIDE antigen and the cancer or cancer cell of the subject expresses or is predicted to express the gene associated with the somatic alteration encompassed by the HLA-PEPTIDE antigen. By way of example only, prior to administering an ABP that selectively binds to RAS G12D neoantigen HLA- A* 11 : 01 _VVV GADGV GK (SEQ ID NO: 29366) (“SNA30”), the cancer or cancer cell of the subject, or a biological sample from the subject is determined to comprise HLA-A* 11:01 and the cancer or cancer cell of the subject expresses RAS, e.g., KRAS, NRAS, or HRAS. In some embodiments, prior to administering the ABP to the subject, the cancer, cancer cell, or biological sample of the subject is determined to comprise the somatic mutation in the gene that is associated with the HLA-PEPTIDE antigen, and the subject is determined to express the HLA Class I subtype comprised in the HLA-PEPTIDE antigen. By way of example only, prior to administering an ABP that selectively binds to RAS G12D neoantigen HLA- A* 11 : 01 _VVVGADGV GK (SEQ ID NO: 29366) (“SNA30”), the cancer or cancer cell of the subject, or a biological sample from the subject is determined to comprise HLA-A* 11:01 and a RAS G12D mutation. In some embodiments, a biological sample obtained from the subject is determined to be positive for the HLA-PEPTIDE antigen or HLA Class I subtype comprised in the HLA-PEPTIDE antigen. In some embodiments, a cancer or cancer cell of the subject is determined to express the gene associated with the somatic alteration, the mutation, or both the gene and the somatic alteration, above a predefined threshold. In some embodiments, loss of the HLA Class I subtype in the cancer or cancer cell of the subject is not detected.

[00398] Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom. In some embodiments, the biological sample comprises a tumor sample, e.g., a solid tumor sample. In some embodiments, the solid tumor sample is a fresh tumor sample. In some embodiments, the solid tumor sample is a frozen tumor sample. In some embodiments, the tumor sample is a formalin-fixed, paraffin- embedded (FFPE) sample. In some embodiments, the tumor sample is a tumor biopsy or resection preserved in an agent formulated to prevent RNA degradation in the sample. Such agents are known in the art, and include, but are not limited to, RNAlater. In some embodiments, the biological sample is a liquid sample. In particular embodiments, the liquid sample is a blood sample. In particular embodiments, the blood sample is a whole blood sample. In particular embodiments, the blood sample is a plasma sample. In particular embodiments, the blood sample is a serum sample.

[00399] By way of example only, if a cancer, cancer cell, or biological sample of the subject is determined to comprise a CREB3L1 V414I mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 10755. By way of other example only, if the subject is determined to express the HLA Class I allele HLA-A*02:06, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 10755. By way of yet other example only, if the subject is determined to express the HLA Class I allele HLA-A*02:06 and a cancer, cancer cell, or biological sample of the subject is determined to comprise a CREB3L1 V414I mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 10755. [90400] By way of example only, if a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12C mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 14954 and 14955. By way of other example only, if the subject is determined to express the HLA Class I allele HLA-A*02:01, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 14954 and 14955. By way of yet other example only, if the subject is determined to express the HLA Class I allele HLA-A*02:01 and a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12C mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 14954 and 14955.

[00401] By way of example only, if a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12D mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19865. By way of other example only, if the subject is determined to express the HLA Class I allele HLA-A* 11 :01, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19865. By way of yet other example only, if the subject is determined to express the HLA Class I allele HLA-A* 11:01 and a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12D mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19865.

[00402] By way of example only, if a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12V mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19,976. By way of other example only, if the subject is determined to express the HLA Class I allele HLA-A* 11 :01, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19,976. By way of yet other example only, if the subject is determined to express the HLA Class I allele HLA-A* 11:01 and a cancer, cancer cell, or biological sample of the subject is determined to comprise a RAS G12V mutation, the subject may be selected for treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 19,976.

[00403] Expression of the antigen, presence of the somatic mutation in the gene associated with the antigen, or expression of the HLA Class I subtype comprised in the antigen can be determined by any means known in the art.

[00404] Expression or presence of the antigen can be determined at the RNA or protein level by any means known in the art. Exemplary methods include, but are not limited to RNASeq, microarray, PCR, Nanostring, in situ hybridization (ISH), Mass spectrometry, or immunohistochemistry (IHC). Thresholds for positivity of gene expression is established by several methods, including: (1) predicted probability of presentation of the epitope by the HLA allele at various gene expression levels, (2) correlation of gene expression and HLA epitope presentation as measured by mass spectrometry, and/or (3) clinical benefits of ABP- based immunotherapy attained for patients expressing the genes at various levels.

[ 00405 ] For example, presence of the somatic mutation associated with the antigen can be determined by sequencing. In some embodiments, polynucleotides are isolated from the biological sample and sequenced. The polynucleotides can comprise DNA. The polynucleotides can comprise cDNA. The polynucleotides can comprise RNA. The sequencing can comprise whole exome sequencing, whole genome sequencing, targeted sequencing of a panel of cancer genes, or targeted sequencing of a single cancer gene. Exemplary gene panels include, but are not limited to FoundationOne, FoundationOne CDx, Guardant 360, Guardant OMNI, and MSK IMPACT. Presence of the somatic mutation associated with the antigen can also be determined by PCR based assays such as cobas® KRAS Mutation Test. Presence of the somatic mutation associated with the antigen can also be determined by mass-spec based assays such as MassARRAY, described in Ibarrola- Villava, Maider et al. “Determination of somatic oncogenic mutations linked to target-based therapies using MassARRAY technology.” Oncotarget y ol. 7,16 (2016): 22543-55. doi: 10.18632/oncotarget.8002, which is hereby incorporated by reference in its entirety. [004061 For example, presence of the HLA Class I subtype in the subject or biological sample of the subject can be determined by sequencing, e.g., next generation sequencing (NGS) of the HLA genes and analysis with a bioinformatic tool such as OptiType, standard sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) technologies, or any other methods known in the art.

Combination Therapies

[00407] In some embodiments, an ABP and/or cell provided herein is administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with an ABP and/or cell provided herein. In some embodiments, the additional therapeutic agent is an ABP.

Diagnostic Methods

[00408] Also provided are methods for predicting and/or detecting the presence of a given HLA-PEPTIDE on a cell from a subject. Such methods may be used, for example, to predict and evaluate responsiveness to treatment with an ABP and/or cell provided herein.

