Antigen binding proteins targeting shared antigens

Abstract

Antigen binding proteins that bind to HLA-PEPTIDE and HLA-PEPTOID targets are provided. Methods of identifying HLA-PEPTIDE targets are also provided, as well as methods of identifying one or more antigen binding proteins that bind to a given HLA-PEPTIDE target.SOLUTION: An isolated antigen binding protein (ABP) that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE target is provided, wherein the HLA-PEPTIDE target may comprise a particular HLA restriction peptide having a defined amino acid sequence complexed with a particular HLA subtype.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 611,403, filed December 28, 2017, and U.S. Provisional Application No. 62 / 756,508, filed November 6, 2018, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy has a creation date of December 28, 2018, is named 41174WO_CRF_sequencelisting.txt, and is 25,492,888 bytes in size. [Background technology]

[0003] The immune system employs two types of adaptive immune responses to confer antigen-specific protection against pathogens: humoral and cell-mediated responses, which involve the specific recognition of pathogen antigens via B and T lymphocytes, respectively.

[0004] T lymphocytes are antigen-specific effectors of cell-mediated immunity and therefore play a central role in the body's defense against diseases mediated by intracellular pathogens, such as viruses, intracellular bacteria, mycoplasma, and intracellular parasites, as well as against cancer cells by directly lysing affected cells. The specificity of T lymphocyte responses is conferred and activated through T cell receptors (TCRs) that bind to major histocompatibility complex (MHC) molecules on the surface of affected cells. T cell receptors are antigen-specific receptors clonally distributed on individual T lymphocytes, and their repertoire of antigen specificities is generated through a somatic gene rearrangement mechanism similar to that involved in the generation of antibody gene repertoires. T cell receptors comprise heterodimers of transmembrane molecules, with the major type consisting of a small subset of α-β and γ-δ polypeptide dimers. T lymphocyte receptor subunits comprise immunoglobulin-like variable and constant regions in the extracellular domain, a short hinge region containing cysteines that promote pairing of the α and β chains, a transmembrane region, and a short cytoplasmic region. Signaling elicited by the TCR is mediated indirectly through CD3-ζ (i.e., an associated multisubunit complex containing the signaling subunit).

[0005] T lymphocyte receptors do not normally recognize native antigens, but instead recognize complexes displayed on the cell surface. These complexes contain fragments of intracellularly processed antigens in association with major histocompatibility complex (MHC) molecules to present peptide antigens. Major histocompatibility complex genes are highly polymorphic among populations of a species, containing multiple common alleles for each individual gene. In humans, MHCs are called human leukocyte antigens (HLA).

[0006] Major histocompatibility complex class I molecules are expressed on the surface of nearly all nucleated cells in the body. They are dimeric molecules containing a transmembrane heavy chain with a peptide antigen-binding cleft and a smaller extracellular chain called β2-microglobulin. MHC class I molecules present peptides derived from the degradation of cytoplasmic proteins by the proteasome, a multiunit structure within the cytoplasm (Niedermann G., 2002. Curr Top Microbiol Immunol. 268:91-136 (Non-Patent Document 1); for bacterial antigen processing, see Wick MJ, and Ljunggren H G., 1999. Immunol Rev. 172:153-62 (Non-Patent Document 2)). The cleaved peptides are transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) and bind to the groove of the assembled MHC class I molecule. The resulting MHC / peptide complex is then transported to the plasma membrane, enabling antigen presentation to T lymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7 (Non-Patent Document 3); Yewdell J W. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8 (Non-Patent Document 4)). Alternatively, the cleaved peptides may be loaded onto MHC class I molecules in a TAP-independent manner, or may present extracellularly derived proteins through the process of cross-presentation. Therefore, a specific MHC / peptide complex presents a new protein structure on the cell surface, which may become a target for new antigen-binding proteins (e.g., antibodies or TCRs) once the identity of the complex's structure (peptide sequence and MHC subtype) is determined.

[0007] Tumor cells can express antigens, and thus present such antigens on the surface of tumor cells. Such tumor-associated antigens can be used to develop new immunotherapy reagents for specifically targeting tumor cells. For example, tumor-associated antigens can be used to identify therapeutic antigen-binding proteins, such as TCRs, antibodies, or antigen-binding fragments. Such tumor-associated antigens can also be used in pharmaceutical compositions, such as vaccines. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Niedermann G.,2002.Curr Top Microbiol Immunol.268:91-136 [Non-patent document 2] Wick MJ, and Ljunggren H G., 1999. Immunol Rev. 172:153-62 [Non-patent document 3] Yewdell J W.,2001.Trends Cell Biol.11:294-7 [Non-patent document 4] Yewdell J W. and Bennink J R.,2001.Curr Opin Immunol.13:13-8 Summary of the Invention

[0009] Provided herein is an isolated antigen binding protein (ABP) that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide being located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and the HLA class I molecule is of HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY, or the HLA class I molecule is of HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA, or the HLA class I molecule is of HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY, or the HLA class I molecule is of HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence HSEVGLPVY.

[0010] In some embodiments, the HLA-restricted peptide is about 5-15 amino acids in length. In some embodiments, the HLA-restricted peptide is about 8-12 amino acids in length. In some embodiments, the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY, or the HLA class I molecule is HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA, or the HLA class I molecule is HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence ASSLPTTMNY, or the HLA class I molecule is HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence HSEVGLPVY.

[0011] In some embodiments, the ABP comprises an antibody or an antigen-binding fragment thereof.

[0012] In some aspects of the ABP comprising the antibody or antigen-binding fragment thereof, the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY. In some embodiments, the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY.

[0013] In some embodiments, the ABP is: and a CDR-H3 comprising a sequence selected from TIFF2026004411000002.tif47156.

[0014] In some embodiments, the ABP is: and a CDR-L3 comprising a sequence selected from TIFF2026004411000003.tif26159.

[0015] In some embodiments, the ABP comprises CDR-H3 and CDR-L3 from an scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01.

[0016] In some embodiments, the ABP comprises all three heavy chain CDRs and all three light chain CDRs from an scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01.

[0017] In some embodiments, the ABP is: Contains a VH sequence selected from TIFF2026004411000004.tif194165TIFF2026004411000005.tif186165.

[0018] In some embodiments, the ABP is: Contains a VL sequence selected from TIFF2026004411000006.tif48165TIFF2026004411000007.tif209166.

[0019] In some embodiments, the ABP comprises the VH and VL sequences from scFvs designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, and G5R4-P4B01.

[0020] In some embodiments, the ABP binds to any one or more of amino acids 2-8 on the restriction peptide EVDPIGHVY.

[0021] In some embodiments of the ABP comprising the antibody or antigen-binding fragment thereof, the HLA class I molecule is of the HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA. In some embodiments of the ABP comprising the antibody or antigen-binding fragment thereof, the HLA class I molecule is of the HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA.

[0022] In some embodiments, the ABP is: and a CDR-H3 comprising a sequence selected from TIFF2026004411000008.tif47163.

[0023] In some embodiments, the ABP is: and a CDR-L3 comprising a sequence selected from TIFF2026004411000009.tif26157.

[0024] In some embodiments, the ABP comprises the CDR-H3 and CDR-L3 from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11.

[0025] In some embodiments, the ABP comprises all three heavy chain CDRs and all three light chain CDRs from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11.

[0026] In some embodiments, the ABP is: Contains a VH sequence selected from TIFF2026004411000010.tif33165, TIFF2026004411000011.tif245166, and TIFF2026004411000012.tif85165.

[0027] In some embodiments, the ABP is: Contains VL sequences selected from TIFF2026004411000013.tif150165TIFF2026004411000014.tif92165.

[0028] In some embodiments, the ABP comprises the VH and VL sequences from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11.

[0029] In some embodiments, the ABP binds to any one or more of amino acid positions 1 to 5 of the restriction peptide AIFPGAVPAA. In some embodiments, the ABP binds to one or both of amino acid positions 4 and 5 of the restriction peptide AIFPGAVPAA.

[0030] In some embodiments, the ABP binds to any one or more of amino acids 45-60 of HLA subtype A*02:01.

[0031] In some embodiments, the ABP binds to one or more of amino acids 56, 59, 60, 63, 64, 66, 67, 70, 73, 74, 132, 150-153, 155, 156, 158-160, 162-164, 166-168, 170, and 171 of the HLA subtype A*02:01.

[0032] In some embodiments of the ABP comprising the antibody or antigen-binding fragment thereof, the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments of the ABP comprising the antibody or antigen-binding fragment thereof, the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence ASSLPTTMNY.

[0033] In some embodiments, the ABP is: and a CDR-H3 comprising a sequence selected from TIFF2026004411000015.tif40151.

[0034] In some embodiments, the ABP is: and a CDR-L3 comprising a sequence selected from TIFF2026004411000016.tif26157.

[0035] In some embodiments, the ABP comprises the CDR-H3 and CDR-L3 from an scFv designated R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08.

[0036] In some embodiments, the ABP is R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A0 2, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08.

[0037] In some embodiments, the ABP is: It contains a VH sequence selected from TIFF2026004411000017.tif106166, TIFF2026004411000018.tif245165, and TIFF2026004411000019.tif26164.

[0038] In some embodiments, the ABP is: Contains a VL sequence selected from TIFF2026004411000020.tif209166TIFF2026004411000021.tif48166.

[0039] In some embodiments, the ABP comprises the VH and VL sequences from an scFv designated R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08.

[0040] In some embodiments, the ABP binds to any one or more of amino acids 4, 6, and 7 of the restriction peptide ASSLPTTMNY.

[0041] In some embodiments, the ABP binds to any one or more of amino acids 49-56 of HLA subtype A*01:01.

[0042] Also provided herein is an isolated antigen binding protein (ABP) that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 portion of the HLA class I molecule, and the HLA-PEPTIDE target is selected from Table A.

[0043] In some embodiments, the HLA-restricted peptide is about 5-15 amino acids in length. In some embodiments, the HLA-restricted peptide is about 8-12 amino acids in length.

[0044] In some embodiments, the ABP comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding protein is linked to a scaffold, optionally the scaffold comprises serum albumin or Fc, optionally the Fc is human and is an IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgD, IgE, or IgM isotype Fc. In some embodiments, the antigen-binding protein is linked to the scaffold via a linker, optionally the linker is a peptide linker, optionally the peptide linker is the hinge region of a human antibody. 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. In some embodiments, the antigen binding protein comprises one or more antibody complementarity determining regions (CDRs), optionally 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 antibody, a human antibody, or a chimeric antibody. In some embodiments, the antigen binding protein has multispecificity, optionally bispecificity. In some embodiments, the antigen binding protein binds to multiple antigens or multiple epitopes on a single antigen. 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 a human IgG class and subclass selected from IgG1, IgG4, IgG2, and IgG3. In some embodiments, the antigen binding protein comprises one or more modifications that increase half-life. In some embodiments, the antigen binding protein comprises a modified Fc, optionally wherein the modified Fc comprises one or more half-life enhancing mutations, and optionally wherein the one or more half-life enhancing mutations is YTE.

[0045] In some embodiments of the isolated ABP, the ABP comprises a T cell receptor (TCR) or an antigen-binding portion thereof. In some embodiments, the TCR or antigen-binding portion thereof comprises a TCR variable region. In some embodiments, the TCR or antigen-binding portion thereof comprises one or more TCR complementarity-determining regions (CDRs).

[0046] In some embodiments, the TCR is an alpha chain and a beta chain. In some embodiments, the TCR comprises a gamma chain and a delta chain.

[0047] In some embodiments, the antigen binding protein is part of a chimeric antigen receptor (CAR), which comprises 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 immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3) chain.

[0048] In some embodiments, the ABP further comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain, hi some embodiments, the transmembrane domain comprises the transmembrane portion of CD28.

[0049] In some embodiments, the ABP further comprises an intracellular signaling domain of a T cell costimulatory molecule, hi some embodiments, the T cell costimulatory molecule is CD28, 4-1BB, OX-40, ICOS, or any combination thereof.

[0050] In some embodiments of an ABP comprising a TCR or antigen-binding portion thereof, the HLA class I molecule is of HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments, the HLA class I molecule is of HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence ASSLPTTMNY. In some embodiments, the ABP comprises a TCR alpha CDR3 sequence selected from Table 15. In some embodiments, the ABP comprises a TCR beta CDR3 sequence selected from Table 15. In some embodiments, the ABP comprises an alpha CDR3 and a beta CDR3 sequence from any one of TCR clonotype ID#s: 1-344. In some embodiments, the ABP comprises a TCR alpha variable (TRAV) amino acid sequence, a TCR alpha binding (TRAJ) amino acid sequence, a TCR beta variable (TRBV) amino acid sequence, a TCR beta diversity (TRBD) amino acid sequence, and a TCR beta binding (TRBJ) amino acid sequence, wherein each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequence of any one of the TCR clonotypes selected from TCR clonotype ID#s: 1-344.

[0051] In some embodiments, the ABP comprises a TCR alpha constant (TRAC) amino acid sequence. In some embodiments, the ABP comprises a TCR beta constant (TRBC) amino acid sequence.

[0052] In some embodiments, the ABP comprises a TCRα VJ sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an αVJ sequence selected from Table 16. In some embodiments, the ABP comprises a TCRβ V(D)J sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a βV(D)J sequence selected from Table 16. In some embodiments, the ABP comprises a TCRα VJ amino acid sequence and a TCRβ V(D)J amino acid sequence, wherein each of the TCRα VJ and TCRβ V(D)J amino acid sequences is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding TCRα VJ and TCRβ V(D)J amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 1-344.

[0053] In some embodiments of an ABP comprising a TCR or antigen-binding portion thereof, the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence HSEVGLPVY. In some embodiments, the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence HSEVGLPVY.

[0054] In some embodiments, the ABP comprises a TCR alpha CDR3 sequence selected from Table 18. In some embodiments, the ABP comprises a TCR beta CDR3 sequence selected from Table 18. In some embodiments, the ABP comprises an alpha CDR3 and a beta CDR3 sequence from any one of TCR clonotype ID#s: 345-447. In some embodiments, the ABP comprises a TCR alpha variable (TRAV) amino acid sequence, a TCR alpha binding (TRAJ) amino acid sequence, a TCR beta variable (TRBV) amino acid sequence, a TCR beta diversity (TRBD) amino acid sequence, and a TCR beta binding (TRBJ) amino acid sequence, wherein each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 345-447. In some embodiments, the ABP comprises a TCR alpha constant (TRAC) amino acid sequence. In some embodiments, the ABP comprises a TCR beta constant (TRBC) amino acid sequence.

[0055] In some embodiments, the ABP comprises a TCRα VJ sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an αVJ sequence selected from Table 19. In some embodiments, the ABP comprises a TCRβ V(D)J sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a βV(D)J sequence selected from Table 19. In some embodiments, the ABP comprises a TCRα VJ amino acid sequence and a TCRβ V(D)J amino acid sequence, wherein each of the TCRα VJ and TCRβ V(D)J amino acid sequences is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding TCRα VJ and TCRβ V(D)J amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 345-447.

[0056] Also provided herein is an isolated HLA-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide being located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and the HLA-PEPTIDE target is selected from Table A.

[0057] In some embodiments, the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY, the HLA class I molecule is of the HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA, or the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments, the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY, the HLA class I molecule is of the HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA, or the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence ASSLPTTMNY.

[0058] In some embodiments, the HLA-restricted peptide is about 5-15 amino acids in length. In some embodiments, the HLA-restricted peptide is about 8-12 amino acids in length.

[0059] In some embodiments, the association of an HLA subtype with the restricted peptide stabilizes the non-covalent association of the β2 microglobulin subunit of the HLA subtype with the α subunit of the HLA subtype. In some embodiments, the stabilized association of the β2 microglobulin subunit of an HLA subtype with the α subunit of the HLA subtype is demonstrated by the conditional peptide exchange.

[0060] In some embodiments, the isolated HLA-PEPTIDE target further comprises an affinity tag. In some embodiments, the affinity tag is a biotin tag. In some embodiments, the isolated HLA-PEPTIDE target is complexed with a detectable label. In some embodiments, the detectable label comprises a β2 microglobulin binding molecule. In some embodiments, the β2 microglobulin binding molecule is a labeled antibody. In some embodiments, the labeled antibody is a fluorochrome-labeled antibody.

[0061] Also provided herein are compositions comprising the HLA-PEPTIDE targets described herein attached to a solid support. In some embodiments, the solid support comprises a bead, a well, a membrane, a tube, a column, a plate, sepharose, a magnetic bead, or a chip.

[0062] In some embodiments, the HLA-PEPTIDE target comprises a first member of an affinity binding pair and the solid support comprises a second member of the affinity binding pair, hi some embodiments, the first member is streptavidin and the second member is biotin.

[0063] Also provided herein is a reaction mixture comprising an isolated and purified α subunit of an HLA subtype from an HLA-PEPTIDE target listed in Table A, an isolated and purified β2-microglobulin subunit of an HLA subtype, a restriction peptide isolated and purified from the HLA-PEPTIDE target listed in Table A, and a reaction buffer.

[0064] Also provided herein is a reaction mixture comprising an isolated HLA-PEPTIDE target described herein and a plurality of T cells isolated from a human subject. In some embodiments, the T cells are CD8+ T cells.

[0065] Also provided herein is an isolated polynucleotide comprising a first nucleic acid sequence encoding an HLA-restricted peptide described herein, the first nucleic acid sequence operably linked to a promoter, and a second nucleic acid sequence encoding an HLA subtype described herein, wherein the second nucleic acid is operably linked to the same or a different promoter as the first nucleic acid sequence, and the encoded peptide and encoded HLA subtype form an HLA / peptide complex described herein.

[0066] Also provided herein are kits for expressing a stable HLA-PEPTIDE target described herein, the kit comprising: a first construct comprising a first nucleic acid sequence encoding an HLA-restricted peptide described herein, the first nucleic acid sequence operably linked to a promoter; and instructions for use in expressing the stable HLA-PEPTIDE complex. In some embodiments, the first construct further comprises a second nucleic acid sequence encoding an HLA subtype as defined herein. In some embodiments, the second nucleic acid sequence is operably linked to the same or a different promoter. In some embodiments, the kit further comprises a second construct comprising a second nucleic acid sequence encoding an HLA subtype as described herein. In some embodiments, one or both of the first and second constructs are lentiviral vector constructs.

[0067] Also provided herein are host cells comprising the heterologous HLA-PEPTIDE targets described herein. Also provided herein are host cells that express an HLA subtype defined by any one of the targets in Table A. Also provided herein are host cells comprising a polynucleotide encoding an HLA-restricted peptide described in Table A, e.g., a polynucleotide encoding an HLA-restricted peptide described herein.

[0068] In some embodiments, the host cell does not contain endogenous MHC. In some embodiments, the host cell contains exogenous HLA. In some embodiments, the host cell is a K562 or A375 cell.