[00409] In some embodiments, a blood or tumor sample is obtained from a subject and the fraction of cells expressing HLA-PEPTIDE is determined. In some aspects, the relative amount of HLA-PEPTIDE expressed by such cells is determined. The fraction of cells expressing HLA-PEPTIDE and the relative amount of HLA-PEPTIDE expressed by such cells can be determined by any suitable method. In some embodiments, flow cytometry is used to make such measurements. In some embodiments, fluorescence assisted cell sorting (FACS) is used to make such measurement. See Li et al., J. Autoimmunity , 2003, 21:83-92 for methods of evaluating expression of HLA-PEPTIDE in peripheral blood.

[00410] In some embodiments, detecting the presence of a given HLA-PEPTIDE on a cell from a subject is performed using immunoprecipitation and mass spectrometry. This can be performed by obtaining a tumor sample (e.g., a frozen tumor sample) such as a primary tumor specimen and applying immunoprecipitation to isolate one or more peptides. The HLA alleles of the tumor sample can be determined experimentally or obtained from a third party source. The one or more peptides can be subjected to mass spectrometry (MS) to determine their sequence(s). The spectra from the MS can then be searched against a database. An example is provided in the Examples section below.

[00411] In some embodiments, predicting the presence of a given HLA-PEPTIDE on a cell from a subject is performed using a computer-based model applied to the peptide sequence and/or RNA measurements of one or more genes comprising that peptide sequence (e.g.,

RNA seq or RT-PCR, or nanostring) from a tumor sample. The model used can be as described in international patent application no. PCT/US2016/067159, herein incorporated by reference, in its entirety, for all purposes.

Kits

[00412] Also provided are kits comprising an ABP and/or cell provided herein. The kits may be used for the treatment, prevention, and/or diagnosis of a disease or disorder, as described herein.

[00413] In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. The containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is by itself, or when combined with another composition, effective for treating, preventing and/or diagnosing a disease or disorder. The container may have a sterile access port. For example, if the container is an intravenous solution bag or a vial, it may have a port that can be pierced by a needle. At least one active agent in the composition is an ABP provided herein. The label or package insert indicates that the composition is used for treating the selected condition.

[00414] In some embodiments, the kit comprises (a) a first container with a first composition contained therein, wherein the first composition comprises an ABP and/or cell provided herein; and (b) a second container with a second composition contained therein, wherein the second composition comprises a further therapeutic agent. The kit in this embodiment can further comprise a package insert indicating that the compositions can be used to treat a particular condition, e.g., cancer.

[00415] Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable excipient. In some aspects, the excipient is a buffer. The kit may further include other materials desirable from a commercial and user standpoint, including filters, needles, and syringes.

EXAMPLES

[00416] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[00417] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);

Remington's Pharmaceutical Sciences , 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 ^rd Ed. (Plenum Press) Vols A and B(1992).

Example 1: Identification of Shared HLA-PEPTIDE neoantigens [00418] We identified shared HLA-PEPTIDE neoantigens using a series of steps. We obtained a list of common driver mutations classified as “confirmed somatic” from the COSMIC database. For each mutation, we generated candidate neoepitopes (8 to 11-mer peptides), used a TPM of 100, and ran our EDGE prediction model (a model trained on HLA presented peptides sequenced by MS/MS, as described in international patent application publicationsWO/2017/106638, WO/2018/195357, and WO/2018/208856, each herein incorporated by reference, in their entirety, for all purposes) across all modeled HLA alleles. Note that each peptide contained at least one mutant amino acid and was not a self-peptide. We then recorded any peptide with an HLA allele that has an EDGE score >0.001. The results are shown in Table A. A total of 10261 shared HLA-PEPTIDE neoantigen sequences were thus identified and are described in SEQ ID NO: 10,755-21,015. The corresponding HLA allele(s) for each sequence are shown.

[00419] The initial list provided in Table A was further analyzed for level of neoantigen/HLA prevalence in the patient population. “Antigen/HLA prevalence” or “is calculated as the frequency of a given mutation “(A)” in a given population multiplied by the frequency of an HLA allele “(B)” in the given population. Antigen/HLA prevalence can also refer to mutation/HLA prevalence or neoantigen/HLA prevalence. As part of this analysis, for each mutation, its (A) frequency was obtained across common tumor types in TCGA and recorded at its highest frequency amongst tumor types. (B) For each HLA allele in EDGE, the HLA allele frequency TCGA (a predominantly Caucasian population) was recorded. HLA allele frequencies are described in more detail in Shukla, S. A. etal. {Nat. Biotechnol. 33, 1152-11582015), herein incorporated by reference for all purposes. The neoantigen/HLA prevalence was calculated as (A) multiplied by (B). Any restricted peptide/HLA pair in Table A that is >0.1% prevalence using this methodology is identified with a “Most Common 1” (2387/10261).

[00420] Additionally, we characterized the prevalence of cancer driver mutations across a large cohort of patient samples representative of the advanced cancer patient population relevant for potential clinical studies. EDGE prediction was performed using the publicly- released AACR Genie v4.1 dataset, which has over 40,000 patients sequenced on NGS cancer gene panels ranging from 50 to 500 genes, from major academic cancer centers including Dana-Farber, Johns Hopkins, MD Anderson, MSKCC, and Vanderbilt. We selected base substitution and indel mutations in lung, microsatellite stable colon, and pancreatic cancers, and required coverage across multiple gene panels. We analyzed each neoantigen peptide paired with each of greater than 90 Class I HLA alleles covered in our EDGE antigen presentation prediction model and recorded the epitopes and corresponding HLA alleles with an EDGE probability of HLA presentation score of >0.001. We then determined the neoantigen/HLA prevalence of those peptide with an EDGE score >0.001, calculated as A*B, where A is the highest frequency of the mutation amongst the three tumor types and B is the HLA allele frequency. We used HLA allele frequencies representative of the population in the USA by examining HLA alleles from the TCGA population and tabulating the frequency for each HLA allele (Shukla, S. A. et al. ^~ ). Peptides and corresponding HLA alleles that demonstrated a neoantigen/HLA prevalence >0.01% from the analysis are described in SEQ ID NO:21, 016-29, 357 and referred to as AACR GENIE Results.

Example 2: Validation of Shared HLA-PEPTIDE Neoantigen Presentation

[00421] Mass spectrometry (MS) validation of candidate shared HLA-PEPTIDE neoantigens was performed using targeted mass spectrometry methods. Nearly 500 frozen resected lung, colorectal and pancreatic tumor samples were homogenized and used for both RNASeq transcriptome sequencing and immunoprecipitation of the HLA/peptide complexes. A peptide target list was generated for each sample by analysis of the transcriptome, whereby recurrent cancer driver mutations, as defined in the AACR Genie v4.1 dataset, were identified and RNA expression levels assessed. The EDGE model of antigen presentation was then applied to the mutation sequence and expression data to prioritize peptides for the targeting list. The peptides from the HLA molecules were eluted and collected using size exclusion to isolate the presented peptides prior to mass spectrometry. Synthetic heavy labeled peptide with the same amino acid sequence was co-loaded with each sample for targeted mass spectrometry. Both coelution of the heavy labeled peptide with the experimental peptide and analysis of the fragmentation pattern we were used to validate a candidate epitope. Mass spectrometry analysis methods are described in more detail in Gillete etal. {Nat Methods. 2013 Jan;10(l):28-34), herein incorporated by reference in its entirety for all purposes.