[0069] In some embodiments, the host cells are cultured cells derived from a tumor cell line. In some embodiments, the tumor cell line expresses an HLA subtype defined by any one of the targets in Table A. In some embodiments, the tumor cell line expresses an HLA subtype defined by any one of the targets in Table A. For example, the tumor cell line may express the ABCB5 and HLA subtype HLA-C*16:01 genes as defined in target #1 of Table A. In some embodiments, the tumor cell line is selected from a database or catalog of tumor cell lines. The selection may be based on known expression of a gene target from any of the targets listed in Table A, or the selection may be based on known expression of an HLA subtype from any of the targets listed in Table A, or the selection may be based on known expression of a gene target and an HLA subtype from any of the targets listed in Table A. One exemplary catalog of tumor cell lines includes, for example, the American Type Culture Collection (ATCC). The ATCC is available at https: / / www.atcc.org / Products / Cells_and_Microorganisms / By_Disease__Model / Cancer / Tumor_Cell_Panels / Panels_by_Tissue_Type.aspx. Another exemplary catalog of tumor cell lines based on HLA type and HLA expression is described in Boegel, Sebastian et al. "A Catalog of HLA Type, HLA Expression, and Neo-Epitope Candidates in Human Cancer Cell Lines," Oncoimmunology 3.8(2014):e954893.PMC.Web.8 Oct. 2018, which is incorporated by reference in its entirety. 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, and NCI-H146.

[0070] Also provided herein is a cell culture system comprising a host cell as defined herein and a cell culture medium. In some embodiments, the host cell expresses an HLA subtype defined by any one of the targets in Table A, and the cell culture medium comprises a restriction peptide defined by the target in Table A. In some embodiments, the host cell is a K562 cell comprising an exogenous HLA, the exogenous HLA is an HLA subtype defined by any one of the targets in Table A, and the cell culture medium comprises a restriction peptide defined by the target in Table A.

[0071] In some embodiments of the ABP, the antigen binding protein binds to the HLA-PEPTIDE target through contact points with an HLA class I molecule and through contact points with an HLA-restricted peptide of the HLA-PEPTIDE target. In some embodiments of the ABP, binding of the ABP to the amino acid positions on the restricted peptide or HLA subtype, or to the contact points, or to residues that directly or indirectly affect binding of the HLA-PEPTIDE target to the ABP is determined by positional scanning, hydrogen-deuterium exchange, or protein crystallography.

[0072] In some embodiments, the ABP may be for use as a pharmaceutical. In some embodiments, the ABP may be for use in treating cancer. Optionally, the cancer expresses or is predicted to express an HLA-PEPTIDE target. In some embodiments, the ABP may be for use in treating cancer. The cancer is selected from solid tumors and hematological tumors.

[0073] Also provided herein are ABPs that are conservatively modified variants of the ABPs described herein. Also provided herein are antigen binding proteins (ABPs) that compete for binding with the antigen binding proteins described herein. Also provided herein are antigen binding proteins (ABPs) that bind to the same HLA-PEPTIDE epitope as the antigen binding proteins described herein.

[0074] Also provided herein are engineered cells that express a receptor, comprising an antigen binding protein described herein. In some embodiments, the engineered cells are T cells, optionally cytotoxic T cells (CTLs). In some embodiments of the engineered cells, the antigen binding protein is expressed from a heterologous promoter.

[0075] Also provided herein is an isolated polynucleotide or series of polynucleotides that encodes an antigen binding protein or antigen binding portion thereof described herein.

[0076] Also provided herein is an isolated polynucleotide or series of polynucleotides that encode the HLA / peptide targets described herein.

[0077] Also provided herein is a vector or a series of vectors that include a polynucleotide or series of polynucleotides described herein.

[0078] Also provided herein is a host cell comprising a polynucleotide or set of polynucleotides described herein, or a vector or set of vectors described herein, optionally wherein the host cell is a CHO or HEK293, or optionally wherein the host cell is a T cell.

[0079] Also provided herein is a method comprising expressing the antigen binding protein using a host cell as described herein and isolating the expressed antigen binding protein.

[0080] Also provided herein are pharmaceutical compositions comprising an antigen binding protein described herein and a pharmaceutically acceptable excipient.

[0081] Also provided herein are methods of treating cancer in a subject, comprising administering to the subject an effective amount of a preceding antigen binding protein described herein or a pharmaceutical composition described herein, optionally wherein the cancer is selected from a solid tumor and a hematological tumor. In some embodiments, the cancer expresses or is predicted to express an HLA-PEPTIDE target.

[0082] Also provided herein are kits comprising an antigen binding protein described herein or a pharmaceutical composition described herein and instructions for use.

[0083] Also provided herein are compositions comprising at least one HLA-PEPTIDE target described herein and an adjuvant.

[0084] Also provided herein are compositions comprising at least one HLA-PEPTIDE target described herein and a pharmaceutically acceptable excipient.

[0085] Also provided herein are compositions comprising an amino acid sequence comprising at least one HLA-PEPTIDE-targeted polypeptide disclosed in Table A, optionally wherein the amino acid sequence consists essentially of or consists of the polypeptide.

[0086] Also provided herein is a virus comprising an isolated polynucleotide or set of polynucleotides described herein. In some embodiments, the virus is a filamentous phage.

[0087] Also provided herein is a yeast cell comprising an isolated polynucleotide or sequence of polynucleotides described herein.

[0088] Also provided herein is a method for identifying the antigen binding proteins described herein, the method comprising providing at least one HLA-PEPTIDE target listed in Table A and allowing the at least one target to bind to the antigen binding protein, thereby identifying the antigen binding protein.

[0089] In some embodiments, the antigen binding protein is present in a phage display library comprising a plurality of individual antigen binding proteins, hi some embodiments, the phage display library is substantially free of antigen binding proteins that non-specifically bind to the HLA of the HLA-PEPTIDE target.

[0090] In some embodiments, the antigen binding protein is present within a TCR library comprising a plurality of individual TCRs or antigen binding fragments thereof.

[0091] In some embodiments, the binding step is carried out more than once, optionally at least three times.

[0092] In some embodiments, the methods further comprise contacting the antigen binding protein with one or more peptide-HLA complexes distinct from the HLA-PEPTIDE target to determine whether the antigen binding protein selectively binds to the HLA-PEPTIDE target, optionally wherein selectivity is determined by measuring the binding affinity of the antigen binding protein to a soluble target HLA-PEPTIDE complex relative to the binding affinity of a soluble HLA-PEPTIDE complex distinct from the target complex, and optionally wherein selectivity is determined by measuring the binding affinity of the antigen binding protein to a target HLA-PEPTIDE complex expressed on the surface of one or more cells relative to a HLA-PEPTIDE complex distinct from the target complex expressed on the surface of one or more cells.

[0093] Also provided herein is a method for identifying an antigen binding protein described herein, comprising obtaining at least one HLA-PEPTIDE target listed in Table A, administering the HLA-PEPTIDE target, optionally in combination with an adjuvant, to a subject, and isolating the antigen binding protein from the subject.

[0094] In some embodiments, isolating the antigen binding protein comprises screening the serum of the subject to identify the antigen binding protein.

[0095] In some embodiments, the methods further comprise contacting the antigen binding protein with one or more peptide-HLA complexes distinct from the HLA-PEPTIDE target to determine whether the antigen binding protein selectively binds to the HLA-PEPTIDE target, wherein optionally, selectivity is determined by measuring the binding affinity of the antigen binding protein to a soluble target HLA-PEPTIDE complex relative to the binding affinity of a soluble HLA-PEPTIDE complex distinct from the target complex, and optionally, selectivity is determined by measuring the binding affinity of the antigen binding protein to a target HLA-PEPTIDE complex expressed on the surface of one or more cells relative to a HLA-PEPTIDE complex distinct from the target complex expressed on the surface of one or more cells.

[0096] In some embodiments, the subject is a mouse, rabbit, or llama.

[0097] In some embodiments, isolating the antigen binding protein comprises isolating B cells from the subject that express the antigen binding protein, and optionally directly cloning a sequence encoding the antigen binding protein from the isolated B cells. In some embodiments, the method further comprises generating a hybridoma using the B cells. In some embodiments, the method further comprises cloning CDRs from the B cells. In some embodiments, the method further comprises immortalizing the B cells, optionally via Epstein-Barr virus (EBV) transformation. In some embodiments, the method further comprises generating a library comprising the antigen binding protein of B cells, the library optionally being phage display or yeast display.

[0098] In some embodiments, the method further comprises humanizing the antigen binding protein.

[0099] Also provided herein is a method for identifying an antigen binding protein described herein, comprising obtaining a cell comprising said antigen binding protein, contacting said cell with an HLA multimer comprising at least one HLA-PEPTIDE target listed in Table A, and identifying said antigen binding protein via binding between said HLA multimer and said antigen binding protein.

[0100] Also provided herein is a method for identifying an antigen binding protein described herein, comprising obtaining one or more cells comprising the antigen binding protein, activating the one or more cells with at least one HLA-PEPTIDE target listed in Table A presented on a natural or artificial antigen-presenting cell (APC), and identifying the antigen binding protein by selecting one or more cells activated by interaction with at least one HLA-PEPTIDE target listed in Table A. In some embodiments, the cells are T cells, optionally CTLs. In some embodiments, the method further comprises isolating the cells, optionally using flow cytometry, magnetic separation, or single-cell separation. In some embodiments, the method further comprises sequencing the antigen binding protein.

[0101] Also provided herein is a method for identifying an antigen binding protein described herein, comprising providing at least one HLA-PEPTIDE target listed in Table A and using the target to identify the antigen binding protein. [The present invention 1001] 1. An isolated antigen binding protein (ABP) that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide being located in the peptide-binding groove of an α1 / α2 heterodimer portion of the HLA class I molecule; and a. the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY; b. the HLA class I molecule is of HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA; c. the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY; or d. The HLA class I molecule is of HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence HSEVGLPVY; The isolated ABP. [The present invention 1002] 1001. The isolated ABP of the present invention, wherein said HLA-restricted peptide is about 5-15 amino acids in length. [The present invention 1003] 1002. The isolated ABP of the present invention, wherein said HLA-restricted peptide is about 8 to 12 amino acids in length. [The present invention 1004] a. the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY; b. the HLA class I molecule is of HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA; c. the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide consists of the sequence ASSLPTTMNY; or d. The HLA class I molecule is of the HLA subtype A*01:01, and the HLA-restricted peptide consists of the sequence HSEVGLPVY. An isolated ABP according to any one of 1001 to 1003 of the present invention. [The present invention 1005] Any of the preceding isolated ABPs of the invention, wherein said ABP comprises an antibody or an antigen-binding fragment thereof. [The present invention 1006] 1005. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype B*35:01, and said HLA-restricted peptide comprises said sequence EVDPIGHVY. [The present invention 1007] 1006. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype B*35:01, and said HLA-restricted peptide consists of said sequence EVDPIGHVY. [The present invention 1008] The ABP may be: The isolated ABP of the present invention 1006 or 1007, comprising a CDR-H3 comprising a sequence selected from TIFF2026004411000022.tif48163. [The present invention 1009] The ABP may be: An isolated ABP of any of 1006 to 1008 of the present invention, comprising a CDR-L3 comprising a sequence selected from TIFF2026004411000023.tif33157. [The present invention 1010] The isolated ABP of any of claims 1006 to 1009, wherein the ABP comprises the CDR-H3 and the CDR-L3 from an scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01. [The present invention 1011] 10. The isolated ABP of any of claims 1006 to 1010, wherein the ABP comprises all three heavy chain CDRs and all three light chain CDRs from an scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01. [The present invention 1012] The ABP may be: An isolated ABP of any of 1006 to 1011 of the present invention, comprising a VH sequence selected from TIFF2026004411000024.tif231158TIFF2026004411000025.tif158158. [The present invention 1013] The ABP may be: An isolated ABP of any of claims 1006 to 1012 of the present invention, comprising a VL sequence selected from TIFF2026004411000026.tif55157TIFF2026004411000027.tif201164. [The present invention 1014] The isolated ABP of any of claims 1006 to 1013, wherein the ABP comprises the VH sequence and the VL sequence from scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, and G5R4-P4B01. [The present invention 1015] 15. The isolated ABP of any of claims 1006 to 1014, wherein the ABP binds to any one or more of amino acids 2 to 8 on the restriction peptide EVDPIGHVY. [The present invention 1016] 1005. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*02:01, and said HLA-restricted peptide comprises said sequence AIFPGAVPAA. [The present invention 1017] 1016. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*02:01, and said HLA-restricted peptide consists of said sequence AIFPGAVPAA. [The present invention 1018] The ABP may be: The isolated ABP of the present invention 1016 or 1017, comprising a CDR-H3 comprising a sequence selected from TIFF2026004411000028.tif48152. [The present invention 1019] The ABP may be: An isolated ABP of any of 1016 to 1018 of the present invention, comprising a CDR-L3 comprising a sequence selected from TIFF2026004411000029.tif33157. [The present invention 1020] The isolated ABP of any of claims 1016 to 1019, wherein the ABP comprises the CDR-H3 and the CDR-L3 from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. [The present invention 1021] 10. The isolated ABP of any of claims 1016 to 1020, wherein the ABP comprises all three heavy chain CDRs and all three light chain CDRs from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. [The present invention 1022] The ABP may be: An isolated ABP of any of 1016 to 1021 of the present invention, comprising a VH sequence selected from TIFF2026004411000030.tif201158TIFF2026004411000031.tif165158. [The present invention 1023] The ABP may be: An isolated ABP of any of 1016 to 1022 of the present invention, comprising a VL sequence selected from TIFF2026004411000032.tif48157TIFF2026004411000033.tif194158. [The present invention 1024] The isolated ABP of any of claims 1016 to 1023, wherein the ABP comprises the VH sequence and the VL sequence from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. [The present invention 1025] 10. The isolated ABP of any of claims 1016 to 1024, wherein the ABP binds to any one or more of amino acid positions 1 to 5 of the restriction peptide AIFPGAVPAA. [The present invention 1026] 1025. An isolated ABP of the present invention, wherein said ABP binds to one or both of amino acid positions 4 and 5 of said restriction peptide AIFPGAVPAA. [The present invention 1027] 1016. The isolated ABP of any one of claims 1016 to 1026, wherein the ABP binds to any one or more of amino acids 45 to 60 of HLA subtype A*02:01. [The present invention 1028] The isolated ABP of any of 1016 to 1027 of the present invention, wherein the ABP binds to one or more of amino acids at positions 56, 59, 60, 63, 64, 66, 67, 70, 73, 74, 132, 150 to 153, 155, 156, 158 to 160, 162 to 164, 166 to 168, 170, and 171 of HLA subtype A*02:01. [The present invention 1029] 1005. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*01:01, and said HLA-restricted peptide comprises said sequence ASSLPTTMNY. [The present invention 1030] 1029. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*01:01, and said HLA-restricted peptide consists of said sequence ASSLPTTMNY. [The present invention 1031] The ABP may be: 1029 or 1030, an isolated ABP of the present invention, comprising a CDR-H3 comprising a sequence selected from TIFF2026004411000034.tif47146. [The present invention 1032] The ABP may be: An isolated ABP of any of 1029 to 1031 of the present invention, comprising a CDR-L3 comprising a sequence selected from TIFF2026004411000035.tif33155. [The present invention 1033] The ABP is R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G The isolated ABP of any of claims 1029 to 1032, comprising the CDR-H3 and the CDR-L3 from an scFv designated R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08. [The present invention 1034] The ABP is R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10 -An isolated ABP of any of claims 1029 to 1033, comprising all three heavy chain CDRs and all three light chain CDRs from an scFv designated R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08. [This invention 1035] The ABP may be: An isolated ABP of any of 1029 to 1034 of the present invention, comprising a VH sequence selected from TIFF2026004411000036.tif158158 and TIFF2026004411000037.tif231158. [The present invention 1036] The ABP may be: An isolated ABP of any of 1029 to 1035 of the present invention, comprising a VL sequence selected from TIFF2026004411000038.tif216158TIFF2026004411000039.tif41164. [This invention 1037] The ABP is R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R An isolated ABP of any of claims 1029 to 1036, comprising the VH sequence and the VL sequence from an scFv designated R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08. [The present invention 1038] The isolated ABP of any one of 1029 to 1037, wherein the ABP binds to any one or more of amino acids at positions 4, 6, and 7 of the restriction peptide ASSLPTTMNY. [This invention 1039] 1038. The isolated ABP of any of claims 1029 to 1038, wherein said ABP binds to any one or more of amino acids 49 to 56 of HLA subtype A*01:01. [The present invention 1040] 1. An isolated antigen binding protein (ABP) that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide being located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and the HLA-PEPTIDE target is selected from Table A. [The present invention 1041] 1040. The isolated ABP of the present invention, wherein said HLA-restricted peptide is about 5-15 amino acids in length. [The present invention 1042] 1041. The isolated ABP of the present invention, wherein said HLA-restricted peptide is about 8 to 12 amino acids in length. [This invention 1043] 1043. The isolated ABP of any of claims 1040 to 1042, wherein the ABP comprises an antibody or an antigen-binding fragment thereof. [This invention 1044] Any of the antigen binding proteins of the present invention, wherein the antigen binding protein is linked to a scaffold, and optionally the scaffold comprises serum albumin or Fc, and optionally the Fc is human and is an IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgD, IgE, or IgM isotype Fc. [This invention 1045] 10. Any of the antigen binding proteins of the invention, wherein the antigen binding protein is linked to the scaffold via a linker, optionally said linker is a peptide linker, optionally said peptide linker is a hinge region of a human antibody. [The present invention 1046] Any of the antigen-binding proteins of the present invention, comprising 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. [This invention 1047] Any of the antigen-binding proteins of the present invention comprising an scFv fragment. [This invention 1048] Any of the antigen binding proteins of the invention described above, comprising one or more antibody complementarity determining regions (CDRs), optionally six antibody CDRs. [This invention 1049] Any of the antigen-binding proteins of the invention, including antibodies. [The present invention 1050] Any of the antigen-binding proteins of the present invention that are monoclonal antibodies. [This invention 1051] Any of the antigen-binding proteins of the present invention which are humanized antibodies, human antibodies, or chimeric antibodies. [This invention 1052] Any of the antigen-binding proteins of the invention which have multispecificity, optionally bispecificity. [This invention 1053] Any of the antigen-binding proteins of the invention which bind to multiple antigens or multiple epitopes on a single antigen. [This invention 1054] Any of the antigen-binding proteins of the present invention, comprising a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. [This invention 1055] Any of the antigen-binding proteins of the present invention described above, comprising a heavy chain constant region of human IgG class and subclass selected from IgG1, IgG4, IgG2, and IgG3. [The present invention 1056] Any of the antigen binding proteins of the invention described above, which comprise a modification that increases its half-life. [This invention 1057] Any of the aforementioned antigen binding proteins of the invention, wherein said antigen binding protein comprises a modified Fc, optionally said modified Fc comprising one or more half-life increasing mutations, optionally wherein said one or more half-life increasing mutations is YTE. [This invention 1058] Any of the preceding isolated ABPs of the present invention, wherein said ABP comprises a T cell receptor (TCR) or an antigen-binding portion thereof. [This invention 1059] 1058. The antigen-binding protein of the present invention, wherein said TCR or antigen-binding portion thereof comprises a TCR variable region. [The present invention 1060] 1058. The antigen binding protein of claim 1059, wherein said TCR or antigen binding portion thereof comprises one or more TCR complementarity determining regions (CDRs). [This invention 1061] The antigen-binding protein of any of claims 1058 to 1060, wherein the TCR comprises an α chain and a β chain. [This invention 1062] The antigen-binding protein of any of claims 1058 to 1061, wherein the TCR comprises a γ chain and a δ chain. [This invention 1063] Any of the antigen binding proteins of the present invention, wherein the antigen binding protein is part of a chimeric antigen receptor (CAR) comprising an extracellular portion comprising the antigen binding protein and an intracellular signaling domain. [This invention 1064] 1063. The antigen-binding protein of the present invention, wherein the antigen-binding protein comprises an scFv and the intracellular signaling domain comprises an ITAM. [This invention 1065] 1063 or 1064, an antigen-binding protein of the present invention, wherein the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3) chain. [The present invention 1066] The antigen-binding protein of any of claims 1063 to 1065, further comprising a transmembrane domain linking said extracellular domain and said intracellular signaling domain. [This invention 1067] 1066. The antigen-binding protein of the present invention, wherein said transmembrane domain comprises the transmembrane portion of CD28. [The present invention 1068] The antigen-binding protein of any one of 1063 to 1067, further comprising an intracellular signaling domain of a T cell costimulatory molecule. [The present invention 1069] 1068. The antigen binding protein of the present invention, wherein said T cell costimulatory molecule is CD28, 4-1BB, OX-40, ICOS, or any combination thereof. [The present invention 1070] 1069. The isolated ABP of any one of claims 1058 to 1069, wherein the HLA class I molecule is of the HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. [This invention 1071] 1070. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*01:01, and said HLA-restricted peptide consists of said sequence ASSLPTTMNY. [This invention 1072] 1072. The isolated ABP of claim 1070 or 1071, wherein said ABP comprises a TCRα CDR3 sequence selected from Table 15. [This invention 1073] The isolated ABP of any of claims 1070 to 1072, wherein the ABP comprises a TCRβ CDR3 sequence selected from Table 15. [This invention 1074] 1074. The isolated ABP of any of claims 1070 to 1073, wherein said ABP comprises αCDR3 and βCDR3 sequences from any one of TCR clonotype ID#: 1 to 344. [This invention 1075] The isolated ABP of any of claims 1070 to 1074, wherein the ABP comprises a TCR alpha variable (TRAV) amino acid sequence, a TCR alpha binding (TRAJ) amino acid sequence, a TCR beta variable (TRBV) amino acid sequence, a TCR beta diversity (TRBD) amino acid sequence, and a TCR beta binding (TRBJ) amino acid sequence, and each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences of any one of TCR clonotypes selected from TCR clonotype ID#s: 1 to 344. [This invention 1076] 1070 to 1075. The isolated ABP of any of claims 1070 to 1075, wherein the ABP comprises a TCR alpha constant (TRAC) amino acid sequence. [This invention 1077] 1076. The isolated ABP of any of claims 1070 to 1076, wherein the ABP comprises a TCRβ constant (TRBC) amino acid sequence. [This invention 1078] The isolated ABP of any of claims 1070 to 1077, wherein the ABP comprises a TCRαVJ sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an αVJ sequence selected from Table 16. [This invention 1079] The isolated ABP of any of claims 1070 to 1078, wherein the ABP comprises a TCRβ V(D)J sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a β V(D)J sequence selected from Table 16. [The present invention 1080] An isolated ABP of any of the present inventions 1070 to 1079, wherein the ABP comprises a TCRαVJ amino acid sequence and a TCRβV(D)J amino acid sequence, and each of the TCRαVJ and TCRβV(D)J amino acid sequences is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding TCRαVJ and TCRβV(D)J amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 1 to 344. [This invention 1081] 1069. The isolated ABP of any of claims 1058 to 1069, wherein said HLA class I molecule is of HLA subtype A*01:01 and said HLA-restricted peptide comprises the sequence HSEVGLPVY. [This invention 1082] 1081. The isolated ABP of the present invention, wherein said HLA class I molecule is of HLA subtype A*01:01, and said HLA-restricted peptide consists of said sequence HSEVGLPVY. [This invention 1083] The isolated ABP of claim 1081 or 1082, wherein said ABP comprises a TCRα CDR3 sequence selected from Table 18. [This invention 1084] The isolated ABP of any of claims 1081 to 1083, wherein the ABP comprises a TCRβ CDR3 sequence selected from Table 18. [This invention 1085] 108. The isolated ABP of any one of claims 1081 to 1084, wherein the ABP comprises αCDR3 and βCDR3 sequences from any one of TCR clonotype ID#s: 345 to 447. [The present invention 1086] The isolated ABP of any of claims 1081 to 1085, wherein the ABP comprises a TCR alpha variable (TRAV) amino acid sequence, a TCR alpha binding (TRAJ) amino acid sequence, a TCR beta variable (TRBV) amino acid sequence, a TCR beta diversity (TRBD) amino acid sequence, and a TCR beta binding (TRBJ) amino acid sequence, and each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 345 to 447. [This invention 1087] The isolated ABP of any of 1081 to 1086 of the present invention, wherein the ABP comprises a TCR alpha constant (TRAC) amino acid sequence. [This invention 1088] The isolated ABP of any of 1081 to 1087 of the present invention, wherein the ABP comprises a TCRβ constant (TRBC) amino acid sequence. [This invention 1089] The isolated ABP of any of claims 1081 to 1088, wherein the ABP comprises a TCRαVJ sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an αVJ sequence selected from Table 19. [The present invention 1090] The isolated ABP of any of claims 1081 to 1089, wherein the ABP comprises a TCRβ V(D)J sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a β V(D)J sequence selected from Table 19. [This invention 1091] An isolated ABP of any of the present inventions 1081 to 1090, wherein the ABP comprises a TCRαVJ amino acid sequence and a TCRβV(D)J amino acid sequence, and each of the TCRαVJ and TCRβV(D)J amino acid sequences is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding TCRαVJ and TCRβV(D)J amino acid sequences of any one of the TCR clonotypes selected from TCR clonotype ID#s: 345 to 447. [This invention 1092] 1. An isolated HLA-PEPTIDE target, the HLA-PEPTIDE target comprising an HLA-restricted peptide complexed with an HLA class I molecule, the HLA-restricted peptide being located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and the HLA-PEPTIDE target being selected from Table A. [This invention 1093] a. the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY; b. the HLA class I molecule is of HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA; or the HLA class I molecule is of HLA subtype A*01:01, and the HLA-restricted peptide comprises the sequence ASSLPTTMNY; An isolated HLA-PEPTIDE target of the present invention 1092. [This invention 1094] a. the HLA class I molecule is of the HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY; b. the HLA class I molecule is of the HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA; or c. the HLA class I molecule is of the HLA subtype A*01:01, and the HLA-restricted peptide consists of the sequence ASSLPTTMNY; An isolated HLA-PEPTIDE target of the present invention 1093. [This invention 1095] 1092-1094. The isolated HLA-PEPTIDE target of any of claims 1092-1094, wherein said HLA-restricted peptide is about 5-15 amino acids in length. [This invention 1096] 1092-1095. The isolated HLA-PEPTIDE target of any of claims 1092-1095, wherein said HLA-restricted peptide is about 8-12 amino acids in length. [This invention 1097] 1096. The isolated HLA-PEPTIDE target of any of claims 1092 to 1096, wherein association of said HLA subtype with said restriction peptide stabilizes a non-covalent association between a β2 microglobulin subunit of said HLA subtype and an α subunit of said HLA subtype. [This invention 1098] 1097. An isolated HLA-PEPTIDE target of the present invention, wherein the stabilized association between said β2 microglobulin subunit of said HLA subtype and said α subunit of said HLA subtype is demonstrated by conditional peptide exchange. [This invention 1099] The isolated HLA-PEPTIDE target of any of the preceding inventions, further comprising an affinity tag. [The present invention 1100] 1099. The isolated HLA-PEPTIDE target of the present invention, wherein said affinity tag is a biotin tag. [The present invention 1101] Any of the foregoing isolated HLA-PEPTIDE targets of the present invention complexed with a detectable label. [The present invention 1102] 1101. The isolated HLA-PEPTIDE target of this invention, wherein said detectable label comprises a β2 microglobulin binding molecule. [The present invention 1103] 1102. The isolated HLA-PEPTIDE target of claim 1102, wherein said β2 microglobulin binding molecule is a labeled antibody. [The present invention 1104] 1103. The isolated HLA-PEPTIDE target of the present invention, wherein said labeled antibody is a fluorochrome-labeled antibody. [This invention 1105] A composition comprising any of the preceding HLA-PEPTIDE targets of the invention attached to a solid support. [The present invention 1106] 1105. The composition of claim 1105, wherein said solid support comprises a bead, a well, a membrane, a tube, a column, a plate, sepharose, a magnetic bead, or a chip. [This invention 1107] 1107. The composition of any one of claims 1105 to 1106, wherein said HLA-PEPTIDE target comprises a first member of an affinity binding pair and said solid support comprises a second member of said affinity binding pair. [This invention 1108] 1107. The composition of claim 1107, wherein said first member is streptavidin and said second member is biotin. [This invention 1109] a. Isolated and purified alpha subunits of HLA subtypes from the HLA-PEPTIDE targets listed in Table A; a. isolated and purified β2-microglobulin subunits of said HLA subtypes; b. Restricted peptides isolated and purified from the HLA-PEPTIDE targets listed in Table A; c. Reaction buffer and A reaction mixture comprising: [The present invention 1110] a. any isolated HLA-PEPTIDE target of the preceding invention; b. A plurality of T cells isolated from a human subject; A reaction mixture comprising: [The present invention 1111] 1110. The reaction mixture of claim 1110, wherein said T cells are CD8+ T cells. [The present invention 1112] A first nucleic acid sequence encoding an HLA-restricted peptide as defined in any one of claims 1092 to 1094 of the present invention, said first nucleic acid sequence being operably linked to a promoter; a second nucleic acid sequence encoding an HLA subtype defined in any one of claims 1092 to 1094 of the present invention; wherein the second nucleic acid is operably linked to the promoter that is the same as or different from the promoter of the first nucleic acid sequence, and the encoded peptide and the encoded HLA subtype form an HLA / peptide complex as defined in any one of claims 1092 to 1094 of the present invention. [The present invention 1113] A kit for expressing a stable HLA-PEPTIDE target, comprising: a first construct comprising a first nucleic acid sequence encoding an HLA-restricted peptide as defined in any one of claims 1092 to 1094 of the present invention, operably linked to a promoter; Instructions for use in expressing stable HLA-PEPTIDE complexes The kit comprises: [This invention 1114] The kit of present invention 1113, wherein the first construct further comprises a second nucleic acid sequence encoding an HLA subtype defined in any one of present inventions 1092 to 1094. [This invention 1115] 1114. The kit of claim 1114, wherein said second nucleic acid sequence is operably linked to the same promoter or a different promoter. [The present invention 1116] The kit of invention 1113, further comprising a second construct comprising a second nucleic acid sequence encoding an HLA subtype as defined in any one of inventions 1092 to 1094. [This invention 1117] The kit of any one of claims 1113 to 1116, wherein one or both of the first and second constructs is a lentiviral vector construct. [This invention 1118] A host cell comprising any one of the heterologous HLA-PEPTIDE targets of the present invention 1092 to 1094. [This invention 1119] A host cell expressing an HLA subtype defined by any one of the targets in Table A. [The present invention 1120] A host cell comprising a polynucleotide encoding an HLA-restricted peptide listed in Table A, for example, a polynucleotide encoding any one of HLA-restricted peptides 1092 to 1094 of the present invention. [This invention 1121] A host cell of the present invention 1120 that does not contain endogenous MHC. [This invention 1122] 1121. A host cell of the present invention comprising an exogenous HLA. [This invention 1123] The host cell of the present invention 1122, which is a K562 or A375 cell. [This invention 1124] The host cell of any of the preceding inventions, which is a cultured cell derived from a tumor cell line. [This invention 1125] The host cell of the present invention 1124, wherein said tumor cell line expresses an HLA subtype defined by any one of the targets in Table A. [Invention 1126] The host cell of the present invention 1124, wherein 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, and NCI-H146. [This invention 1127] a. any of the preceding host cells of the invention; b. Cell culture medium and A cell culture system comprising: [This invention 1128] 1127. The cell culture system of claim 1127, wherein said host cells express an HLA subtype defined by any one of the targets in Table A, and said cell culture medium comprises a restriction peptide defined by said target in Table A. [This invention 1129] 1127. The host cell of claim 1127, wherein the host cell is a K562 cell comprising an exogenous HLA, the exogenous HLA being an HLA subtype defined by any one of the targets in Table A, and the cell culture medium comprises a restriction peptide defined by the target in Table A. [The present invention 1130] Any of the ABPs of the invention, wherein the antigen binding protein binds to the HLA-PEPTIDE target through contact points with the HLA class I molecule and through contact points with the HLA-restricted peptide of the HLA-PEPTIDE target. [This invention 1131] 1039, or 1130, wherein the binding of said ABP to said amino acid position on said restricted peptide or HLA subtype, or to said contact point, is determined by positional scanning, hydrogen-deuterium exchange, or protein crystallography. [This invention 1132] Any of the antigen-binding proteins of the present invention described above for use as a pharmaceutical. [This invention 1133] 2. Any of the antigen binding proteins of the present invention for use in the treatment of cancer, optionally wherein said cancer expresses or is predicted to express an HLA-PEPTIDE target. [This invention 1134] 10. The antigen-binding protein of any of the preceding claims for use in treating cancer, wherein said cancer is selected from a solid tumor and a haematological tumor. [This invention 1135] An ABP that is a conservatively modified variant of any of the preceding ABPs of the invention. [This invention 1136] An antigen-binding protein (ABP) that competes for binding with any of the antigen-binding proteins of the present invention. [This invention 1137] An antigen binding protein (ABP) that binds to the same HLA-PEPTIDE epitope as any of the antigen binding proteins of the present invention. [This invention 1138] An engineered cell expressing a receptor comprising any of the preceding antigen binding proteins of the invention. [This invention 1139] The engineered cells of the present invention 1138 are T cells, optionally cytotoxic T cells (CTLs). [This invention 1140] 1139. The engineered cell of claim 1138 or 1139, wherein said antigen binding protein is expressed from a heterologous promoter. [This invention 1141] An isolated polynucleotide or series of polynucleotides encoding any of the antigen-binding proteins or antigen-binding portions thereof of the present invention. [This invention 1142] An isolated polynucleotide or series of polynucleotides encoding any of the HLA / peptide targets of the present invention. [This invention 1143] A vector or a series of vectors comprising a polynucleotide or a series of polynucleotides of the present invention 1141 or 1142. [This invention 1144] A host cell comprising any of the preceding polynucleotides or series of polynucleotides of the invention or the vector or series of vectors of the invention 1143, optionally wherein the host cell is a CHO or HEK293, or optionally wherein the host cell is a T cell. [Invention 1145] 1144。 A method of producing an antigen binding protein, the method comprising expressing said antigen binding protein using a host cell of the present invention 1144, and isolating said expressed antigen binding protein. [Invention 1146] A pharmaceutical composition comprising any of the preceding antigen binding proteins of the invention and a pharmaceutically acceptable excipient. [This invention 1147] A method of treating cancer in a subject, comprising administering to said subject an effective amount of any of the preceding antigen binding proteins of the invention or pharmaceutical composition of invention 1146, optionally wherein said cancer is selected from a solid tumor and a hematological tumor. [This invention 1148] 1147. The method of claim 1147, wherein said cancer expresses or is predicted to express an HLA-PEPTIDE target. [This invention 1149] A kit comprising any of the preceding antigen binding proteins of the invention or pharmaceutical compositions of the invention 1146 and instructions for use. [This invention 1150] A composition comprising at least one HLA-PEPTIDE target of the present invention 1092 and an adjuvant. [This invention 1151] A composition comprising at least one HLA-PEPTIDE target of the present invention 1092 and a pharmaceutically acceptable excipient. [This invention 1152] A composition comprising an amino acid sequence comprising at least one HLA-PEPTIDE-targeted polypeptide disclosed in Table A, optionally wherein said amino acid sequence consists essentially of or consists of said polypeptide. [This invention 1153] A virus comprising any of the preceding isolated polynucleotides or sequence of polynucleotides of the invention. [This invention 1154] The virus of the present invention 1153, which is a filamentous phage. [Invention 1155] A yeast cell comprising any of the preceding isolated polynucleotides or sequence of polynucleotides of the invention. [Invention 1156] A method of identifying an antigen binding protein of any of the preceding inventions, comprising providing at least one HLA-PEPTIDE target listed in Table A and allowing said at least one target to bind to said antigen binding protein, thereby identifying said antigen binding protein. [This invention 1157] 1156. The method of claim 1156, wherein said antigen binding protein is present in a phage display library comprising a plurality of individual antigen binding proteins. [This invention 1158] 1157. The method of claim 1157, wherein said phage display library is substantially free of antigen binding proteins that non-specifically bind to said HLA of said HLA-PEPTIDE target. [This invention 1159] 1156. The method of claim 1156, wherein said antigen binding protein is present in a TCR library comprising a plurality of individual TCRs or antigen binding fragments thereof. [The present invention 1160] 1159. The method of any of claims 1156 to 1159, wherein said binding step is carried out more than once, optionally at least three times. [This invention 1161] contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE target to determine whether the antigen binding protein selectively binds to the HLA-PEPTIDE target; Optionally, selectivity is determined by measuring the binding affinity of said antigen binding protein to a soluble target HLA-PEPTIDE complex relative to the binding affinity to a soluble HLA-PEPTIDE complex that is different from the target complex; Optionally, the selectivity is determined by measuring the binding affinity of said antigen binding protein to a target HLA-PEPTIDE complex expressed on the surface of one or more cells against an HLA-PEPTIDE complex that is different from the target complex expressed on the surface of one or more cells. Any of the methods of the present invention 1156 to 1160. [This invention 1162] 1. A method of identifying an antigen binding protein of any of the preceding inventions, comprising: obtaining at least one HLA-PEPTIDE target listed in Table A and administering said HLA-PEPTIDE target, optionally in combination with an adjuvant, to a subject; isolating said antigen binding protein from said subject; The method comprising: [This invention 1163] 1163. The method of claim 1162, wherein isolating said antigen binding protein comprises screening serum of said subject to identify said antigen binding protein. [This invention 1164] contacting the antigen binding protein with one or more peptide-HLA complexes that are distinct from the HLA-PEPTIDE target to determine whether the antigen binding protein selectively binds to the HLA-PEPTIDE target; Optionally, selectivity is determined by measuring the binding affinity of said antigen binding protein to a soluble target HLA-PEPTIDE complex relative to the binding affinity to a soluble HLA-PEPTIDE complex that is different from the target complex; Optionally, the selectivity is determined by measuring the binding affinity of said antigen binding protein to a target HLA-PEPTIDE complex expressed on the surface of one or more cells against an HLA-PEPTIDE complex that is different from the target complex expressed on the surface of one or more cells. The method of the present invention 1162. [Invention 1165] 1163. The method of claim 1162, wherein the subject is a mouse, rabbit, or llama. [Invention 1166] 1163. The method of claim 1162, wherein isolating said antigen binding protein comprises isolating B cells from said subject which express said antigen binding protein, and optionally directly cloning a sequence encoding said antigen binding protein from said isolated B cells. [This invention 1167] The method of claim 1166, further comprising using said B cells to produce hybridomas. [Invention 1168] The method of claim 1166, further comprising cloning CDRs from said B cells. [This invention 1169] 116. The method of claim 1166, further comprising immortalizing said B cells, optionally via EBV transformation. [This invention 1170] 116. The method of claim 1166, further comprising generating a library comprising said antigen binding proteins of said B cells, optionally wherein said library is a phage display or yeast display. [This invention 1171] 1163. The method of claim 1162, further comprising humanizing said antigen-binding protein. [This invention 1172] 1. A method of identifying an antigen binding protein of any of the preceding inventions, comprising: obtaining a cell comprising said antigen-binding protein; contacting said cells with an HLA multimer comprising at least one HLA-PEPTIDE target listed in Table A; identifying said antigen binding protein via binding between said HLA multimer and said antigen binding protein; The method comprising: [This invention 1173] 1. A method of identifying an antigen binding protein of any of the preceding inventions, comprising: obtaining one or more cells comprising said antigen binding protein; activating said one or more cells with at least one HLA-PEPTIDE target listed in Table A presented on a natural or artificial antigen-presenting cell (APC); identifying said antigen binding protein by selecting one or more cells activated by interaction with at least one HLA-PEPTIDE target listed in Table A; The method comprising: [This invention 1174] 1174. The method of claim 1172 or 1173, wherein said cell is a T cell, optionally a CTL. [This invention 1175] 1174. The method of claim 1172 or 1173, further comprising isolating said cells, optionally using flow cytometry, magnetic separation or single cell separation. [Invention 1176] 1175. The method of claim 1175, further comprising sequencing said antigen binding protein. [This invention 1177] A method of identifying an antigen binding protein of any of the preceding inventions, comprising providing at least one HLA-PEPTIDE target listed in Table A and identifying said antigen binding protein using said target. [Brief explanation of the drawings]