Shared HLA-PEPTIDE neoantigens from driver mutations validated in this manner with sufficient prevalence for further consideration are summarized in Table 5A below, along with sample tumor type and associated HLA alleles. Table 5A: Expression of MS-validated HLA-PEPTIDE neoantigens and detected by MS/MS, it was inferred to be presented by the highest scoring HLA allele by EDGE or both alleles if the scores were sufficiently close [00422] Selected HLA-PEPTIDE neoantigens were also validated by in vitro assay.

Briefly, cell lines were engineered to express a single specific HLA alleles and inducibly express a candidate shared neoantigen according to methods described below.

[00423] Materials and Methods for validation of selected HLA-PEPTIDE neoantiens by in vitro assay.

[00424] Single HLA allele expressing K562 cell lines were created by traditional transfection methods using reagent kits and the instructions provided.

[00425] To create virus particles for transduction of the HLA genes into K562 cells the plasmids were transfection into Phoenix-ampho cells. Phoenix-ampho cells were introduced into 6 well plates at a density of 5x10 ⁵ cells per well and incubated at 37C overnight prior to transfection. 10ug of purified DNA was mixed with lOuL Plus Reagent and brought to 100uL with pre-warmed Opti-MEM media. Lipofectamine reagent was prepared by mixing 8uL of Lipofectamine with 92uL of the pre-warmed Opti-MEM. Both mixtures were incubated at room temperature for 15 prior to mixing the 100uL of Lipofectamine reagent with the 100uL of DNA solution and allowing the combined solution to incubate at room temperature for another 15min. The Phoenix-ampho cells were washed gently by aspirating the media and adding 6mL of pre-warmed Opti-MEM media to wash the cells. The media was removed from the plated cells. 800uL of the pre-warmed Opti-MEM was added to the DNA/Lipofectamine mixture to make lmL and that solution was added to the plated cells. After the plate was incubated for 3hrs at 37C, 3mL of complete media was added and the cells were incubated overnight at 37C. The complete media was exchanged after the incubation and the cells incubated for another 2 days. Virus particles were collected after the supernatant was passed through a 45um filter into a new 6 well plate. 20uL of Plus reagent and 8uL of Lipofectamine was added to each well with a 15 min room temperature incubation after each addition.

[00426] K562 cells were suspended complete media at a concentration of 5x10 ⁶ per mL. 100uL of K562 cells were added to each well of the 6 well plate containing the virus particles. The plate was centrifuged at 700xg for 20 min and the cells were incubated for 6 hrs at 37C. Cells and virus were collected and transferred to T25 flasks with the addition of 7mL of complete media. The cells were incubated for 3 days prior to a media change which included selection with Puromyocin antibiotic. Live cells were collected and passaged to create stocks of K562 cells expressing a single HLA allele. Overall 25 of these cell lines were created, each with a different HLA expressed, to provide a reagent pool for future experiments.

[00427] A shared neoantigen cassette was created to express 20 shared neoantigens with the mutation centered in a 25mer amino acid chain and was created with no linkers between the entries. This cassette was subcloned into a lentiviral Tet-One Inducible Expression vector system (Clonetech) and lenitvirus was produced in 293T cells by contransfecting the shared neoantigen expression vector with ViraPower (Thermo) packaging plasmids according to manufacturer’s specifications. Single HLA Allele expressing K562 cell lines were then transduced with this virus as described above and single cell clones were characterized for shared neoantigen expression. Briefly, expression of the shared neoantigen cassette was placed under control of a doxy cy cline (DOX)-controlled TRE3G promoter, where administration of DOX leads to expression of the neoantigens via stabilization of the Tet-On 3G transactivator protein that is constitutively expressed on the same plasmid. The TREG3 promoter - Tet-On 3G transactivator system allows titration of DOX to control the level of expression. As shown in FIG. 7, expression of a representative neoantigen increased as the concentration of DOX administered increased, demonstrating regulatable expression.

[004281 Cells containing both the single HLA allele and the shared neoantigen cassette were grown to ~2.5x10 ⁸ cells and pelleted into 15mL vials. Additionally, cells were plated in limited dilution to prepare single clones of the HLA/Cassette pairing. These single clones were tested to achieve a variety of expression levels of the cassette. Use of cell lines with differing expression levels of the cassette allows for analysis of the system at close to endogenous expression levels. Single clones were also grown to ~2.5x10 ⁸ cells and pelleted into 15mL vials. All pellets were washed 2x with cold PBS and frozen to allow for processing for mass spectrometry detection of HLA peptides. Expression levels of the HLA and the cassette was performed using SmartSeq or Taqman assays with appropriate probes. [004291 For isolation of HLA peptides, each cell pellet was lysed with lysis buffer and centrifuged at 20,000 x g for 1 hr to clarify the lysate and the HLA peptide complexes were enriched as previously described (see Section Error! Reference source not found.). Heavy peptides — peptides synthesized with amino acids containing isotopically heavy amino acids - - were added to the peptides prior to analysis by MS to aid in confirmation of the identity of the peptides detected.

[00430] As shown in FIG. 8, a representative KRAS GUV peptide VVGAVGVGK (SEQ ID NO: 29362) was observed by mass-spectrometry in a HLA-A* 11:01 expressing K562 cell line, in a DOX-dependent manner (FIG. 8, top panels). Detection of the heavy peptide control standard was equivalent (FIG. 8, bottom panels). Thus, validation of HLA-specific presentation of predicted neoantigens was demonstrated using the single-HLA K562 in vitro system.

[00431] The in vitro system described above was used to validate HLA-specific presentation of predicted neoantigens.

[00432] Results are shown in Table 5B, below.

Table 5B: Validated HLA-PEPTIDE neoantigens by in vitro assay

[00433] We further evaluated the MS data with respect to mutations for which peptides were not detected in order to assess the value of narrowly targeting patients with specific HLAs for treatment, e.g. , requiring patients to have at least one validated or predicted HLA allele that presents a restricted peptide disclosed herein.