[0102] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0103] [Figure 1] 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 [Figure 2] Exemplary constructs for cloning TCRs into expression systems for therapeutic development are depicted. [Figure 3] Target and minipool negative control designs for HLA-PEPTIDE target "G5" are shown. [Figure 4] Target and minipool negative control designs for HLA-PEPTIDE targets "G8" and "G10" are shown. [Figure 5] A and B show the HLA stability results of the G5 counterscreen "minipool" and G5 target. [Figure 6] A-E show HLA stability results for the G5 "full" pool counterscreen peptides. [Figure 7] A and B show the HLA stability results of the counterscreen peptide and G8 target. [Figure 8]A and B show the HLA stability results of the G10 counterscreen "minipool" and G10 target. [Figure 9] AD show HLA stability results for additional G8 and G10 "full" pool counterscreen peptides. [Figure 10] AC show the results of phage supernatant ELISA, suggesting progressive enrichment of G5-, G8-, and G10-binding phages with successive panning rounds. [Figure 11] FIG. 1 shows a flow chart describing the antibody selection process, including criteria and intended uses for scFv, Fab, and IgG formats. [Figure 12] A, B, and C depict the results of biolayer interferometry (BLI) performed on Fab clone G5-P7A05 against HLA-PEPTIDE target B*35:01-EVDPIGHVY, Fab clones R3G8-P2C10 and G8-P1C11 against HLA-PEPTIDE target A*02:01-AIFPGAVPAA, and Fab clone R3G10-P1B07 against HLA-PEPTIDE target A*01:01-ASSLPTTMNY. [Figure 13] 1 shows the general experimental design of a position scanning experiment. [Figure 14] Figure 1A shows the stability results of G5 position mutant-HLA. Figure 1B shows the binding affinity of Fab clone G5-P7A05 to G5 position mutant-HLA. [Figure 15] Figure 1A shows the stability results of G8 mutant-HLA. Figure 1B shows the binding affinity of Fab clone G8-P2C10 to G8 mutant-HLA. [Figure 16] Figure 1A shows the stability results of the G10 position mutant-HLA. Figure 1B shows the binding affinity of Fab clone G10-P1B07 to the G10 position mutant-HLA. [Figure 17] A, B, and C show representative examples of antibodies binding to either G5, G8, or G10-presenting K562 cells as detected by flow cytometry. [Figure 18] AC show histogram plots of binding of K562 cells to generated target-specific antibodies. [Figure 19] AC show histogram plots of cell binding assays using HLA subtypes of selected HLA-PEPTIDE targets and tumor cell lines expressing the target genes. [Figure 20] A and B show the number of target-specific T cells (A) and the number of target-specific TCR clonotypes (B) from the donors tested. [Figure 21] A shows an exemplary heatmap of scFv G8-P1H08 visualized across the HLA portion of the HLA-PEPTIDE target G8 using a concatenated perturbation view. B shows an example of HDX data from scFv G8-P1H08 plotted on the crystal structure PDB5bs0. [Figure 22] A shows a heat map of the HLA α1 helix of all ABPs tested against the HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA). B shows a heat map of the HLA α2 helix of all ABPs tested against the HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA). C shows a heat map of the results across the restricted peptide AIFPGAVPAA for all ABPs tested. [Figure 23] A shows an exemplary heatmap of scFv R3G10-P2G11 visualized across the HLA portion of the HLA-PEPTIDE target G10 using a concatenated perturbation view. B shows an example of HDX data from scFv R3G10-P2G11 plotted on the crystal structure PDB5bs0. [Figure 24]A shows the resulting heatmap across the HLA α1 helix of all ABPs tested against the HLA-PEPTIDE target G10 (HLA-A*01:01_ASSLPTTMNY). B shows the resulting heatmap across the HLA α2 helix of all ABPs tested against the HLA-PEPTIDE target G10 (HLA-A*01:01_ASSLPTTMNY). C shows the resulting heatmap across the restricted peptide ASSLPTTMNY for all ABPs tested. [Figure 25] Exemplary spectral data for the peptide EVDPIGHVY is depicted, including peptide fragmentation information as well as information related to the patient sample (e.g., HLA type). [Figure 26] Exemplary spectral data for the peptide AIFPGAVPAA is depicted, including peptide fragmentation information as well as information related to the patient sample (e.g., HLA type). [Figure 27] Exemplary spectral data for the peptide ASSLPTTMNY is depicted, including peptide fragmentation information as well as information related to the patient sample (e.g., HLA type). [Figure 28] A and B depict size exclusion chromatography fractions (A) and SDS-PAGE analysis of chromatography fractions under reducing conditions (B). [Figure 29] An exemplary crystal micrograph of a complex containing Fab clone G8-P1C11 and HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8") is depicted. [Figure 30] The overall structure of the complex formed by binding of Fab clone G8-P1C11 to the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8") is depicted. [Figure 31]A refined electron density region of the crystal structure of Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8") is depicted. The depicted region corresponds to the constraint peptide AIFPGAVPAA. [Figure 32] A LigPlot of the interaction between HLA and the restricted peptide is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 33] Plots of interacting residues between the Fab VH and VL chains and the restraining peptide are depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 34] A LigPlot of the interaction between the restraining peptide and the Fab chain is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 35] A LigPlot of the interaction between the Fab VH chain and HLA is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 36] A LigPlot of the interaction between the Fab VL chain and HLA is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 37] A summary of the interface in the PiSA analysis of the interaction between HLA and the restricted peptide is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 38]A PiSA analysis of the interacting residues between HLA and the restricted peptide is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 39] A Pisa analysis of the interacting residues between the Fab VH chain and the restraining peptide is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 40] A Pisa analysis of the interacting residues between the Fab VL chain and the restraining peptide is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 41] A summary of the interface in the Pisa analysis of the interaction between the Fab VH chain and HLA is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 42] A Pisa analysis of the interacting residues between the Fab VH chain and HLA is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 43] A summary of the interface in the Pisa analysis of the interaction between the Fab VL chain and HLA is depicted. This crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 44] A Pisa analysis of the interacting residues between the Fab VL chain and HLA is depicted. The crystal structure corresponds to Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 45](A) depicts an exemplary heat map of the HLA portion of the G8 HLA-PEPTIDE complex incubated with scFv clone G8-P1C11. The entire image is visualized using the integrated perturbation view. (B) depicts example HDX data from scFv G8-P1C11 plotted on the crystal structure of Fab clone G8-P1C11 in complex with the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"). [Figure 46] The binding affinity of Fab clone G8-P1C11 to G8 position mutant-HLA is depicted. [Figure 47] 1 shows a histogram plot of K562 cell binding to G8-P1C11 (ie, a target-specific antibody against the HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8")). DETAILED DESCRIPTION OF THE INVENTION

[0104] Detailed Description Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings commonly understood by those of ordinary skill in the art. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ease of reference. The inclusion of such definitions herein should not necessarily be construed as representing a deviation from a definition commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies, e.g., commonly employed 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. Procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise noted.

[0105] As used herein, the singular forms "a," "an," and "the" include 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 stated.

[0106] As used herein, the term "comprising" specifically includes embodiments "consisting of" and "consisting essentially of" the listed elements, unless specifically indicated otherwise. For example, a multispecific ABP "comprising a diabody" includes a multispecific ABP "consisting of a diabody" as well as a multispecific ABP "consisting essentially of a diabody."

[0107] The term "about" refers to and encompasses the indicated value, as well as a range of values ​​above and below that value. In certain embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" refers to the specified value(s) ± one standard deviation around that value(s).

[0108] The term "immunoglobulin" refers to a class of structurally related proteins comprising roughly two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an "intact immunoglobulin," all four 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 heavy chain constant region (C H The heavy chain constant region typically comprises C H1 , C H2 , and C H3 Each light chain typically comprises three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region typically comprises C L It has one domain, abbreviated as .

[0109] The term "antigen binding protein" or "ABP" is used herein in its broadest sense and includes a specific type of molecule that comprises one or more antigen binding domains that specifically bind to an antigen or epitope.

[0110] 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. ABPs include whole antibodies (such as whole immunoglobulins), antibody fragments, ABP fragments, and multispecific antibodies. In some embodiments, the ABP comprises an surrogate scaffold. In some embodiments, the ABP consists of an surrogate scaffold. In some embodiments, the ABP consists essentially of an surrogate 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, the ABP comprises a TCR or an antigen-binding portion thereof. In some embodiments, the ABP consists of a TCR or an antigen-binding portion thereof. In some embodiments, the ABP consists essentially of a TCR or an antigen-binding portion thereof. In some embodiments, a CAR comprises an ABP. An "HLA-PEPTIDE ABP," "anti-HLA-PEPTIDE ABP," or "ABP specific for HLA-PEPTIDE" is an ABP as provided herein that specifically binds to the antigen HLA-PEPTIDE. ABPs comprise proteins that comprise 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., an antibody) or a T cell (e.g., a TCR).

[0111] The term "antibody" is used herein in the broadest sense and includes both polyclonal and monoclonal antibodies. Examples include intact antibodies and functional (antigen-binding) antibody fragments, such as fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specific binding to antigen, single-chain antibody fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also 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 specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also includes intact or whole antibodies of any class or subclass, including IgG and its subclasses IgM, IgE, IgA, and IgD.

[0112] As used herein, "variable region" refers to a variable nucleotide sequence resulting from a recombination event and may comprise the V, J, and / or D regions of an immunoglobulin or T cell receptor (TCR) sequence derived from a B cell or T cell (e.g., an activated T cell or an activated B cell).

[0113] The term "antigen-binding domain" refers to the portion of an ABP that has the ability to specifically bind to an antigen or epitope. H -V LAn antigen-binding domain formed by a dimer is one example of an antigen-binding domain. Another example of an antigen-binding domain is an antigen-binding domain formed by diversifying a specific loop from the tenth fibronectin type III domain of an Adnectin. The antigen-binding domain may comprise antibody CDR1, 2, and 3 from the heavy chain, in that order, or antibody CDR1, 2, and 3 from the light chain, in that order. The antigen-binding domain may comprise TCR CDRs (e.g., αCDR1, αCDR2, αCDR3, βCDR1, βCDR2, and βCDR3). TCR CDRs are described herein.

[0114] Antibody V H Area and V L The region may be further subdivided into hypervariable regions (also called hypervariable regions (HVRs) or complementarity determining regions (CDRs)), and these HVRs are interspersed with highly conserved regions, which are called framework regions (FRs). H and V L An antibody typically contains 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 CDRs of an antibody are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated by reference in its entirety.

[0115] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0116] Heavy chains from any vertebrate species can be assigned to one of five classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also called α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. The subclasses expressed in humans are as follows: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0117] The amino acid sequence boundaries of antibody CDRs can be determined by those skilled in the art using any of a number of known numbering schemes, including those compiled by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. 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 these references is incorporated by reference in its entirety.

[0118] The positions of antibody CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat and Chothia schemes, are provided in Table 20. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0119] Antibody CDRs can be assigned using, for example, ABP numbering software. For example, in the case of Abnum, this software is available at www.bioinf.org.uk / abs / abnum / . Antibody CDRs are described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety.

[0120] Table 20: Residues in the CDRs according to Kabat and Chothia numbering scheme TIFF2026004411000040.tif46142 * The C-terminus of CDR-H1 varies between H32 and H34 depending on the CDR length when numbered using the Kabat numbering convention.

[0121] The "EU numbering scheme" is typically used when referring to residues in the ABP heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless otherwise specified, the EU numbering scheme is used when referring to residues in the ABP heavy chain constant region described herein.

[0122] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a naturally occurring antibody, and having a heavy chain with an Fc region. For example, a "full length antibody," when used to refer to an IgG molecule, would be an antibody comprising two heavy chains and two light chains.

[0123] The amino acid sequence boundaries of TCR CDRs 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 described in LeFranc, M.-P, Immunol Today. 1997 Nov; 18(11):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 Facts Book; and M.-P. Lefranc / Developmental and Comparative Immunology 27 (2003) 55-77, all of which are incorporated by reference in their entirety.

[0124] An "ABP fragment" includes a portion of an intact ABP, such as the antigen-binding or variable region of the intact ABP. Examples of ABP fragments include Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments. Fragments of ABPs include antibody fragments. Examples of antibody fragments include Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, scFv-Fc fragments, and TCR fragments.

[0125] An "Fv" fragment comprises a dimer of one heavy-chain and one light-chain variable domain in non-covalent association.

[0126] The "Fab" fragment contains not only the heavy and light chain variable domains, but also the constant domain of the light chain and the first constant domain of the heavy chain (C H1 Fab fragments may be produced, for example, by recombinant methods or by papain digestion of full-length ABP.

[0127] The "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. "F(ab')2" can be produced, for example, by recombinant methods or by pepsin digestion of intact ABP. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0128] "Single-chain Fv" or "sFv" or "scFv" fragments contain a VFv fragment in a single polypeptide chain. H Domain and V L With domain. V H and V L are typically linked via a peptide linker. Source: Pluckthun A. (1994). Any suitable linker can be used. In some embodiments, the linker is (GGGGS) n and in some embodiments, n=1, 2, 3, 4, 5, or 6. Source: ABPs from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal ABPs, vol. 113 (pp. 269-315). Springer-Verlag, New York. The document is incorporated by reference in its entirety.

[0129] An "scFv-Fc" fragment comprises an scFv attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. The orientation of the variable domains within the scFv (i.e., V H -V L or V L -V H ) depending on V H or V L can be followed by an Fc domain. Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0130] The term "single-domain antibody" refers to a molecule in which one variable domain of the ABP specifically binds to an antigen without the intervention of other variable domains. 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.

[0131] The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and specific proteins of the complement system. The structures of various immunoglobulin Fc regions and the glycosylation sites contained within those structures are known in the art. Source: Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or a modified Fc region as described in the art or elsewhere in this disclosure.

[0132] The term "alternative scaffold" refers to a molecule in which one or more regions can be diversified to generate one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain is a domain that binds to an antigen or epitope with the same specificity and affinity as an ABP. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), β-sandwich (e.g., iMabs), lipocalin (e.g., Anticalins®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, protein A (e.g., Affibody®), ankyrin repeat (e.g., DARPins), gamma-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., tetranectin), 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. Alternative scaffolds are a type of ABP.

[0133] A "multispecific ABP" refers to an ABP that comprises two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single HLA-PEPTIDE molecule expressed by a cell) or epitopes on different antigens (e.g., different HLA-PEPTIDE molecules, or an HLA-PEPTIDE molecule and a non-HLA-PEPTIDE molecule, expressed by the same cell). In some embodiments, the multispecific ABP binds to two different epitopes (i.e., is a "bispecific ABP"). In some embodiments, the multispecific ABP binds to three different epitopes (i.e., is a "trispecific ABP").

[0134] A "monospecific ABP" is an ABP that has 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 is bivalent (i.e., has two antigen-binding domains) but recognizes the same epitope in each of the two antigen-binding domains. The binding specificity can be in any suitable valency.

[0135] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies includes antibodies that are substantially similar and bind to the same epitope(s), excluding variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes selecting 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 modified, for example, to improve its affinity for the target ("affinity maturation"), humanize the antibody, improve its production in cell culture, and / or reduce its immunogenicity in a subject.

[0136] 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, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0137] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, e.g., a mouse, rat, rabbit, chicken, or non-human primate antibody with the desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced with the corresponding framework region residues from the donor antibody. It is also possible to include residues in the humanized antibody that are not found in either the recipient or 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.

[0138] A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or one derived from a non-human source that utilizes the human antibody repertoire or sequences encoding human antibodies (e.g., obtained from a human source or designed de novo). Human antibody specifically excludes humanized antibodies.

[0139] "Affinity" refers to the strength of the sum 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 otherwise specified, "affinity" as used herein refers to the inherent binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an ABP and an antigen or epitope). The affinity of molecule X for partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are discussed 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®).

[0140] The terms "bind," "specific binding," "specifically binds," "specifically binds," "selectively binds," and "selectively binds" refer to binding of an ABP to a target molecule that is significantly different from a nonspecific or nonselective interaction (e.g., interaction with a non-target molecule) of a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen. 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 by the target molecule. In this case, specific binding is indicated if binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 50% of its affinity for the HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 40% of its affinity for the HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 30% of its affinity for HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 20% of its affinity for HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 10% of its affinity for HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 1% of its affinity for HLA-PEPTIDE. In some embodiments, the affinity of the HLA-PEPTIDE ABP for the non-target molecule is less than about 0.1% of its affinity for HLA-PEPTIDE.

[0141] As used herein, "k d " (sec -1 The term k ) refers to the dissociation rate constant of a particular ABP-antigen interaction. This value is off Also called the value.

[0142] As used herein, "k a " (M -1 ×sec -1 The term k ) refers to the binding rate constant for a particular ABP-antigen interaction. This value is on Also called the value.

[0143] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. D =k d / k a In some embodiments, K D The affinity of an ABP is described in terms of the K corresponding to the interaction between such an ABP and its antigen. For clarity, as known in the art, K D A smaller value indicates a higher affinity interaction, and K D Higher values ​​indicate lower affinity interactions.

[0144] As used herein, "K A " (M -1 The term K ) refers to the binding equilibrium constant for a particular ABP-antigen interaction. A =k a / k d .

[0145] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecule(s), eg, a therapeutic agent (such as a cytokine) or a diagnostic agent.

[0146] "Fc effector function" refers to a biological activity mediated by the Fc region of an ABP having an Fc region. These activities vary depending on the isotype. Examples of ABP effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate ABP-dependent cellular cytotoxicity (ADCC), and ABP-dependent cellular phagocytosis (ADCP).

[0147] The terms "compete with" or "competes with," when used herein in the context of two or more ABPs, indicate that the two or more ABPs compete for binding to an antigen (e.g., HLA-PEPTIDE). In one exemplary assay, an HLA-PEPTIDE is applied to a surface and contacted with a first HLA-PEPTIDE ABP, followed by the addition of a second HLA-PEPTIDE ABP. In another exemplary assay, a first HLA-PEPTIDE ABP is applied to a surface and contacted with an HLA-PEPTIDE, followed by the addition of a second HLA-PEPTIDE ABP. In either assay, ABPs compete with each other if the presence of the first HLA-PEPTIDE ABP reduces the binding of the second HLA-PEPTIDE ABP. The term "competes with" also encompasses combinations of ABPs in which one ABP reduces the binding of another ABP, but where competition is not observed when the ABPs are added in the reverse order. On the other hand, in some embodiments, the first and second ABPs inhibit each other's binding, regardless of the order in which the ABPs are added. In some embodiments, an ABP reduces the 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%. The concentration of ABP used in a competition assay can be selected by one of skill in the art based on the affinity of the ABP for the HLA-PEPTIDE and the valency of the ABP. The assays described in this definition are exemplary, and one of skill in the art can use any suitable assay to determine whether 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.

[0148] The term "epitope" refers to a portion of an antigen that specifically binds to an ABP. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and charge characteristics. In this regard, a distinction is made between conformational and nonconformational epitopes in that binding to the former conformational epitopes (i.e., but not the latter nonconformational epitopes) may be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination (e.g., ABP binding studies with HLA-PEPTIDE variants with different point mutations or chimeric HLA-PEPTIDE variants).

[0149] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage.Alignment to determine the percentage (%) of amino acid sequence identity can be achieved in a variety of ways within the scope of those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software.Appropriate parameters for aligning sequences (for example, any algorithm required to achieve maximum alignment across the entire length of the sequences being compared) can be determined by those skilled in the art.

[0150] "Conservative substitution" or "conservative amino acid substitution" refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. As examples, the amino acid groups provided in Tables 21-23 are considered conservative substitutions for each other in some embodiments.

[0151] Table 21: Selected groups of amino acids that, in certain embodiments, are considered conservative substitutions for one another TIFF2026004411000041.tif32128

[0152] Table 22: Additional amino acid groups that, in certain embodiments, are considered conservative substitutions for one another TIFF2026004411000042.tif32128

[0153] Table 23: Further selected amino acid groups that are considered conservative substitutions for one another in certain embodiments. TIFF2026004411000043.tif43128

[0154] Additional conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W.H. Freeman & Co., New York, NY. ABPs generated by making one or more conservative substitutions of amino acid residues in a parent ABP are referred to as "conservatively modified variants."

[0155] The term "amino acid" refers to the 20 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 (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 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).

[0156] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term encompasses vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are 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."

[0157] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include "transformants" (or "transformed cells") and "transformants" (or "transfected cells"), which include the primary transformed or transfected cell, respectively, and their derived progeny. Such progeny may not necessarily be completely identical in nucleic acid content to the parent cell and may contain mutations.

[0158] The term "treat" (and variations thereof, such as "treating" 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 may be performed both prophylactically and during the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of the direct or indirect pathological effects of disease, prevention of metastasis, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0159] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[0160] As used herein, the term "subject" refers to 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's disease or condition can be treated with the ABPs provided herein. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a viral infection.

[0161] The term "package insert" is used to refer to instructions typically included in a commercially available package (e.g., a kit) of a therapeutic or diagnostic product, including information regarding directions, availability, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic product.

[0162] 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 when referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematological malignancy.

[0163] The term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredients contained therein can be made effective to treat a subject, and which does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0164] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0165] The terms "increase" and "activation" refer to a 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 increment in the recited variable.

[0166] The terms "reduce" and "inhibit" refer to a 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 decrement in the recited variable.

[0167] The term "stimulate" refers to activating receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and stimulates a receptor.

[0168] The term "antagonize" refers to inhibiting a biological response associated with receptor activation by inhibiting receptor signaling. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0169] The terms "nucleic acid" and "polynucleotide" are sometimes used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can include, but are not limited to, coding or non-coding regions of a gene or gene fragment, loci 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. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Exemplary modified nucleotides include, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, and N6-substituted adenine. Examples include denine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthioN6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, quosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0170] Isolated HLA-PEPTIDE targets The major histocompatibility complex (MHC) is a complex of antigens encoded by a group of linked genetic loci. These antigens are collectively referred to as H-2 in mice and HLA in humans. The two major classes of MHC antigens, class I and class II, each contain a series of cell surface glycoproteins that play a role in determining tissue type and transplant compatibility. In the transplant response, cytotoxic T cells (CTLs) respond primarily to class I glycoproteins, while helper T cells respond primarily to class II glycoproteins.

[0171] Human major histocompatibility complex (MHC) class I molecules (interchangeably referred to herein as HLA class I molecules) are expressed on the surface of nearly all cells. These molecules function in presenting peptides, most of which are derived from endogenously synthesized proteins, to, for example, CD8+ T cells via interaction with the α-β T cell receptor. Class I MHC molecules contain heterodimers composed of a 46 kDa α chain noncovalently associated with a 12 kDa light chain β2-microglobulin. The α chain generally comprises α1 and α2 domains, which form a groove for presenting HLA-restricted peptides and an α3 transmembrane domain that interacts with the CD8 coreceptor on T cells. Figure 1 (Prior Art) depicts the general structure of a class I HLA molecule. Some TCRs can bind to MHC class I independently of the 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).

[0172] Class I MHC-restricted peptides (also referred to herein interchangeably as HLA-restricted antigens, HLA-restricted peptides, MHC-restricted antigens, restricted peptides, or peptides) typically bind to the α1-α2 groove of the heavy chain via approximately two or three anchor residues that interact with the corresponding binding pocket of the MHC molecule. The β-2 microglobulin chain plays a critical role in intracellular trafficking, peptide binding, and structural stability of MHC class I. 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 β2 microglobulin results in protein maturation and transport to the cell surface.

[0173] Binding of a given HLA subtype to an HLA-restricted peptide results in the formation of a complex with a unique and novel surface that can be specifically recognized by, for example, a TCR on a T cell or an ABP, such as an antibody or antigen-binding fragment thereof. HLA complexed with an HLA-restricted peptide is referred to herein as an HLA-PEPTIDE or HLA-PEPTIDE target. In some cases, the restricted peptide is located within the α1 / α2 groove of the HLA molecule. In some cases, the restricted peptide binds to the α1 / α2 groove of the HLA molecule via approximately two or three anchor residues that interact with the corresponding binding pocket of the HLA molecule.

[0174] Thus, provided herein are antigens that include HLA-PEPTIDE targets, which can include specific HLA-restricted peptides with defined amino acid sequences complexed with specific HLA subtypes.

[0175] The HLA-PEPTIDE targets identified herein can be useful for cancer immunotherapy. In some embodiments, the HLA-PEPTIDE targets identified herein are presented on the surface of tumor cells. The HLA-PEPTIDE targets identified herein can be expressed by tumor cells in human subjects. The HLA-PEPTIDE targets identified herein can be expressed by tumor cells in a population of human subjects. For example, the HLA-PEPTIDE targets identified herein can be shared antigens that are commonly expressed in a population of human subjects with cancer.

[0176] The HLA-PEPTIDE targets identified herein may have a prevalence in individual tumor types. The prevalence of individual tumor types was approximately 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, It can be 5%, 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 of individual tumor types can be about 0.1% to 100%, 0.2 to 50%, 0.5 to 25%, or 1 to 10%.

[0177] Preferably, HLA-PEPTIDE targets are not typically expressed in most normal tissues. For example, an HLA-PEPTIDE target may not be expressed in tissues from a Genotype-Tissue Expression (GTEx) project, or may be expressed only in immune-privileged or non-essential tissues. Exemplary immune-privileged or non-essential tissues include testis, minor salivary glands, cervix, and thyroid. In some cases, an HLA-PEPTIDE target may be considered not expressed in essential or non-immune-privileged tissues if the median expression of the gene from which the restriction peptide is derived is less than 0.5 RPKM (transcript reads (kilobases) / million mapped reads) across all GTEx samples, if the gene is not expressed above 10 RPKM across all GTEx samples, or if the gene is expressed at 5 RPKM or higher in no more than two samples from all essential tissue samples, or any combination thereof.

[0178] Exemplary HLA Class I Subtypes of HLA-PEPTIDE Targets Many MHC haplotypes exist in humans (interchangeably referred to herein as MHC subtypes, HLA subtypes, MHC types, and HLA types). Exemplary HLA subtypes include, by way of example only, HLA-A*01:01, HLA-A*02:01, HLA-A*02:03, HLA-A*02:04, HLA-A*02:07, HLA-A*03:01, HLA-A*03:02, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-A*25:01, HLA -A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*68:01, HLA-A*68:02, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*15: 01, HLA-B*15:03, HLA-B*18:01, HLA-B*27:02, HLA-B*27:05, HLA-B*35:01, HLA-B*35:03, HLA-B* 37:01, HLA-B*38:01, HLA-B*39:01, HLA-B*40:01, HLA-B*40:02, HLA-B*44:02, HLA-B*44:03, HLA -B*46:01, HLA-B*49:01, HLA-B*51:01, HLA-B*54:01, HLA-B*55:01, HLA-B*56:01, HLA-B*57:01, HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05: Examples of HLA alleles include HLA-C*01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*07:06, HLA-C*12:03, HLA-C*14:02, HLA-C*16:01, HLA-C*16:02, HLA-C*16:04, and all subtypes thereof (such as 4-digit, 6-digit, and 8-digit subtypes). As known to those skilled in the art, there are allelic variants of the above HLA types, all of which are encompassed by the present invention. A complete list of HLA class alleles can be found at http: / / hla.alleles.org / alleles / .For example, a complete list of HLA class I alleles can be found at http: / / hla.alleles.org / alleles / class1.html.

[0179] HLA-restricted peptides The HLA-restricted peptide as a "restricted peptide" (interchangeably referred to herein) can be a peptide fragment of a tumor-specific gene, such as a cancer-specific gene. Preferably, the cancer-specific gene is expressed in cancer samples. Genes that are abnormally expressed in cancer samples can be identified through databases. Examples of databases include, but are not limited to: The Cancer Geonome Atlas (TCGA) Research Network: http: / / cancergenome.nih.gov / ; the International Cancer Genome Consortium: https: / / dcc.icgc.org / . In some embodiments, for a cancer-specific gene, expression of at least 10 RPKM is observed in at least five samples in the TCGA database. The cancer-specific gene may have an observable bimodal distribution.

[0180] A cancer-specific gene may be observed to be expressed at more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transcripts per minute (TPM) in at least one TCGA tumor tissue. In a preferred embodiment, a cancer-specific gene is observed to be expressed at more than 100 TPM in at least one TCGA tumor tissue. In some cases, a bimodal expression distribution across TCGA samples has been observed for a cancer-specific gene. Without being bound by theory, such a bimodal expression pattern is consistent with a biological model in which minimal expression is seen at baseline in all tumor samples and higher expression is seen in a subset of tumors experiencing epigenetic dysregulation.

[0181] Preferably, a cancer-specific gene is not typically expressed in most normal tissues. For example, a cancer-specific gene may not be expressed in tissues from a Genotype-Tissue Expression (GTEx) project, or may be expressed only in immune-privileged or non-essential tissues. Examples of immune-privileged or non-essential tissues include testis, minor salivary glands, cervix, and thyroid. In some cases, a cancer-specific gene may be considered not expressed in essential or non-immune-privileged tissues if the median expression of the cancer-specific gene is less than 0.5 RPKM (transcript reads (kilobases) / million mapped reads) across all GTEx samples, if the gene is not expressed at more than 10 RPKM across all GTEX samples, if the gene is expressed at 5 RPKM or more in no more than two samples across all essential tissue samples, or any combination thereof.

[0182] In some embodiments, the cancer-specific genes meet the following criteria based on GTEx assessment: (1) median GTEx expression in brain, heart, or lung is less than 0.1 transcripts per million (TPM), with no sample exceeding 5 TPM, and (2) median GTEx expression in other essential organs (excluding testis, thyroid, and minor salivary glands) is less than 2 TPM, with no sample exceeding 10 TPM.

[0183] In some embodiments, the cancer-specific gene is unlikely to be generally expressed in immune cells, e.g., the cancer-specific gene is not an interferon family gene, is not an eye-related gene, is not an olfactory or taste receptor gene, or is not a gene associated with circadian cycles (e.g., is not a CLOCK, PERIOD, or CRY gene).

[0184] Preferably, the restricted peptide is one that can be presented on the surface of the tumor.

[0185] The restricted peptide residue size can be 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 certain embodiments, the restricted peptide size is about 8, about 9, about 10, about 11, or about 12 amino acid residues. The restricted peptide can be about 5-15 amino acids in length, preferably about 7-12 amino acids in length, or more preferably about 8-11 amino acids in length.

[0186] Exemplary HLA-PEPTIDE Targets Examples of HLA-PEPTIDE targets are shown in Table A. Each row of Table A lists the HLA alleles and corresponding HLA-restricted peptide sequences for each complex. The peptide sequence may consist of each sequence shown in each row of Table A. Alternatively, the peptide sequence may include each sequence shown in each row of Table A. Alternatively, the peptide sequence may consist essentially of each sequence shown in each row of Table A.

[0187] In some embodiments, the HLA-PEPTIDE target is a target shown in Table A.

[0188] In some embodiments, the HLA-restricted peptide is not derived from a gene selected from WT1 or MART1.

[0189] HLA class I molecules that do not associate with a restrictive peptide ligand are generally unstable. Therefore, the association of a restrictive peptide with the α1 / α2 groove of an HLA molecule may stabilize the noncovalent association of the β2-microglobulin subunit of an HLA subtype with the α subunit of an HLA subtype.

[0190] The stability of the non-covalent association between the β2 microglobulin subunit of HLA subtype and the α subunit of HLA subtype can be determined by any suitable means.For example, this stability can be evaluated by dissolving the insoluble aggregate of HLA molecule in high concentration urea (for example, about 8M urea), and determining the ability of HLA molecule to refold in the presence of limiting peptide during urea removal, for example, by dialysis urea removal.This refolding approach is described, for example, in Proc.Natl.Acad.Sci.USA Vol.89, pp.3429-3433, April 1992, and this document is incorporated herein by reference in its entirety.

[0191] In another example, such stability may be assessed using conditional HLA class I ligands. Conditional HLA class I ligands are generally designed as short, restricted peptides that bind to the α1 / α2 groove of HLA molecules, stabilizing the association of the β2 and α subunits of HLA class I molecules, and include one or more amino acid modifications that allow the restricted peptide to be cleaved upon exposure to a conditional stimulus. When the conditional ligand is cleaved, the β2 and α subunits of the HLA molecule dissociate unless the conditional ligand is replaced with a restricted peptide that binds to the α1 / α2 groove and stabilizes the HLA molecule. Conditional ligands can be designed by introducing amino acid modifications into known or predicted high-affinity HLA peptide ligands. For HLA alleles for which structural information is available, it is also possible to select positions for introducing amino acid modifications by exploiting the accessibility of side chain water. The use of conditional HLA ligands may be advantageous in some cases. This is because it allows batch preparation of stable HLA-peptide complexes, which can be used to investigate test-limiting peptides in a high-throughput manner.Conditional HLA class I ligands and methods for producing them are described, for example, in Proc Natl Acad Sci US A. 2008 Mar 11; 105(10): 3831-3836; Proc Natl Acad Sci US A. 2008 Mar 11; 105(10): 3825-3830; J Exp Med. 2018 May 7; 215(5): 1493-1504; Choo, J.A. Let al. Bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides. Angew Chem Int Ed Engl 53, 13390-13394(2014); Amore, 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 Cells. Characterization. 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, CX Let 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.

[0192] Thus, in some embodiments, the ability of the HLA-restricted peptides described herein (e.g., those described in Table A) to stabilize the association of the β2 and α subunits of HLA molecules is assessed by performing a conditional ligand-mediated exchange reaction and an HLA stability assay. HLA stability can be assayed using any suitable method, including, for example, mass spectrometry, immunoassays (such as ELISA), size exclusion chromatography, HLA multimer staining followed by flow cytometric evaluation of T cells, etc.

[0193] Another exemplary method for evaluating the stability of the non-covalent association between the β2 microglobulin subunit of HLA subtype and the α subunit of HLA subtype also includes the peptide exchange using dipeptide.The peptide exchange using dipeptide is described in, for example, Proc Natl Acad Sci US A.2013 Sep 17,110(38):15383-8;Proc Natl Acad Sci US A.2015 Jan 6,112(1):202-7, and this document is incorporated herein by reference in its entirety.

[0194] Useful antigens are provided herein, including HLA-PEPTIDE targets, which may include specific HLA-restricted peptides having defined amino acid sequences complexed with specific HLA subtype alleles.