[00434] For example, in the case of KRAS, we counted the number of patient samples in which KRAS restricted peptides for particular HLA alleles were detected or not detected. (When the same peptide was predicted to be presented by multiple HLA alleles for a patient and detected by MS/MS, it was inferred to be presented by the highest scoring HLA allele by EDGE or both alleles if the scores were sufficiently close). Results are presented in Table 6. Based on these results, several common HLA alleles are not expected to present a given KRAS restricted peptide and these KRAS restricted peptide/HLA pairs can be excluded for purposes of selection criteria for immunotherapy design and patient selection in this instance. For example, Table 7 directed to selected HLA-PEPTIDE neoantigen targets for immunotherapy does not include predicted HLA-PEPTIDE neoantigen HLA-A*02:01_RAS G12D, on the basis that the restricted peptide was not detected in 17 samples tested, and likewise did not include G12V/A*02:01 on the basis that the peptide was not detected in 9 samples tested. In contrast, neoantigen/HLA pair G12D/A* 11:01 was considered validated on the basis that the peptide was detected in 1/5 samples tested, and likewise G12V/A* 11:01 was considered validated on the basis that the peptide was detected in 2/6 samples tested. [00435] These results highlight the importance of identifying the relevant restricted peptide/HLA pairs for proper HLA type selection in patient slection for treatment with a shared neoantigen immunotherapy, such as that described in Table 7. Specifically, several common KRAS restricted peptide/HLA pairs were excluded for purposes of selection criteria in this case as the MS data suggested a shared HLA-PEPTIDE neoantigen ABP would unlikely provide a benefit to a patient with that predicted KRAS neoantigen/HLA pair ( e.g ., G12D/A*02:01 or G12V/A*02:01).

Table 6

Example 3: Selection of Shared HLA-PEPTIDE neoantigens for immunotherapy [00436] A selection of clinically useful HLA-PEPTIDE neoantigen targets for immunotherapy (“GO-005”) containing 20 shared HLA-PEPTIDE neoantigens was constructed. Table 7 describes features of the HLA-PEPTIDE neoantigens selected for the selection. Shared HLA-PEPTIDE neoantigens directly detected on the surface of tumor cells by mass spectrometry, as described above in Table 5, were included in the cassette and the HLA of the epitope was added to the eligible HLA list for the mutations. HLA-PEPTIDE neoantigens not independently verified as being presented in our assays were considered validated and added to the cassette if there was compelling literature evidence of tumor presentation ( e.g ., tumor-infiltrating lymphocytes (TIL) recognizing the neoantigen). KRAS G12D presented by HLA-C*08:02 was considered validated and added based on literature evidence of adoptive cell therapy targeting this HLA-PEPTIDE neoantigen causing tumor regression in a patient with CRC (Tran et al. N Engl JMed. 2016 Dec 8; 375(23): 2255- 2262.). HLA-PEPTIDE neoantigens with validated HLA alleles occupied 6 out of 20 slots. [00437] Additional, rarer HLA-PEPTIDE neoantigens predicted to be presented by tumor cells, but not yet validated by MS, were used to complement the initial set. Mutations with high EDGE scores were prioritized for inclusion as predicted HLA-PEPTIDE neoantigens given the strong dependence we observed between EDGE score and probability of detection of candidate shared HLA-PEPTIDE neoantigen peptides by targeted mass spectrometry (MS) validation experiments described herein. Results showing the correlation between EDGE score and the probability of detection of candidate shared HLA-PEPTIDE neoantigen peptides by targeted MS are shown in FIG. 4. Specifically, predicted HLA-PEPTIDE neoantigens with an EDGE HLA presentation score of at least 0.3 and the highest cumulative neoantigen/HLA prevalence across NSCLC, CRC and Pancreatic cancer were included in the selection. Combined HLA frequency was required to be at least 5 - 10% (e.g., there are 11% of the American population harboring HLA alleles B1501 or B 1503). Of note, KRAS and NRAS harbors the same sequence around codons 12, 13, and 61. Validated HLAs, predicted HLAs with an EDGE score of at least 0.3, the mean EDGE score of the predicted HLAs, and neoantigen/HLA prevalence in the three cancer populations are also presented in Table 7.

[00438] Table 7 (below) depicts 20 exemplary shared HLA-PEPTIDE neoantigens comprising a cancer-related mutation and a particular HLA Class I allele, based on EDGE Score and prevalence in cancer patient populations. The exemplary shared HLA-PEPTIDE neoantigens are particularly useful targets for cancer immunotherapy, e.g., by treatment with an ABP that selectively binds the HLA-PEPTIDE neoantigen.

Table 7: Selected Shared HLA-PEPTIDE neoantigens

[00439] Additionally, we determined the total population of patients with at least one HLA allele identified (i.e., either validated or predicted) to present at least one shared neoantigen from Table 7 (i.e., the HLA-PEPTIDE neoantigen comprising both the mutation and the HLA allele, referred to herein as GO-005 targeted patient population) and compared it to the population of patients with the mutations (agnostic of whether the patient had the identified allele). To estimate the GO-005 targeted patient population, we collected patient mutation data from AACR Genie. As such patients do not have matching HLA alleles, we sampled HLA alleles from the TCGA population and paired it to the AACR Genie dataset. Then given a tumor type, any patient from AACR Genie with matching both mutation and HLA was labeled positive, and any patient that doesn’t meet the criteria was labeled negative. The percent positives give the overall addressable patient population, per tumor type, in Table 16.

[00440] It can be readily appreciated from Table 16 that only a subset of patients who carry a particular mutation also carry the HLA allele likely to present that mutation as a HLA- PEPTIDE neoantigen. Patients with the mutation, but without the appropriate HLA allele are less likely to benefit from therapy. As an example, whereas an estimated -60% of pancreatic cancer patients carry appropriate mutations/neoantigens, more than 2 out of 3 of these patients do not carry the corresponding HLA allele(s). Therefore, an ABP-based immunotherapy strategy that considers the relevant mutation and HLA allele pairs as proposed will target just those patients who may benefit. Thus, consideration of epitope presentation by validated or high-scoring predicted HLA is an important step in determining the potential efficacy of a shared HLA-PEPTIDE neoantigen ABP.

Table 16: Neoantigen/HLA Prevalence in Target Populations

Example 4: Evaluation of immune response induction by Shared HLA-PEPTIDE neoantigens

[00441] We evaluated whether HLA-PEPTIDE neoantigens induce an immune response in patients. We obtained dissociated tumor cells from a patient with lung adenocarcinoma. Tumor cells were sequenced to determine the patient’s HLA and identify mutations. The patient expressed HLA- A* 11:01 and we identified the KRAS G12V mutation in the tumor. Simultaneously, we sorted and expanded CD45+ cells from the tumor which represent tumor infiltrating lymphocytes (TIL). Expanded TILs were stained with mutated peptide HLA- A* 11 :01 tetramers to assess immunogenicity of this mutation in the patient. FIG. 5 shows the flow cytometry gating strategy on CD8+ cells (FIG. 5A) and the staining of CD8+ cells by KRAS-G12V/ HLA-A* 11:01 tetramer (FIG. 5B). A large portion (greater than 66%) of CD8+ T cells demonstrate binding to the KRAS G12V:HLA*1101 tetramer, indicating the ability of CD8+ T cells to recognize the HLA-PEPTIDE neoantigen and indicating a pre- existing immune response to the HLA-PEPTIDE neoantigen.

Example 5: Selection of Shared HLA-PEPTIDE neoantigens and Patient Populations for HLA-PEPTIDE neoantigen-specific immunotherapy [00442] An ABP to a HLA-PEPTIDE neoantigen as described in Table 7, Table A, the AACR GENIE Results, or SEQ ID NOs 29358-29364 described herein (SEQ ID NOs: 10,755-29,364) is administered to a cancer patient. The ABP is administered to a patient, e.g., to treat cancer. In certain instances the patient is selected, e.g, using a companion diagnostic or a commonly use cancer gene panel NGS assay such as FoundationOne, FoundationOne CDx, Guardant 360, Guardant OMNI, or MSK IMPACT. Exemplary patient selection criteria are described below.

Patient Selection

[00443] Patient selection for ABP administration is performed by consideration of tumor gene expression, somatic mutation status, and patient HLA type. Specifically, a patient is considered eligible for the ABP-based immunotherapy therapy if the patient has cancer, and if:

(a)one or more cells of the patient expresses or is known to express an HLA Class I molecule as described in any one of SEQ ID NOs: 10,755 to 29,364. In such cases, the patient may be administered an ABP that targets a HLA-PEPTIDE neoantigen described herein, such that the HLA-PEPTIDE neoantigen comprises the same HLA Class I molecule expressed by the one or more cells of the patient. By way of example only, a patient is considered eligible for ABP-based immunotherapy by administration of an ABP that selectively binds to RAS G12D neoantigen HLA-

A* 11 : 01 _VVV GADGV GK (SEQ ID NO: 29366) (“SNA30”) if one or more cells of the patient expresses or is known to express HLA-A* 11:01.

(b) one or more cells of the patient expresses or is known to express an HLA Class I molecule as described in any one of SEQ ID NOs: 10,755 to 29,364, and the cancer expresses or is predicted to express a gene associated with a somatic mutation. By way of example only, a patient is considered eligible for ABP-based immunotherapy by administration of an ABP that selectively binds to RAS G12D neoantigen HLA- A* 11 : 01 _VVV GADGV GK (SEQ ID NO: 29366) (“SNA30”) if one or more cells of the patient expresses or is known to express HLA-A* 11:01 and the cancer expresses or is predicted to express KRAS.

(c) one or more cells of the patient expresses or is known to express an HLA Class I molecule as described in any one of SEQ ID NOs: 10,755 to 29,364, and the patient tumor or tumor nucleic acid carries the somatic mutation associated with the SEQ ID NO. By way of example only, a patient is considered eligible for ABP-based immunotherapy by administration of an ABP that selectively binds to RAS G12D neoantigen HLA-A* 11 :01_VVVGADGVGK (SEQ ID NO: 29366) (“SNA30”) if one or more cells of the patient expresses or is known to express HLA-A* 11:01 and a tumor sample from the patient harbors the RAS G12D somatic mutation.

(d) Same as (b) or (c), but also requiring that the patient tumor expresses the gene with the mutation above a certain threshold (e.g., 1 TPM or 10 TPM), or

(e) Same as (b) or (c), but also requiring that the patient tumor expresses the mutation above a certain threshold (e.g., at least 1 mutated read observed at the level of RNA)

(f) Same as (b) or (c), but also requiring both additional criteria in (c) and (d)

(g) Any of the above, but also optionally requiring that loss of the presenting HLA allele is not detected in the tumor

[00444] Gene expression may be measured at the RNA or protein level by methods including, but not limited to RNASeq, microarray, PCR, Nanostring, ISH, Mass spectrometry, or IHC. Thresholds for positivity of gene expression is established by several methods, including: (1) predicted probability of presentation of the epitope by the HLA allele at various gene expression levels, (2) correlation of gene expression and HLA epitope presentation as measured by mass spectrometry, and/or (3) clinical benefits of ABP-based immunotherapy attained for patients expressing the genes at various levels.

[00445] Somatic mutational status may be assessed by any of the established methods, including exome sequencing (NGS DNASeq), targeted exome sequencing (panel of genes), transcriptome sequencing (RNASeq), Sanger sequencing, PCR-based genotyping assays (e.g., Taqman or droplet digital PCR), Mass-spectrometry based methods (e.g., by Sequenom), next generation sequencing, massively parallel sequencing, or any other method known to those skilled in the art.

Example 6: Identification of antibodies and antigen binding fragments thereof that bind exemplary RAS HLA-PEPTIDE neoantigens.

[00446] Overview

[00447] The following exemplification demonstrates that antibodies (Abs) can be identified that recognize HLA-PEPTIDE neoantigens disclosed herein. The overall epitope that is recognized by such Abs generally comprises a composite surface of both the peptide as well as the HLA protein presenting that particular peptide. Abs that recognize HLA complexes in a peptide-specific manner are often referred to as T cell receptor (TCR)-like Abs or TCR- mimetic Abs. As a proof of concept, TCR-like Abs were generated to the following exemplary HLA-PEPTIDE neoantigens: a RAS_G12D neoantigen described in SEQ ID NO: 19,865, corresponding to HLA-PEPTIDE HLA-A* 11 :01_ VVVGADGVGK (SEQ ID NO: 19865) (designated “SNA30”), a RAS_G12V neoantigen described in SEQ ID NO: 19,976, corresponding to HLA-PEPTIDE HLA-A* 11 :01_VVVGAVGVGK (SEQ ID NO: 19976) (designated “SNA6”); and a RAS_G12C neoantigen described in SEQ ID NO: 14954 and 14955, corresponding to HLA-PEPTIDE HLA-A*02:01_KLVVVGACGV (SEQ ID NO: 14954) (designated “SNA31”). Cell surface presentation of these HLA-PEPTIDE neoantigens was confirmed as described above.

[00448] HLA-PEPTIDE neoantigen target complexes and counterscreen peptide-HLA complexes

[00449] The HLA-PEPTIDE neoantigen targets SNA30, SNA6, and SNA31, as well as counterscreen negative control peptide-HLAs, were produced recombinantly using conditional ligands for HLA molecules using established methods. The counterscreen HLA- peptides were designed such that (A) the negative control peptide was known to be presented by the same HLA subtype (i.e. the HLA-related controls) or (B) the negative control peptides were known to be presented by a different HLA subtype.