[0195] The HLA-PEPTIDE target may be isolated and / or in substantially pure form. For example, the HLA-PEPTIDE target may be isolated from its natural environment or may be produced by a technological process. In some cases, the HLA-PEPTIDE target is provided in a form that is substantially free of other peptides or proteins.

[0196] The HLA-PEPTIDE target may be provided in a soluble form, or optionally as a recombinant HLA-PEPTIDE target complex. Those skilled in the art can use any suitable method for producing and purifying a recombinant HLA-PEPTIDE target. Suitable methods include, for example, the use of E. coli expression systems, insect cells, and the like. Other methods, such as synthetic production using cell-free systems, are also included. Examples of suitable cell-free systems are described in WO2017089756, which is incorporated herein by reference in its entirety.

[0197] Also provided herein are compositions that include HLA-PEPTIDE targets.

[0198] In some cases, the composition comprises an HLA-PEPTIDE target attached to a solid support. Examples of solid supports include, but are not limited to, beads, wells, membranes, tubes, columns, plates, sepharose, magnetic beads, and chips. Exemplary solid supports are described, for example, in Catalysts 2018, 8, 92; doi:10.3390 / catal8020092, which is incorporated herein by reference in its entirety.

[0199] The HLA-PEPTIDE target can be attached to the solid support by any suitable method known in the art. In some cases, the HLA-PEPTIDE target is covalently linked to the solid support.

[0200] In some cases, HLA-PEPTIDE targets are attached to solid supports via affinity binding pairs. Affinity binding pairs typically involve specific interactions between two molecules. A ligand with affinity for its binding partner molecule can be covalently attached to the solid support. This ligand is then used as a bait for immobilization. Common affinity binding pairs include, for example, streptavidin and biotin, avidin and biotin, polyhistidine tags containing metal ions such as copper, nickel, zinc, and cobalt, and the like.

[0201] The HLA-PEPTIDE target may comprise a detectable label.

[0202] Pharmaceutical compositions comprising HLA-PEPTIDE targets.

[0203] The composition containing an HLA-PEPTIDE target may be a pharmaceutical composition. Such a composition may contain multiple HLA-PEPTIDE targets. Exemplary pharmaceutical compositions are described herein. The composition may be capable of eliciting an immune response. The composition may include an adjuvant. Examples of suitable adjuvants include, but are not limited to, 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, and Montanide ISA. Adjuvants include 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, β-glucan, Pam3Cys, and Aquila's QS21 Stimulon (Aquila Biotech, Worcester, Mass., USA), derived from saponins, mycobacterial extracts, synthetic bacterial cell wall mimics, and other proprietary adjuvants (e.g., Ribi's Detox, Quil, or Superfos). Useful adjuvants include incomplete Freund's or GM-CSF. Several immunological adjuvants specific for dendritic cells and their preparation (e.g., MF59) have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines may also be used.Some cytokines, such as TNF-α, are directly involved in influencing dendritic cell migration to lymphoid tissues and accelerate 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 by reference in its entirety), while others act as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418). Furthermore, the expression of intracellular proteins on the HLA surface and their processing into peptides for presentation on HLA can be enhanced by interferon-gamma (IFN-γ). Sources: 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 by reference herein in their entireties.

[0204] HLA-PEPTIDE ABP Also provided herein are ABPs that specifically bind to the HLA-PEPTIDE targets disclosed herein.

[0205] The HLA-PEPTIDE target may be expressed on the surface of any suitable target cell, including a tumor cell.

[0206] The ABP is capable of specifically binding to a human leukocyte antigen (HLA)-PEPTIDE target, which comprises an HLA-restricted peptide complexed with an HLA class I molecule, and the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule.

[0207] In some embodiments, the ABP does not bind to HLA class I in the absence of the HLA-restricted peptide. In some embodiments, the ABP does not bind to the HLA-restricted peptide in the absence of human MHC class I. In some embodiments, the ABP binds to tumor cells that display human MHC class I complexed with the HLA-restricted peptide. Optionally, the HLA-restricted peptide is a tumor antigen that characterizes the cancer.

[0208] ABPs can bind to portions of the HLA-PEPTIDE complex (i.e., HLA and peptides representing portions of the complex), and when bound together, create new targets and protein surfaces for interaction and binding with the ABP, distinct from the surfaces presented by the peptide alone or the HLA subtype alone. Binding of HLA to a peptide typically creates new targets and protein surfaces, although these targets and protein surfaces do not exist in the absence of the portions of the HLA-PEPTIDE complex.

[0209] An ABP can specifically bind to a complex comprising an HLA and an HLA-restricted peptide (HLA-PEPTIDE), e.g., derived from a tumor. In some embodiments, the ABP does not bind to an HLA in the absence of an HLA-restricted peptide derived from a tumor. In some embodiments, the ABP does not bind to an HLA-restricted peptide derived from a tumor in the absence of an HLA. In some embodiments, the ABP binds to a complex comprising an HLA and an HLA-restricted peptide when the complex is presented in nature by a cell, such as a tumor cell.

[0210] In some embodiments, the ABPs provided herein modulate the binding of HLA-PEPTIDE to one or more ligands of HLA-PEPTIDE.

[0211] The ABP may specifically bind to any of the HLA-PEPTIDE targets disclosed in Table A. In some embodiments, the HLA-restricted peptide is not derived from a gene selected from WT1 or MART1.

[0212] In more particular embodiments, the ABP is: HLA subtype B*35:01 complexed with an HLA-restricted peptide containing the sequence EVDPIGHVY; HLA subtype A*02:01 complexed with an HLA-restricted peptide containing the sequence AIFPGAVPAA; and HLA subtype A*01:01 complexed with an HLA-restricted peptide containing the sequence ASSLPTTMNY. The peptide binds specifically to an HLA-PEPTIDE target selected from any of the following:

[0213] In a more particular embodiment, the ABP is: The antibody specifically binds to an HLA-PEPTIDE target selected from HLA subtype B*35:01 complexed with an HLA-restricted peptide consisting essentially of the sequence EVDPIGHVY; HLA subtype A*02:01 complexed with an HLA-restricted peptide consisting essentially of the sequence AIFPGAVPAA; and HLA subtype A*01:01 complexed with an HLA-restricted peptide consisting essentially of the sequence ASSLPTTMNY.

[0214] In some embodiments, the ABP is: HLA subtype B*35:01 complexed with an HLA-restricted peptide containing the sequence EVDPIGHVY; HLA subtype A*02:01 complexed with an HLA-restricted peptide containing the sequence AIFPGAVPAA; and HLA subtype A*01:01 complexed with an HLA-restricted peptide containing the sequence ASSLPTTMNY. The peptide binds specifically to an HLA-PEPTIDE target selected from any of the following:

[0215] In some embodiments, the ABP is an ABP that competes with the exemplary ABPs provided herein. In some aspects, the ABP that competes with the exemplary ABPs provided herein binds to the same epitope as the exemplary ABPs provided herein.

[0216] In some embodiments, the ABPs described herein are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or other methods known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein but can be isolated de novo, for example, according to the methods provided herein to obtain the ABP. In some embodiments, variants are derived from any of the sequences provided herein by one or more conservative amino acid substitutions. In some embodiments, variants are derived from any of the sequences provided herein by one or more non-conservative amino acid substitutions. Conservative amino acid substitutions are described herein. Exemplary non-conservative amino acid substitutions include those described in J Immunol. 2008 May 1;180(9):6116-31, which is incorporated herein by reference in its entirety. In preferred embodiments, the non-conservative amino acid substitution does not disrupt or inhibit the biological activity of the functional variant. The non-conservative amino acid substitutions enhance the biological activity of the functional variant, resulting in an enhanced biological activity of the functional variant compared to the parent ABP.

[0217] ABP containing an antibody or an antigen-binding fragment thereof The ABP may comprise an antibody or an antigen-binding fragment thereof.

[0218] In some embodiments, the ABPs provided herein comprise a light chain. In some embodiments, the light chain is a κ light chain. In some embodiments, the light chain is a λ light chain.

[0219] In some embodiments, the ABP provided herein comprises a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2.

[0220] In some embodiments, the ABP provided herein comprises an antibody fragment. In some embodiments, the ABP provided herein consists of an antibody fragment. In some embodiments, the ABP provided herein consists essentially of an antibody fragment. In some embodiments, the ABP fragment is an Fv fragment. In some embodiments, the ABP fragment is a Fab fragment. In some embodiments, the ABP fragment is a F(ab')2 fragment. In some embodiments, the ABP fragment is a Fab' fragment. In some embodiments, the ABP fragment is an scFv (sFv) fragment. In some embodiments, the ABP fragment is an scFv-Fc fragment. In some embodiments, the ABP fragment is a fragment of a single domain ABP.

[0221] In some embodiments, the ABP fragments provided herein are derived from the exemplary ABPs provided herein. In some embodiments, the ABP fragments provided herein are not derived from the exemplary ABPs provided herein, and can be isolated de novo, for example, according to the methods provided herein for obtaining ABP fragments.

[0222] In some embodiments, the ABP fragments provided herein retain the ability to bind to an HLA-PEPTIDE target, as measured by one or more of the assays or biological effects described herein. In some embodiments, the ABP fragments provided herein retain the ability to prevent HLA-PEPTIDE from interacting with one or more of its ligands, as described herein.

[0223] In some embodiments, the ABPs provided herein are monoclonal ABPs. In some embodiments, the ABPs provided herein are polyclonal ABPs.

[0224] In some embodiments, the ABP provided herein comprises a chimeric ABP. In some embodiments, the ABP provided herein consists of a chimeric ABP. In some embodiments, the ABP provided herein consists essentially of a chimeric ABP. In some embodiments, the ABP provided herein comprises a humanized ABP. In some embodiments, the ABP provided herein consists of a humanized ABP. In some embodiments, the ABP provided herein consists essentially of a humanized ABP. In some embodiments, the ABP provided herein comprises a human ABP. In some embodiments, the ABP provided herein consists of a human ABP. In some embodiments, the ABP provided herein consists essentially of a human ABP.

[0225] 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 can be used. In some aspects, the alternative scaffold is selected from Adnectin™, iMab, Anticalin®, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affibody®, DARPin, Affilin, Tetranectin, Fynomer, and Avimer.

[0226] Also disclosed herein are isolated humanized, human, or chimeric ABPs that compete with the ABPs disclosed herein for binding to HLA-PEPTIDEs.

[0227] Also disclosed herein are isolated humanized, human, or chimeric ABPs that bind to HLA-PEPTIDE epitopes bound via the ABPs disclosed herein.

[0228] In certain embodiments, the ABP comprises a human Fc region that includes at least one modification that reduces binding to a human Fc receptor.

[0229] It is known that ABPs are post-translationally modified when expressed in cells. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid at the N-terminus of the heavy and light chains to pyroglutamic acid by pyroglutamylation, glycosylation, oxidation, deamidation, and glycation. Such post-translational modifications are known to occur in various ABPs (Source: Journal of Pharmaceutical Sciences, 2008, Vol. 97, pp. 2426-2447, which is incorporated by reference in its entirety). In some embodiments, the ABP is an ABP or an antigen-binding fragment thereof that has undergone post-translational modification. Examples of ABPs or antigen-binding fragments thereof that have undergone post-translational modification include ABPs or antigen-binding fragments thereof that 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. As is known in the art, such post-translational modifications, when resulting from pyroglutamylation at the N-terminus and lysine deletion at the C-terminus, do not affect the activity of ABP or its fragments (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39, which is incorporated by reference in its entirety).

[0230] Monospecific and multispecific HLA-PEPTIDE ABPs In some embodiments, the ABPs provided herein are monospecific ABPs.

[0231] In some embodiments, the ABPs provided herein are multispecific ABPs.

[0232] In some embodiments, the multispecific ABPs provided herein bind to multiple antigens. In some embodiments, the multispecific ABPs bind to two antigens. In some embodiments, the multispecific ABPs bind to three antigens. In some embodiments, the multispecific ABPs bind to four antigens. In some embodiments, the multispecific ABPs bind to five antigens.

[0233] In some embodiments, the multispecific ABPs provided herein bind to multiple epitopes on the HLA-PEPTIDE antigen. In some embodiments, the multispecific ABPs bind to two epitopes on the HLA-PEPTIDE antigen. In some embodiments, the multispecific ABPs bind to three epitopes on the HLA-PEPTIDE antigen.

[0234] 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.

[0235] In some embodiments, the multispecific ABP comprises an immunoglobulin comprising at least two different heavy chain variable regions, each of which is 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 from each of the two different heavy chain variable regions. Source: Merchant et al., Nature Biotechnol., 1998, 16:677-681, which is incorporated by reference in its entirety.

[0236] In some embodiments, a multispecific ABP comprises an immunoglobulin comprising an ABP or a fragment thereof, wherein the ABP or a fragment thereof is attached to one or more of the N-terminus or C-terminus of a heavy or light chain of such an immunoglobulin. Source: Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163, which is incorporated by reference in its entirety. In some embodiments, such an ABP comprises a tetravalent bispecific ABP.

[0237] In some embodiments, the multispecific ABP comprises a hybrid immunoglobulin with at least two different heavy chain variable regions and at least two different light chain variable regions. Sources: 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.

[0238] In some embodiments, the multispecific ABP comprises immunoglobulin chains modified to reduce the formation of non-multispecific by-products. In some aspects, the ABP comprises one or more "knob-into-hole" modifications, as described in U.S. Patent No. 5,731,168, which is incorporated by reference in its entirety.

[0239] In some embodiments, the multispecific ABP comprises immunoglobulin chains with one or more electrostatic modifications to promote assembly of Fc heteromultimers. See International Publication No. WO2009 / 089004, which is incorporated by reference in its entirety.

[0240] 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.

[0241] In some embodiments, the multispecific ABP comprises heavy and light chain variable domains connected by a polypeptide linker, the length of which is selected to facilitate assembly of the multispecific ABP with the desired multispecificity. For example, monospecific scFvs are typically formed when the heavy and light chain variable domains are linked by a polypeptide linker of more than 12 amino acid residues. See U.S. Patent Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety. In some embodiments, when the polypeptide linker length is shortened to less than 12 amino acid residues, pairing of the heavy and light chain variable domains on the same polypeptide chain is prevented, allowing pairing of the heavy and light chain variable domains of one chain with the complementary domains of another chain. Therefore, the resulting ABP has multispecificity, with the specificity of each binding site contributed by multiple polypeptide chains. Polypeptide chains comprising heavy and light chain variable domains joined by a linker of 3 to 12 amino acid residues mostly form dimers (called diabodies). When the linker is 0 to 2 amino acid residues, trimers (called triabodies) and tetramers (called tetrabodies) predominate. However, the exact type of oligomerization depends not only on the length of the linker but also on the composition of amino acid residues and the order of the variable domains in each polypeptide chain (e.g., V H -Linker-V L Against V L -Linker-V H ) The skilled artisan will be able to select an appropriate linker length based on the desired multispecificity.

[0242] Fc Region and Variants In certain embodiments, the ABPs provided herein comprise an Fc region. The Fc region can be wild-type or a mutant thereof. In certain embodiments, the ABPs provided herein comprise an Fc region with one or more amino acid substitutions, insertions, or deletions compared to a naturally occurring Fc region. In some embodiments, such substitutions, insertions, or deletions result in an ABP with altered stability, glycosylation, or other properties. In some embodiments, such substitutions, insertions, or deletions result in a glycosylated ABP.

[0243] A "variant Fc region" or "engineered Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region through at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide (e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide). As used herein, a variant Fc region preferably has at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology to the native-sequence Fc region and / or the Fc region of a parent polypeptide.

[0244] The term "ABP comprising an Fc region" refers to an ABP comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during purification of the ABP or by recombinantly engineering a nucleic acid encoding the ABP. Thus, an ABP having an Fc region can include an ABP with or without K447.

[0245] In some aspects, the Fc region of the ABPs provided herein is modified to produce ABPs with altered affinity for Fc receptors or that are more immunologically inert. In some embodiments, the ABP variants provided herein retain some, but not all, effector functions. Such ABPs are useful, for example, when the half-life of the ABP is important in vivo, but not when certain effector functions (such as complement activation and ADCC) are unnecessary or deleterious.

[0246] In some embodiments, the Fc region of the ABP provided herein is a human IgG4 Fc region comprising one or more of the hinge-stabilizing mutations S228P and L235E. From: Aalberse et al., Immunology, 2002, 105:9-19, which is incorporated by reference in its entirety. In some embodiments, the IgG4 Fc region comprises one or more of the mutations E233P, F234V, and L235A. From: Armour et al., Mol. Immunol., 2003, 40:585-593, which is incorporated by reference in its entirety. In some embodiments, the IgG4 Fc region comprises a deletion at position G236.

[0247] In some embodiments, the Fc region of the ABP provided herein is a human IgG1 Fc region comprising one or more mutations that reduce Fc receptor binding. In some embodiments, the one or more mutations are at residues selected from S228 (e.g., S228A), L234 (e.g., L234A), L235 (e.g., L235A), D265 (e.g., D265A), and N297 (e.g., N297A). In some embodiments, the ABP comprises a PVA236 mutation. PVA236 refers to the substitution of the amino acid sequence ELLG (amino acids 233-236 of IgG1) or EFLG of IgG4 with PVA. Source: U.S. Patent No. 9,150,641, which is incorporated by reference in its entirety.

[0248] In some embodiments, modifications of the Fc region of the ABPs provided herein are as described in Armour et al., Eur. J. Immunol., 1999, 29:2613-2624; WO 1999 / 058572, and / or UK Patent Application No. 98099518, each of which is incorporated by reference in its entirety.

[0249] In some embodiments, the Fc region of an ABP provided herein is a human IgG2 Fc region comprising one or more of the mutations A330S and P331S.

[0250] In some embodiments, the Fc region of an ABP provided herein comprises an amino acid substitution at one or more positions selected from 238, 265, 269, 270, 297, 327, and 329. Source: U.S. Pat. No. 6,737,056, which is incorporated by reference in its entirety. Such Fc variants include Fc variants with substitutions at two or more of the amino acids at positions 265, 269, 270, 297, and 327, including so-called "DANA" Fc variants in which residues 265 and 297 are substituted with alanine. Source: U.S. Pat. No. 7,332,581, which is incorporated by reference in its entirety. In some embodiments, the ABP comprises an alanine at amino acid position 265. In some embodiments, the ABP comprises an alanine at amino acid position 297.

[0251] In certain embodiments, the ABPs provided herein comprise an Fc region with one or more amino acid substitutions to improve ADCC, including substitutions at one or more of the following positions in the Fc region: 298, 333, 334. In some embodiments, the ABPs provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330, as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010, which is incorporated by reference in its entirety.