[00450] Phage library screening

[00451] The highly diverse SuperHuman 2.0 synthetic naive scFv library from Distributed Bio Inc was used as input material for phage display, which has a 7.6x10 ¹⁰ total diversity on ultra-stable and diverse VH/VL scaffolds. Three to four rounds of bead-based phage panning with the target pHLA complex were conducted using established protocols to identify scFv binders to SNA31, SNA30, and SNA6, respectively. For each round of panning, the phage library was initially depleted with the pooled counterscreened pHLA complexes prior to the binding step with the target pHLAs. The phage titer was determined at every round of panning to establish removal of non-binding phage.

[00452] Generation of PPEs of individual output clones

[00453] Bacterial periplasmic extracts (PPEs) of individual output clones were subsequently generated as described below.

[00454] From each selection, ten microliters of eluted phage were taken and infected with 200 pi of HB2151 bacteria (OD 600 of 0.4) growing exponentially at 37 ° C for 30 minutes. [00455] 1/100 serial dilutions were plated on 2xYT plates containing 100 μg/ml carbenicillin and 1 % glucose and grow overnight at 37°C. Individual colonies were picked into 96 cell-well plates containing 100 mΐ 2xYT, 100 μg/ml carbenicillin and 1 % glucose. Colonies were shaken at 250 rpm overnight at 37°C. Optionally, glycerol stocks of the 96- well plate can be made by adding glycerol to a final concentration of 15 %, and then storing at -80°C.

[00456] To induce scFv expression in E. Coli , ten microliters of bacterial cultures from the individual colonies were transferred to 350 uL Overnight Express Instant TB Medium (Millipore sigma) and the cultures were grown overnight at 30 °C with shaking (700 rpm). Cultures were then centrifuged for 10 min at 3000 x G to pellet the cells and store the supernatant.

[00457] The periplasmic extracts were prepared by resuspending pellets from the overnight cultures in 60 mΐ BugBuster master mix (Millipore Sigma). Cell suspensions were incubated on a shaking platform for 20 min at room temperature. Insoluble cell debris was removed by centrifugation at 3000 x G for 20 min at 4°C. Supernatants were transfered to a fresh 96 well plate and store at -20°C until further analysis.

[00458] Evaluation of PPEs or bacterial supernatants of individual output clones [00459] Periplasmic extracts from individual output clones were evaluated by Carterra surface plasmon resonance (SPR), Meso Scale Discovery (MSD) electrochemiluminescence (ECL) screening, Octet biolayer interferometry (BLI), and flow cytometry.

[00460] MSD evaluation

[00461] MSD screening utilized the Meso Scale Discovery U-PLEX Development Pack, 9- assay (cat. No. K15234N). The pack contains a 10-spot U-PLEX plate with 9 activated spots and 9 unique linkers as well as stop solution and read buffer. Biotinylated pHLA and biotinylated Protein L were each diluted to 33nM using PBS+0.5% BSA. For each plate, 200 μL of the diluted pHLA or protein L was mixed with 300μL of the corresponding Linker (See Tables 12 and 13) and incubated at room temperature for 30 minutes.

Table 12: A*11:01 pHLA conjugation to unique linker for SNA6 and SNA30 screening

Table 13: A*02:01 pHLA conjugation to unique linker for SNA31 screening

[00462] Following the 30minute incubation, 200 μL Stop solution was added to each linker-pHLA solution. They were again incubated for 30 minutes at room temperature.

These volumes were scaled based on the number of plates. The linker-pHLA solutions were then pooled together and further diluted with stop solution to a final lx concentration. The pooled linker-pHLA solution was then coated onto the 10-spot plate at 50μL/well. The plate sealed and stored at 4°C overnight.

[00463] Bacterial supernatant or periplasmic extracts were diluted 10-fold with PBS. The plate was washed 3 times with PBS + 0.05% Tween and samples added as 50μL/well. Plates were shaken at room temperature for 2 hours. The plates were washed as before and 50μL of 250ng/mL sulfo-tag labeled anti-V5 antibody (Abeam, ab27671) was added to each well.

The anti-V5 antibody was sulfo-tag labeled using the MSD Gold Sulfo-tag NHS-Ester Conjugation kit (Meso Scale Discovery, R31 AA-2) at a challenge ratio of 10. The plates were then shaken for 2 hours at room temperature. The plate wash was repeated and 150μL 2x Read Buffer T (Meso Scale Discovery, R92TC-2) was added to all wells and the plate read immediately on the Quickplex SQ 120.

[00464] Results are shown as fold binding to the indicated HLA-PEPTIDE neoantigen target over wildtype antigen (containing the same HLA allele but wild-type sequence) (comprising the HLA allele and the restricted peptide comprising the mutation) as compared to the control antigen comprising the same HLA allele but a restricted peptide containing the corresponding wild-type sequence (WT). [00465] Flow cytometry evaluation (cell-based binding)

[00466] K562 cell line generation:

[00467] The Phoenix- AMPHO cells (ATCC®, CRL-3213™) were cultured in DMEM (Corning™, 17-205-CV) supplemented with 10% FBS (Seradigm, 97068-091) and Glutamax (Gib co™, 35050079). K-562 cells (ATCC®, CRL-243™) were cultured in IMDM (Gibco™, 31980097) supplemented with 10% FBS. Lipofectamine LTX PLUS (Fisher Scientific, 15338100) contains a Lipofectamine reagent and a PLUS reagent. Opti-MEM (Gibco™, 31985062) was purchased from Fisher Scientific.

[00468] Phoenix cells were plated at 5*10e5 cells/well in a 6 well plate and incubated overnight at 37°C. For the transfection, 10 μg plasmid, lOμL Plus reagent and 100 μL Opti- MEM were incubated at room temperature for 15 minutes. Simultaneously, 8 μL Lipofectamine was incubated with 92 μL Opti-MEM at room temperature for 15 minutes. These two reactions were combined and incubated again for 15 minutes at room temperature after which 800 μL Opti-MEM was added. The culture media was aspirated from the Phoenix cells and they were washed with 5 mL pre-warmed Opti-MEM. The Opti-MEM was aspirated from the cells and the lipofectamine mixture was added. The cells were incubated for 3 hours at 37°C and 3 mL complete culture medium was added. The plate was then incubated overnight at 37°C. The media was replaced with Phoenix culture medium and the plate incubated an additional 2 days at 37°C.