[0252] In some embodiments, the ABPs provided herein contain one or more modifications that improve or reduce C1q binding and / or CDC. Sources: U.S. Patent No. 6,194,551; WO 99 / 51642; and Idusogie et al., J. Immunol., 2000, 164:4178-4184, each of which is incorporated by reference in its entirety.

[0253] In some embodiments, the ABPs provided herein comprise one or more modifications to extend half-life. ABPs with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described, for example, in Hinton et al., J. Immunol., 2006, 176:346-356, and U.S. Patent Application Publication No. 2005 / 0014934, each of which is incorporated by reference in its entirety. Such Fc variants include those that modify the following residues in the Fc region of IgG: and 434. In some embodiments, the ABP comprises one or more non-Fc modifications that enhance half-life. Exemplary half-life enhancing non-Fc modifications are described, for example, in U.S. Patent Application Publication No. 20170218078, which is incorporated by reference in its entirety.

[0254] In some embodiments, the ABPs provided herein comprise one or more Fc region variants described in U.S. Patent Nos. 7,371,826, 5,648,260, and 5,624,821; Duncan and Winter, Nature, 1988, 322:738-740; and WO 94 / 29351, each of which is incorporated by reference in its entirety.

[0255] B*35:01_EVDPIGHVY (HLA-PEPTIDE target "G5") specific antibody In some embodiments, the ABP provided herein comprises an antibody or antigen-binding fragment thereof that specifically binds to an HLA-PEPTIDE target, wherein the HLA class I molecule of the HLA-PEPTIDE target is of HLA subtype B*35:01, and the HLA-restricted peptide of the HLA-PEPTIDE target comprises, consists of, or consists essentially of the sequence EVDPIGHVY ("G5").

[0256] CDR An ABP specific for B*35:01_EVDPIGHVY can include one or more antibody complementarity determining region (CDR) sequences, for example, three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0257] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-H3 sequence. The CDR-H3 sequence may be: TIFF2026004411000044.tif55157.

[0258] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-L3 sequence. The CDR-L3 sequence may be: TIFF2026004411000045.tif33156.

[0259] An ABP specific for B*35:01_EVDPIGHVY may comprise a specific heavy chain CDR3 (CDR-H3) sequence and a specific light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP comprises CDR-H3 and CDR-L3 from scFvs designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01. The CDR sequences of identified scFvs that specifically bind to B*35:01_EVDPIGHVY are shown in Table 5. For clarity, each identified scFv hit is designated as a clone name, and each row contains the CDR sequences for that particular clone name. For example, the scFv identified with clone name G5_P7_E7 contains the heavy chain CDR3 sequence CARDGVRYYGMDVW and the light chain CDR3 sequence CMQGLQTPITF.

[0260] An ABP specific for B*35:01_EVDPIGHVY can comprise all six CDRs from scFvs designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01.

[0261] VH The ABP specific for B*35:01_EVDPIGHVY can comprise a VH sequence. TIFF2026004411000046.tif172159TIFF2026004411000047.tif215159.

[0262] VL The ABP specific for B*35:01_EVDPIGHVY can comprise a VL sequence. TIFF2026004411000048.tif231160TIFF2026004411000049.tif27165.

[0263] VH-VL combinations An ABP specific for B*35:01_EVDPIGHVY may comprise a particular VH sequence and a particular VL sequence. In some embodiments, the ABP specific for B*35:01_EVDPIGHVY comprises the VH and VL sequences from scFvs designated G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, and G5R4-P4B01. The VH and VL sequences of identified scFvs that specifically bind to B*35:01_EVDPIGHVY are shown in Table 4. For clarity, each identified scFv hit is designated as a clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv identified with clone name G5_P7_E7 has the VH sequence TIFF2026004411000050.tif19159 and VL sequence Includes TIFF2026004411000051.tif11157.

[0264] A*02:01_AIFPGAVPAA (antibody specific to HLA-PEPTIDE target "G8") In some embodiments, the ABP provided herein comprises an antibody or antigen-binding fragment thereof that specifically binds to an HLA-PEPTIDE target, wherein the HLA class I molecule of the HLA-PEPTIDE target is of HLA subtype A*02:01, and the HLA-restricted peptide of the HLA-PEPTIDE target comprises, consists of, or consists essentially of the sequence AIFPGAVPAA ("G8").

[0265] CDR An ABP specific for A*02:01_AIFPGAVPAA may include one or more antibody complementarity determining region (CDR) sequences, for example, three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0266] The ABP specific for A*02:01_AIFPGAVPAA may comprise a CDR-H3 sequence. The CDR-H3 sequence is TIFF2026004411000052.tif48154.

[0267] The ABP specific for A*02:01_AIFPGAVPAA may comprise a CDR-L3 sequence. The CDR-L3 sequence is TIFF2026004411000053.tif33157.

[0268] ABPs specific for A*02:01_AIFPGAVPAA can comprise specific heavy chain CDR3 (CDR-H3) and light chain CDR3 (CDR-L3) sequences. In some embodiments, the ABP comprises the CDR-H3 and CDR-L3 from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. The CDR sequences of identified scFvs that specifically bind to A*02:01_AIFPGAVPAA are shown in Table 7. For clarity, each identified scFv hit is designated as a clone name, and each row contains the CDR sequences for that particular clone name. For example, the scFv identified with clone name G8-P1A03 contains the heavy chain CDR3 sequence CARDDYGDYVAYFQHW and the light chain CDR3 sequence CQQNYNSVTF.

[0269] An ABP specific for A*02:01_AIFPGAVPAA can comprise all six CDRs from the scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11.

[0270] VH The ABP specific for A*02:01_AIFPGAVPAA may comprise a VH sequence. The VH sequence is It can be selected from TIFF2026004411000054.tif41159TIFF2026004411000055.tif245159TIFF2026004411000056.tif77159.

[0271] VL The ABP specific for A*02:01_AIFPGAVPAA may comprise a VL sequence. TIFF2026004411000057.tif143160TIFF2026004411000058.tif99159.

[0272] VH-VL combinations An ABP specific for A*02:01_AIFPGAVPAA can comprise a particular VH sequence and a particular VL sequence. In some embodiments, an ABP specific for A*02:01_AIFPGAVPAA comprises the VH and VL sequences from an scFv designated G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. The VH and VL sequences of identified scFvs that specifically bind to A*02:01_AIFPGAVPAA are shown in Table 6. For clarity, each identified scFv hit is designated as a clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv identified with clone name G8-P1A03 has the VH sequence TIFF2026004411000059.tif19159 and VL sequence Includes TIFF2026004411000060.tif12159.

[0273] A*01:01 ASSLPTTMNY (HLA-PEPTIDE target "G10") specific antibody In some embodiments, the ABPs provided herein comprise an antibody or antigen-binding fragment thereof that specifically binds to an HLA-PEPTIDE target, wherein the HLA class I molecule of the HLA-PEPTIDE target is of the HLA subtype A*01:01, and the HLA-restricted peptide of the HLA-PEPTIDE target comprises, consists of, or consists essentially of the sequence ASSLPTTMNY ("G10").

[0274] CDR An ABP specific for A*01:01_ASSLPTTMNY may include one or more antibody complementarity determining region (CDR) sequences, for example, three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0275] The ABP specific for A*01:01_ASSLPTTMNY may comprise a CDR-H3 sequence. The CDR-H3 sequence is TIFF2026004411000061.tif47148.

[0276] The ABP specific for A*01:01_ASSLPTTMNY may comprise a CDR-L3 sequence. The CDR-L3 sequence is TIFF2026004411000062.tif33158.

[0277] An ABP specific for A*01:01_ASSLPTTMNY may comprise a specific heavy chain CDR3 (CDR-H3) sequence and a specific light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP comprises the CDR-H3 and CDR-L3 from an scFv designated R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08. The CDR sequences of identified scFvs that specifically bind to A*01:01_ASSLPTTMNY are shown in Table 9. For clarity, each identified scFv hit is designated as a clone name, and each row contains the CDR sequences for that particular clone name. For example, the scFv identified with clone name R3G10-P1A07 contains the heavy chain CDR3 sequence CARDQDTIFGVVITWFDPW and the light chain CDR3 sequence CQQYFTTPYTF.

[0278] The ABPs specific for A*01:01_ASSLPTTMNY are R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, and R3G10-P3E04. , R3G10-P4A02, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08.

[0279] VH The ABP specific for A*01:01_ASSLPTTMNY may comprise a VH sequence. The VH sequence is TIFF2026004411000063.tif158159TIFF2026004411000064.tif230160.

[0280] VL The ABP specific for A*01:01_ASSLPTTMNY may comprise a VL sequence. TIFF2026004411000065.tif225159TIFF2026004411000066.tif33164.

[0281] VH-VL combinations ABPs specific for A*01:01_ASSLPTTMNY may comprise a particular VH sequence and a particular VL sequence. In some embodiments, ABPs specific for A*01:01_ASSLPTTMNY include R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P2G12, R3G10-P2G13, R3G10-P2G14, R3G10-P2G15, R3G10-P2G16, R3G10-P2G17, R3G10-P2G18, R3G10-P2G19, R3G10-P2G20, R3G10-P2G21, R3G10-P2G22, R3G10-P2G30, R3G10-P2G40, R3G10-P2G50, R3G10-P2G60, R3G10-P2G70, R3G10-P2G80, R3G10-P2G90, R3G10-P2G10, R3G10-P2G11, R3G10-P2G12, R3G10-P2G13, R3G10-P2G14, R3G10-P2G15, R3G10-P2G16, R3G10-P2G17, R3G10-P2G18, R3G10-P2G19, R3G10-P2G19, R3G10-P2G19, The VH and VL sequences from scFvs designated R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08 are included. The VH and VL sequences of identified scFvs that specifically bind to A*01:01_ASSLPTTMNY are shown in Table 8. For clarity, each identified scFv hit is designated as a clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv identified with clone name R3G10-P1A07 has the VH sequence TIFF2026004411000067.tif19159 and VL sequence Includes TIFF2026004411000068.tif12157.

[0282] receptor The provided ABP, for example, HLA-PEPTIDE ABP, has a receptor. The receptor can include antigen receptors and other chimeric receptors that specifically bind to the HLA-PEPTIDE target disclosed herein. The receptor can be a T cell receptor (TCR). The receptor can be a chimeric antigen receptor (CAR).

[0283] TCRs can be soluble or membrane-bound. Among antigen receptors, functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), are particularly prominent. Also provided are the use of cells expressing the receptors in cell therapy and adoptive cell therapy (e.g., for the treatment of diseases and disorders, such as cancer, associated with HLA-PEPTIDE expression).

[0284] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US201301493, and the like. 37, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March. 18(2): 160-75. In some embodiments, the CAR used as the antigen receptor is described in U.S. Patent No. 7,446,190, as well as in International Patent Application Publication No. WO / 2014055668(A1). Examples of CARs include those disclosed in any of the aforementioned publications, e.g., WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, U.S. Patent Application Publication No. 2013 / 0149337, U.S. Patent No. 7,446,190, and U.S. Patent No. 8,389,282, in which the antigen-binding portion, e.g., an scFv, is replaced by an antibody provided herein.

[0285] Chimeric receptors include chimeric antigen receptors (CARs). Chimeric receptors such as CARs generally comprise an extracellular antigen-binding domain, which comprises one of the provided anti-HLA-PEPTIDE ABPs, such as an anti-HLA-PEPTIDE antibody, is the provided anti-HLA-PEPTIDE ABP, or is contained within the provided anti-HLA-PEPTIDE ABP. Thus, chimeric receptors (e.g., CARs) typically comprise one or more HLA-PEPTIDE-ABPs (e.g., one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules as described herein) in their extracellular portion. In some embodiments, CARs comprise an HLA-PEPTIDE-binding portion or a portion of an ABP (e.g., an antibody) molecule (e.g., a variable heavy (VH) chain region and / or a variable light (VL) chain region of an antibody, such as an scFv antibody fragment).

[0286] TCR In one embodiment, an ABP provided herein (e.g., an ABP that specifically binds to an HLA-PEPTIDE target disclosed herein) comprises a T cell receptor (TCR). The TCR can be isolated and purified.

[0287] In the majority of T cells, the TCR is a heterodimeric polypeptide consisting of an alpha (α) chain and a beta (β) chain, encoded by TRA and TRB, respectively. The α chain typically contains an α variable region encoded by TRAV, an α binding region encoded by TRAJ, and an α constant region encoded by TRAC. The β chain typically contains a β variable region encoded by TRBV, a β diversity region encoded by TRBD, a β binding region encoded by TRBJ, and a β constant region encoded by TRBC. TCR-α chains are generated by VJ recombination, and β chain receptors are generated by V(D)J recombination. Additional TCR diversity results from junctional diversity. At each junction, some bases (called N and P nucleotides) are deleted, while others are added. A minority of T cells contain both γ and δ chains within their TCRs. The TCR γ chain is generated by V(D)J recombination, and the TCR δ chain is generated by V(D)J recombination (Kenneth Murphy, Paul Travers, and Mark Walport, Janeway's Immunology 7th edition, Garland Science, 2007, which is incorporated herein by reference in its entirety). The antigen-binding site of a TCR typically contains six complementarity-determining regions (CDRs). The α chain contributes three CDRs (αCDR1, αCDR2, and αCDR3). Similarly, the β chain contributes three CDRs (βCDR1, βCDR2, and βCDR3). αCDR3 and βCDR3 are the regions most affected by V(D)J recombination, resulting in fluctuations in the TCR repertoire.

[0288] A TCR can specifically recognize an HLA-PEPTIDE target (e.g., an HLA-PEPTIDE target disclosed in Table A). Therefore, a TCR is considered to be an ABP that specifically binds to an HLA-PEPTIDE. A TCR can be soluble, for example, like an antibody secreted by a B cell. A TCR can also be membrane-bound on a cell, for example, a T cell or a natural killer (NK) cell. Therefore, a TCR may be used in the context of a soluble antibody and / or a membrane-bound CAR.

[0289] Any of the TCRs disclosed herein may comprise an alpha variable region, an alpha joining region, optionally an alpha constant region, optionally a beta variable region, optionally a beta diversity region, a beta joining region, and optionally a beta constant region.

[0290] In some embodiments, the TCR or CAR is a recombinant TCR or CAR. The recombinant TCR or CAR can include any of the TCRs identified herein, but also include one or more modifications. Exemplary modifications, such as amino acid substitutions, are described herein. The amino acid substitutions described herein can be made with reference to the IMGT nomenclature and amino acid numbering. See www.imgt.org.

[0291] The recombinant TCR or CAR may be a human TCR or CAR that contains a fully human sequence, e.g., a naturally occurring human sequence. The recombinant TCR or CAR may retain its naturally occurring human variable domain sequence, but include modifications to the alpha constant region, the beta constant region, or both the alpha and beta constant regions. Such modifications to the TCR constant region may improve TCR assembly and expression for TCR gene therapy, for example, by driving preferential pairing of exogenous TCR chains.

[0292] In some embodiments, the α and β constant regions are modified by replacing the murine constant region sequences with the entire human constant region sequences. Such "murinized" TCRs and methods for making them are described in Cancer Res. 2006 Sep 1;66(17):8878-86, which is incorporated herein by reference in its entirety.

[0293] In some embodiments, the α and β constant regions are modified by replacing specific human residues with mouse residues (human-to-mouse amino acid exchange), by making one or more amino acid substitutions in the human TCR α constant (TRAC) region, TCR β constant (TRBC) region, or TRAC and TRAB region. The one or more amino acid substitutions in the TRAC region can include a Ser substitution at residue 90, an Asp substitution at residue 91, a Val substitution at residue 92, a Pro substitution at residue 93, or any combination thereof. The one or more amino acid substitutions in the human TRBC region can include a Lys substitution at residue 18, an Ala substitution at residue 22, an Ile substitution at residue 133, a His substitution at residue 139, or any combination thereof. Such targeted amino acid substitutions are described in J Immunol June 1, 2010, 184(11)6223-6231, which is incorporated herein by reference in its entirety.

[0294] In some embodiments, human TRAC contains an Asp substitution at residue 210, and human TRBC contains a Lys substitution at residue 134. Such substitutions may facilitate salt bridge formation between the α and β chains and TCR interchain disulfide bond formation. These targeted substitutions are described in J Immunol June 1, 2010, 184(11)6232-6241, which is incorporated herein by reference in its entirety.

[0295] In some embodiments, the human TRAC and human TRBC regions are modified to contain an introduced cysteine ​​that can improve preferential pairing of exogenous TCR chains by forming an additional disulfide bond. For example, human TRAC may contain a Cys substitution at residue 48, and human TRBC may contain a Cys substitution at residue 57. This is described in Cancer Res. 2007 Apr 15;67(8):3898-903 and Blood. 2007 Mar 15;109(6):2331-8, which are incorporated by reference in their entireties.

[0296] Other modifications to the α and β chains may also be made in the recombinant TCR or CAR.

[0297] In some embodiments, the α and β chains are modified by linking the extracellular domains of the α and β chains to a complete human CD3ζ (CD3-zeta) molecule. Such modifications are described in J Immunol June 1, 2008, 180(11)7736-7746; Gene Ther. 2000 Aug;7(16):1369-77; and Open Gene Therapy Journal, 2011, 4:11-22, which are incorporated by reference in their entireties.

[0298] In some embodiments, the alpha chain is modified by introducing hydrophobic amino acid substitutions in the transmembrane region of the alpha chain, as described in J Immunol June 1, 2012, 188(11)5538-5546, which is incorporated herein by reference in its entirety.

[0299] Modifications to the α or β chain can be made by modifying any one of the N-glycosylation sites within the amino acid sequence, as described in J Exp Med. 2009 Feb 16;206(2):463-475, which is incorporated herein by reference in its entirety.

[0300] The α and β chains may each comprise a dimerization domain, e.g., a heterodimerization domain. Such heterodimerization domains may be leucine zippers, 5H3 domains, or hydrophobic proline-rich counter domains, or other similar domains, as known in the art. In one example, the α and β chains can be modified by introducing 30-mer segments into the carboxyl termini of the α and β extracellular domains, thereby allowing the segments to selectively associate to form stable leucine zippers. Such modifications are described in PNAS November 22, 1994. 91(24)11408-11412; https: / / doi.org / 10.1073 / pnas.91.24.11408, which is incorporated herein by reference in its entirety.

[0301] The TCRs identified herein can be modified to contain mutations that result in increased affinity or half-life, including those described in WO2012 / 013913, which is incorporated herein by reference in its entirety.

[0302] A recombinant TCR or CAR may also be a single-chain TCR (scTCR). Such scTCRs may comprise an α chain variable region sequence fused to the N-terminus of a TCR α chain constant region extracellular sequence, a TCR β chain variable region fused to the N-terminus of a TCR β chain constant region extracellular sequence, and a linker sequence connecting the C-terminus of the α segment to the N-terminus of the β segment, or vice versa. In some embodiments, the constant region extracellular sequences of the α and β segments of the scTCR are linked by a disulfide bond. In some embodiments, the length of the linker sequence and the position of the disulfide bond are such that the variable region sequences of the α and β segments are oriented relative to each other substantially similarly to a native αβ T cell receptor. An exemplary scTCR is described in U.S. Patent No. 7,569,664, which is incorporated by reference herein in its entirety.