[00469] The media was collected and filtered through a 0.45 pm filter into a clean 6 well dish. 20 μL Plus reagent was added to each virus suspension and incubated at room temperature for 15 minutes followed by the addition of 8 μL/well of Lipofectamine and another 15 min room temperature incubation. K562 cells were counted and resuspended to 5E6 cells/mL and 100 μL added to each virus suspension. The 6 well plate was centrifuged at 700g for 30 minutes and then incubated at 37°C for 5-6 hours. The cells and virus suspension were then transferred to a T25 flask and 7 mL K562 culture medium was added. The cells were then incubated for three days. The transduced K562 cells were then cultured in medium supplemented with 0.6 μg/mL Puromycin (Invivogen, ant-pr-1) and selection monitored by flow cytometry.

[00470] Flow cytometry methods

[00471] HLA-transduced K562 cells were pulsed the night before with 50 pM of peptide (Genscript) (either the restricted peptide of the target HLA-PEPTIDE neoantigen, or the corresponding peptide fragment containing the wild-type sequence) in IMDM containing 1% FBS in 6 well plates and incubated under standard tissue culture conditions. Cells were harvested, washed in PBS, and stained with eBioscience Fixable Viability Dye eFluor 450 for 15 minutes at room temperature. Following another wash in PBS + 2% FBS, cells were resuspended in bacterial supernatant from scFv expression in E coli. Cells were incubated for 1 hour at 4°C. After another wash, PE-conjugated PE anti-Myc (clone 9E10, Novus NB600- 302PE) 1:500. After incubating at 4C for 45 minutes and washing in PBS + 2% FBS, cells were resuspended in PBS + 2% FBS and analyzed by flow cytometry. Flow cytometric analysis was performed on the Attune NxT Flow Cytometer (Therm oFisher) using the Attune NxT Software. Data was analyzed using FlowJo. Results are shown as fold binding to the target HLA-PEPTIDE neoantigen (comprising the HLA allele and the restricted peptide comprising the mutation) as compared to the control antigen comprising the same HLA allele but a restricted peptide containing the corresponding wild-type sequence (WT).

[00472] Binding affinity evaluation by Carterra biosensor measurements [00473] Binding affinity of selected scFv PPE extracts were assessed via Carterra biosensor measurements according to established methods. Briefly, HC200M surfaces were activated with 1:1:1 (v/v/v) of 100 mM MES pH 5.5/100 mM s-NHS/400 mM EDC for 10 min. Anti- V5 antibody was diluted to 50 μg/mL into 10 mM NaOAc pH 4.3, and then amine coupled to the surfaces for 14 min. The HC200M surfaces were deactivated with 1M Ethanolamine-HCl pH 8.5 for 10 min.

[00474] PPE scFvs (diluted 2X in 1X HBSTE) were captured by their V5-tag for 15 min onto the anti-V5 antibody-coupled surface. Non-regenerative Kinetic Assay was run in IX HBSTE, 0.5 g/L BSA. pHLA analytes (target or control) were injected for 5 min followed by 15 min dissociation in running buffer. All data were double-referenced and Target-pHLA sensorgrams were fitted using a 1 : 1 Langmuir model.

[00475] Results of MSP and flow cytometry evaluation

[00476] For each of the HLA-PEPTIDE neoantigen targets selected for scFv discovery, 50 total clones that exhibited selective binding to the target HLA-PEPTIDE neoantigen as compared to counterscreen antigens were identified (data not shown). Of these, 10-15 scFv clones exhibited selective binding to HLA-transdued K562 cells expressing the target restricted peptide, as compared to the wild-type peptide fragment (data not shown).

[00477] scFv screening for the HLA-PEPTIDE target designated SNA31 yielded several clones that exhibited selective binding (at least two-fold binding) to SNA31 as compared to the comparable wild-type HLA-PEPTIDE (data not shown). [00478] FIG. 2 depicts MSD and cell-based binding data of three exemplary scFv clones specific for the HLA-PEPTIDE neoantigen designated SNA6. These results show that antibodies that specifically bind to SNA6 can be generated. These antibodies exhibit higher binding affinity to SNA6 than to other shared HLA-PEPTIDE neoantigen targets, and higher binding affinity to SNA6 as compared to the corresponding wild-type HLA-PEPTIDE antigen (HLA-A*11:01_VVVGAGGVGK (SEQ ID NO: 29373)).

[00479] FIG. 3 depicts MSD and cell-based binding data of three exemplary scFv clones for the HLA-PEPTIDE neoantigen designated SNA30. These results show that antibodies that specifically bind to SNA30 can be generated. These antibodies exhibit higher binding affinity to SNA30 than to other shared HLA-PEPTIDE neoantigen targets, and higher binding affinity to SNA30 as compared to the corresponding wild-type HLA-PEPTIDE antigen.

[00480] Results of binding affinity assessment using Carterra biosensor

[00481] Kd for selected SNA6 (KRAS_G12V neoantigen HLA-A* 11 :01_ VVVGAVGVGK (SEQ ID NO: 29368)) and SNA30 (KRAS_G12D neoantigen HLA- A* 11 :01_ VVVGADGVGK (SEQ ID NO: 29366) ) scFvs are depicted in Table 15, below.

Table 15: Binding affinities of selected scFv PPEs by Carterra

[00482] Carterra sensograms are depicted in FIG. 6.

[00483] Amino acid sequences of exemplary scFv clones

[00484] VH/VL sequences of the exemplary SNA30 scFv clones are shown in Table 8 [00485] VH/VL sequences of the exemplary SNA6 scFv clones are shown in Table 9. [00486] CDR sequences of the exemplary SNA30 scFv clones are shown in Table 10. [00487] CDR sequences of the exemplary SNA6 scFv clones are shown in Table 11. Example 7: Identification of antibodies and antigen binding fragments thereof that bind target neoantigens

[00488] Neoantigen target complexes and counterscreen peptide-HLA complexes [00489] Neoantigen targets as well as counterscreen negative control peptide-HLAs, are produced recombinantly using conditional ligands for HLA molecules using established methods. The counterscreen HLA-peptides are designed such that (A) the negative control peptide is known to be presented by the same HLA subtype (i.e. the HLA-related controls) or (B) the negative control peptides is known to be presented by a different HLA subtype. [00490] Phage library screening

[00491] An scFv library, e.g., a highly diverse SuperHuman 2.0 synthetic naive scFv library from Distributed Bio Inc, is used as input material for phage display. Three to four rounds of bead-based phage panning with the target pHLA complex are conducted using established protocols to identify scFv binders to a target neoantigen described in Table A or AACR Genie Results. For each round of panning, the phage library is initially depleted with the pooled counterscreened pHLA complexes prior to the binding step with the target pHLAs.

The phage titer is determined at every round of panning to establish removal of non-binding phage. The output phage supernatant is also tested for target binding by ELISA and suggested progressive enrichment of the target binding phage.

[00492] Generation of PPEs of individual output clones

[00493] Bacterial periplasmic extracts (PPEs) of individual output clones are subsequently generated as described in Example 6.

[00494] Evaluation of PPEs or bacterial supernatants of individual output clones [00495] Periplasmic extracts from individual output clones are evaluated by meso scale discovery (MSD) screening and flow cytometry (see Example 6)

[00496] scFv clones that exhibit selective binding to the target HLA-PEPTIDE neoantigen as compared to a corresponding pHLA control comprising the same HLA subtype but the restricted peptide corresponding to wild-type sequence, or to any of the counterscreen pHLAs are isolated and sequenced. VH, VL, and CDR sequences of the isolated scFv clones are determined by any means known in the art.

SEQUENCE TABLES

Table A

[00497] Refer to Sequence Listing, SEQ ID NOS. 10,755-21,015. [00498] Table A includes HLA-PEPTIDE neoantigens, wherein a specific restricted peptide having a specific amino acid sequence is predicted to associate with a given HLA allele with an EDGE score >0.001. The restricted peptide corresponds to a peptide fragment containing a somatic mutation associated with a cancer.

[00499] For clarity, each HLA-PEPTIDE neoantigen in Table A is assigned a unique SEQ ID NO. Each of the above sequence identifiers is associated with the Table identifier (i.e., Table A), the HLA Class I subtype, the gene name corresponding to the restricted peptide, the somatic mutation, whether the prevelance of the peptide:HLA pair was 0.1% or greater (noted as “1”) or less than 0.1% (noted as “0”), and the amino acid sequence of the restricted peptide. For example, the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 10755 is a CREB3L1 V414I neoantigen that is HLA-PEPTIDE target HLA- A* 02 : 06 AADGIYT A (SEQ ID NO: 10755). As indicated by SEQ ID NO: 10755, the restricted peptide AADGIYTA (SEQ ID NO: 10755) contains the V414I mutation in the protein encoded by gene CREB3L1, and the HLA-PEPTIDE target has a prevalence less than 0.1%.

[00500] Table A HLA-PEPTIDE neoantigens are disclosed in PCT/US2019/033830, filed on May 23, 2019, which application is hereby incorporated by reference in its entirety.

AACR GENIE Results

[00501] Refer to Sequence Listing, SEQ ID NOS. 21,016-29,357.

[00502] AACR GENIE results includes HLA-PEPTIDE neoantigens wherein a specific restricted peptide having a specif amino acid sequence is predicted to associate with a given HLA allele with an EDGE score >0.001 and prevalence >0.1%. The restricted peptide corresponds to a peptide fragment containing a somatic mutation associated with a cancer.

[00503] For clarity, each HLA-PEPTIDE neoantigen in the AACR GENIE results is assigned a unique SEQ ID. NO. Each of the above sequence identifiers includes a designation as an AACR GENIE result, the gene name corresponding to the restricted peptide, the type and nature of somatic mutation, the HLA Class I subtype, and the amino acid sequence of the restricted peptide. For the AACR GENIE results, the HLA Class I subtype designation is expressed as a single letter followed by a 4-digit code.

[00504] For clarity, the designation “p ” indicates a change in the protein sequence, the designation “i^number” stands for a frameshift mutation causing a stop codon in [the designated number ] of amino acids, the designation “dup” stands for an in-frame sequence insertion of a sequence flanked by the designated amino acids, and the designation “del” stands for an in-frame sequence deletion of the designated amino acids.

[00505] For example, the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 21016 is a ACVR1 neoantigen carrying point mutation S290L (denoted as “ACVR1 _p.S290L”) that is HLA-PEPTIDE target HL A-A*29 : 02 HYHEMGLL Y (SEQ ID NO: 21016). As indicated by SEQ ID NO: 21016, the restricted peptide HYHEMGLLY (SEQ ID NO: 21016) contains the S290L point mutation in the protein encoded by gene ACVR1.

[00506] For example, the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 25566 is an NF1 neoantigen carrying insertion or deletion mutation Y2285Tfs*5 (denoted as “NF1_p.Y2285Tfs*5”) resulting in an HLA-PEPTIDE target HLA-A* 11 :01_KGPDTTVKF (SEQ ID NO: 25566). As indicated by SEQ ID NO: 25566, the restricted peptide KGPDTTVKF (SEQ ID NO: 25566) contains the substitution Y2285T and subsequent sequence that is frameshifted from the normal reading frame of the NF1 gene, resulting in a stop codon in 5 amino acids.

[00507] For example, the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 22713 is a CDKN2A neoantigen carrying an in-frame sequence insertion T18_A19dup (denoted as “CDKN2A_p.T18_A19dup”), resulting in the HLA-PEPTIDE target HLA-A*68:01_

ATATAAARGR (SEQ ID NO: 22713). As indicated by SEQ ID NO: 22713, the restricted peptide ATATAAARGR (SEQ ID NO: 22713) contains an insertion of amino acids T and A at amino acid positions 18 and 19, and its surrounding sequence in the CDKN2A protein. [00508] For example, the HLA-PEPTIDE neoantigen designated as SEQ ID NO: 23233 is a CTNNB1 neoantigen carrying an in-frame sequence deletion S45del (denoted as “CTNNBl_p.S45del”), resulting in and HLA-PEPTIDE target HLA-A*03:01_TTTAPLSGK (SEQ ID NO: 23233). As indicated by SEQ ID NO: 23233, the restricted peptide TTTAPLSGK (SEQ ID NO: 23233) includes the deletion S45del and its surrounding sequence in the CTNNB1 gene.

SEQ ID NOs 29358- 29364

Table 8: VH/VL sequences of selected scFv clones that bind to RAS G12D HLA-

PEPTIDE neoantipen HLA-A* 11 :01_VVVGADGVGK (SEQ ID NO: 29366)

(“SNA30”)

Table 9: VH/VL sequences of selected scFv clones that bind to RAS G12V HLA-

PEPTIDE neoantipen HLA-A* 11 :01_VVVGAVGVGK (SEQ ID NO: 29368)

(“SNA6”)

Table 10: CDR sequences of selected scFv clones that bind to RAS G12D HLA-

PEPTIDE neoantipen HLA-A*11:01 VVVGADGVGK (SEQ ID NO: 29366) (“SNA30”)

Table 11: CDR sequences of selected scFv clones that bind to RAS G12V HLA-

PEPTIDE neoantipen HLA-A* 11 :01 _VVVGAVGVGK (SEQ ID NO: 29368)

(“SNA6”) References

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