[0303] In some cases, the variable regions of the scTCR may be covalently linked by a short peptide linker, such as those described in Gene Therapy volume 7, pages 1369-1377 (2000). The short peptide linker may be a serine-rich linker or a glycine-rich linker. For example, the linker may be (Gly4Ser)3, as described in Cancer Gene Therapy (2004) 11, 487-496, which is incorporated by reference in its entirety.

[0304] A recombinant TCR or its antigen-binding fragment can be expressed as a fusion protein. For example, the TCR or its antigen-binding fragment can be fused to a toxin. Such a fusion protein is described in Cancer Res. 2002 Mar 15;62(6):1757-60. The TCR or its antigen-binding fragment can be fused to the Fc region of an antibody. Such a fusion protein is described in J Immunol May 1, 2017, 198(1 Supplement)120.9.

[0305] In some embodiments, the recombinant receptor, e.g., TCR or CAR (e.g., antibody portion thereof), further comprises a spacer, which may be or may comprise at least a portion of an immunoglobulin constant region or a mutant or modified form thereof (e.g., a hinge region, such as an IgG4 hinge region, and / or a CH1 / CL and / or Fc region). In some embodiments, the constant region or constant portion is derived from human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region functions as a spacer region between the antigen recognition component, e.g., an scFv, and the transmembrane domain. The length of the spacer may be such that it enhances the responsiveness of cells after antigen binding compared to when the spacer is absent. In some examples, the length of the spacer is about 12 amino acids or less. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids (including any integer between any endpoints of the recited ranges). In some embodiments, the spacer region has no more than about 12 amino acids, no more than about 119 amino acids, or no more than about 229 amino acids. Exemplary spacers include an IgG4 hinge only, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, or International Patent Application Publication No. WO2014031687. In some embodiments, the constant region or constant moiety is IgD.

[0306] The antigen recognition domain of a receptor (e.g., a TCR or a CAR) can be linked to one or more intracellular signaling components (e.g., signaling components). In the case of a CAR, these intracellular signaling components mimic activation via an antigen receptor complex (e.g., a TCR complex) and / or signaling via another cell surface receptor. Thus, in some embodiments, an HLA-PEPTIDE-specific binding component (e.g., an ABP, such as an antibody or TCR) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the receptor (e.g., a CAR) is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution, thereby avoiding binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimizing interaction with other members of the receptor complex.

[0307] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain in some aspects is derived from a membrane-bound or transmembrane protein. Transmembrane regions include those derived from the α, β, or ζ chain (i.e., comprising at least the transmembrane region(s)) of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain in some embodiments is a synthetic transmembrane domain. In some aspects, synthetic transmembrane domains are dominated by hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at both ends of the synthetic transmembrane domain. In some embodiments, the linkage is via a linker, spacer, and / or transmembrane domain(s).

[0308] Prominent intracellular signaling domains are those that mimic or approximate signaling via natural antigen receptors, signaling via such receptors in combination with costimulatory receptors, and / or signaling via costimulatory receptors alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2-10 amino acids in length, such as one containing glycine and serine (e.g., a glycine-serine doublet), is present between the transmembrane domain and the cytoplasmic signaling domain of the receptor to form the link.

[0309] A receptor, e.g., a TCR or CAR, can comprise at least one intracellular signaling component. In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as the TCR CD3 chain, which mediates T cell activation and cytotoxicity, e.g., the CD3ζ chain. Thus, in some embodiments, an HLA-PEPTIDE-binding ABP (e.g., an antibody) is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., a CAR, further comprises one or more additional molecules, e.g., a portion of Fc receptor-gamma, CD8, CD4, CD25, or CD16. For example, in some embodiments, the CAR comprises a chimeric molecule between CD3ζ or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0310] In some embodiments, upon ligation of the TCR or CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the receptor. For example, in some situations, the receptor induces a T cell function, e.g., cytolytic activity or T helper activity (such as secretion of cytokines or other factors). In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., if it transmits an effector function signal. In some embodiments, the intracellular signaling domain(s) include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, the cytoplasmic sequence of a co-receptor that, in its natural context, acts in concert with such receptor to initiate signaling following antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capability.

[0311] In the context of native TCRs, full activation typically requires not only TCR-mediated signaling but also a costimulatory signal. Thus, in some embodiments, the receptor also includes components for generating a secondary or costimulatory signal to promote full activation. In other embodiments, the receptor does not include components for generating a costimulatory signal. In some aspects, additional receptors are expressed in the same cell and provide components for generating a secondary or costimulatory signal.

[0312] In some embodiments, T cell activation has been described to be mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a receptor contains one or both of such signaling components.

[0313] In some embodiments, the receptor comprises a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. The stimulatory primary cytoplasmic signaling sequence may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that comprise primary cytoplasmic signaling sequences include those derived from TCR or CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) of the CAR comprises a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0314] In some embodiments, the receptor comprises the signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same receptor comprises both an activating component and a costimulatory component.

[0315] In some embodiments, the activation domain is contained in one receptor, but the costimulatory component is provided by another receptor that recognizes a different antigen. In some embodiments, the receptor includes an activating or stimulatory receptor and a costimulatory receptor, both of which are expressed in the same cell (Source: WO2014 / 055668). The HLA-PEPTIDE-targeted receptor is, in some aspects, a stimulatory or activation receptor, and in other aspects, a costimulatory receptor. In some embodiments, the cell further includes an inhibitory receptor, such as an iCAR (e.g., Source: Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), e.g., a receptor that recognizes an antigen other than HLA-PEPTIDE, thereby attenuating or inhibiting the activation signal delivered via the HLA-PEPTIDE-targeted receptor by binding of the inhibitory receptor to its ligand, thus, for example, reducing off-target effects.

[0316] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain, hi some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3zeta intracellular domain.

[0317] In some embodiments, the receptor includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary receptors include the intracellular components of CD3ζ, CD28, and 4-1BB.

[0318] In some embodiments, the CAR or other antigen receptor, such as a TCR, further comprises a marker, such as a cell surface marker. This marker can be used to transduce or engineer cells to confirm the expression of the receptor, including, for example, a truncated form of the cell surface receptor, such as truncated EGFR (tEGFR). In some embodiments, the marker includes all or a portion (e.g., a truncated form) of CD34, nerve growth factor receptor (NGFR), or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a cleavable linker sequence or a ribosomal skip sequence (e.g., a linker sequence such as T2A). See WO2014031687. In some embodiments, two proteins may be expressed from the same construct by introducing a construct encoding a CAR and EGFRt separated by a T2A ribosomal switch. The use of EGFRt as a marker allows for the detection of cells expressing such a construct. In some embodiments, the marker, and optionally the linker sequence, can be any of those disclosed in Patent Application Publication No. WO 2014031687. For example, the marker can be a truncated EGFR (tEGFR) optionally linked to a linker sequence, such as a T2A ribosomal skip sequence.

[0319] In some embodiments, the marker is a molecule (eg, a cell surface protein) or portion thereof that is not naturally found on or on the surface of a T cell.

[0320] In some embodiments, the molecule is a non-self molecule (eg, a non-self protein), ie, one that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0321] In some embodiments, the marker serves as a marker for genetic manipulation (e.g., selection of successfully manipulated cells) but does not otherwise serve any therapeutic function and / or effect, while in other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some other desired effect (e.g., a ligand for cells encountered in vivo, e.g., a costimulatory or immune checkpoint molecule to enhance and / or suppress the response of cells upon adoptive transfer and encounter with the ligand).

[0322] A TCR or CAR may contain one or more modified synthetic amino acids in place of one or more naturally occurring amino acids. Exemplary modified amino acids include, but are not limited to, aminocyclohexanecarboxylic acid, norleucine, alpha-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, 3-phenylserine, 3-hydroxyphenylalanine, phenylglycine, alpha-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indolylalanine, cyclohexylalanine, cyclohexylglycine, cyclohexylglycerin ... 2-amino-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tertbutylglycine.

[0323] In some cases, CARs are referred to as first-, second-, and / or third-generation CARs. In some embodiments, a first-generation CAR is a CAR that provides only a CD3 chain-inducing signal upon antigen binding. In some embodiments, a second-generation CAR provides such a signal as well as a costimulatory signal (e.g., one that comprises an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137). In some embodiments, a third-generation CAR in some embodiments is a CAR that comprises multiple costimulatory domains from different costimulatory receptors.

[0324] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv or single-domain VH antibody, and the intracellular domain comprises an ITAM. In some embodiments, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0325] In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. The extracellular domain and the transmembrane may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer as described herein. In some embodiments, the chimeric antigen receptor comprises the intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB.

[0326] In some embodiments, a CAR comprises an antibody (such as an antibody fragment), a transmembrane domain that is or comprises the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that comprises the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some embodiments, a CAR comprises an antibody (such as an antibody fragment), a transmembrane domain that is or comprises the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that comprises the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer that includes a portion of an Ig molecule (such as a human Ig molecule, such as an Ig hinge (e.g., an IgG4 hinge)), such as a hinge-only spacer.

[0327] In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession Number: P10747.1).

[0328] In some embodiments, the chimeric antigen receptor comprises the intracellular domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB.

[0329] In some embodiments, the intracellular signaling domain includes the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof (e.g., the 41 amino acid domain thereof), and / or a species domain thereof having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular domain includes the intracellular costimulatory signaling domain of 41BB or a functional variant thereof, or a portion thereof (e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant thereof, or a portion thereof).

[0330] In some embodiments, the intracellular signaling domain includes the human CD3ζ stimulatory signaling domain or a functional variant thereof, such as the 112AA cytoplasmic domain of human CD3ζ isoform 3 (Accession Number: P20963.2) or the CD3ζ signaling domain described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0331] In some embodiments, the spacer comprises only the hinge region of an IgG (e.g., only an IgG4 or IgG1 hinge). In other embodiments, the spacer is an Ig hinge linked to the CH2 and / or CH3 domains, e.g., an IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked to the CH2 and CH3 domains, e.g., an IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked to the CH3 domain only, e.g., an IgG4 hinge. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker (e.g., a known flexible linker).

[0332] For example, in some embodiments, a CAR comprises an antibody or fragment thereof (e.g., any of the HLA-PEPTIDE antibodies), e.g., a single chain antibody (sdAb, e.g., containing only the VH region) and an scFv as described herein, a spacer such as a spacer comprising an Ig hinge, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, a CAR comprises an antibody or fragment thereof (e.g., any of the HLA-PEPTIDE antibodies), e.g., a sdAb and an scFv as described herein, a spacer such as a spacer comprising an Ig hinge, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3ζ signaling domain.

[0333] TCR targeting specific for A*01:01_ ASSLPTTMNY (SEQ ID NO:) [G10] In some embodiments, the ABPs provided herein comprise a TCR or antigen-binding fragment thereof that specifically binds to an HLA-PEPTIDE target, wherein the HLA class I molecule of the HLA-PEPTIDE target is of HLA subtype A*01:01, and the HLA-restricted peptide of the HLA-PEPTIDE target comprises ASSLPTTMNY ("G10").

[0334] A TCR specific for A*01:01_ASSLPTTMNY may comprise an αCDR3 sequence. The αCDR3 sequence may be any of the αCDR3 sequences in Table 15. The α and βCDR3 sequences of the identified TCR clonotypes are as shown in Table 15.

[0335] A TCR specific for A*01:01_ASSLPTTMNY may comprise a βCDR3 sequence. The βCDR3 sequence may be any of the βCDR3 sequences in Table 15.

[0336] A TCR specific for A*01:01_ASSLPTTMNY may comprise a particular αCDR3 sequence and a particular βCDR3 sequence. For example, a TCR specific for A*01:01_ASSLPTTMNY may comprise an αCDR3 sequence and a βCDR3 sequence from any one of the TCRs identified in Table 15. For clarity, each identified TCR has been assigned a TCR ID number. For example, TCR ID#1 comprises the αCDR3 sequence CAGPGNTGKLIF and the βCDR3 sequence CASSNAGDQPQHF.

[0337] A TCR specific for A*01:01_ASSLPTTMNY may comprise a TRAV, TRAJ, TRBV, optionally a TRBD, and a TRBJ amino acid sequence, optionally a TRAC sequence, and optionally a TRBC sequence. For example, a TCR specific for A*01:01_ASSLPTTMNY may comprise a TRAV, TRAJ, TRBV, TRBD, and a TRBJ amino acid sequence, a TRAC sequence, and a TRBC sequence from any one of the TCRs identified in Table 14. For clarity, each identified TCR has been assigned a TCR ID number. For example, TCR ID#1 assigned to TCR includes the TRAV25 sequence, the TRAJ37 sequence, the TRAC sequence, the TRBV19 sequence, the TRBD1 sequence, the TRBJ1-5 sequence, and the TRBC1 sequence.

[0338] A TCR specific for A*01:01_ASSLPTTMNY may comprise an αVJ sequence. The αVJ sequence may be any of the αVJ sequences in Table 16.

[0339] A TCR specific for A*01:01_ASSLPTTMNY may comprise a βV(D)J sequence. The βV(D)J sequence may be any of the βV(D)J sequences in Table 16.

[0340] A TCR specific for A*01:01_ASSLPTTMNY may comprise an αVJ sequence and a βV(D)J sequence. For example, a TCR specific for A*01:01_ASSLPTTMNY may comprise an αVJ sequence and a βV(D)J sequence from any one of the TCRs identified in Table 16. The full-length αV(J) and full-length βV(D)J sequences of the identified TCR clonotypes are as shown in Table 16. For example, TCRID#1 contains the αV(J) sequence TIFF2026004411000069.tif19158 and βV(D)J sequence Includes TIFF2026004411000070.tif19159.

[0341] A*01:01_HSEVGLPVY target-specific TCR In some embodiments, the ABP provided herein comprises a TCR or antigen-binding fragment thereof that specifically binds to an HLA-PEPTIDE target, wherein the HLA class I molecule of the HLA-PEPTIDE target is of HLA subtype A*01:01, and the HLA-restricted peptide of the HLA-PEPTIDE target comprises HSEVGLPVY.

[0342] A TCR specific for A*01:01_HSEVGLPVY may comprise an αCDR3 sequence, which may be any one of the αCDR3 sequences in Table 18. The α and βCDR3 sequences of the identified TCR clonotypes are as shown in Table 18.

[0343] A TCR specific for A*01:01_HSEVGLPVY may comprise a β CDR3 sequence. The β CDR3 sequence may be any one of the β CDR3 sequences in Table 18.

[0344] A TCR specific for A*01:01_HSEVGLPVY may comprise a particular αCDR3 sequence and a particular βCDR3 sequence. For example, a TCR specific for A*01:01_HSEVGLPVY may comprise the αCDR3 sequence and the βCDR3 sequence from any one of the TCRs identified in Table 18. For clarity, each identified TCR has been assigned a TCR ID number. For example, TCR ID #345 comprises the αCDR3 sequence CAANPGDYKLSF and the βCDR3 sequence CASSSNYEQYF.

[0345] A TCR specific for A*01:01_HSEVGLPVY may comprise the TRAV, TRAJ, TRBV, optionally the TRBD and TRBJ amino acid sequences, optionally the TRAC sequence, and optionally the TRBC sequence. For example, a TCR specific for A*01:01_HSEVGLPVY may comprise the TRAV, TRAJ, TRBV, TRBD, TRBJ amino acid sequences, the TRAC sequence, and the TRBC sequence from any one of the TCRs identified in Table 17. For clarity, each identified TCR has been assigned a TCR ID number. For example, TCR ID # 345 assigned to TCR includes the TRAV13-1 sequence, the TRAJ20 sequence, the TRAC sequence, the TRBV7-9 sequence, the TRBJ2-7 sequence, and the TRBC2 sequence.

[0346] A TCR specific for A*01:01_HSEVGLPVY may comprise an αVJ sequence, which may be any one of the αVJ sequences in Table 19.

[0347] A TCR specific for A*01:01_HSEVGLPVY may comprise a βV(D)J sequence. The βV(D)J sequence may be any of the βV(D)J sequences in Table 19.

[0348] A TCR specific for A*01:01_HSEVGLPVY may comprise an αVJ sequence and a βV(D)J sequence. For example, a TCR specific for A*01:01_HSEVGLPVY may comprise an αVJ sequence and a βV(D)J sequence from any one of the TCRs identified in Table 19. The full-length αV(J) and full-length βV(D)J sequences of the identified TCR clonotypes are as depicted in Table 19. For example, TCR ID # 345 contains the αV(J) sequence TIFF2026004411000071.tif18163 and βV(D)J sequence Includes TIFF2026004411000072.tif19164.

[0349] Engineered cells Also provided are cells, such as cells comprising an antigen receptor, e.g., comprising an extracellular domain comprising an anti-HLA-PEPTIDE ABP (e.g., CAR or TCR) described herein. Also provided are populations of such cells and compositions comprising such cells. In some embodiments, the composition or population is enriched for cells, e.g., HLA-PEPTIDE ABP-expressing cells, comprising at least 1 percent, 5 percent, 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or greater than 99 percent of the total cells in the composition, or for a particular type of cell, such as T cells or CD8+ or CD4+ cells. In some embodiments, the composition comprises at least one cell comprising an antigen receptor disclosed herein. Among the compositions are pharmaceutical compositions and formulations for administration, such as adoptive cell therapy. Therapeutic methods for administering the cells and compositions to a subject, eg, a patient, are also provided.

[0350] Therefore, genetically engineered cells expressing ABPs, including receptors such as TCRs or CARs, are also provided. The cells are generally eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, cells derived from blood, bone marrow, lymph, or lymphoid organs are cells of the immune system (e.g., innate cells) or adaptive immunity (e.g., lymphocytes, including myeloid or lymphocytes, typically T cells and / or NK cells). Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). These cells are typically primary cells, such as those isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, defined by, for example, function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject receiving treatment, the cells can be allogeneic and / or autologous. Prominent among the methods are off-the-shelf methods. In some aspects, e.g., in the case of off-the-shelf technologies, the cells are pluripotent and multipotent, e.g., stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, treating, culturing, and / or manipulating the cells as described herein, and reintroducing them into the same patient, before or after cryopreservation.

[0351] Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells, particularly prominent are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated), effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MALT) cells, spontaneous and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, δ / γ T cells.

[0352] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0353] Cells can be genetically modified to reduce expression or knock out endogenous TCRs. Such modifications are described in Mol Ther Nucleic Acids. 2012 Dec;1(12):e63; Blood. 2011 Aug 11;118(6):1495-503; Blood. 2012 Jun 14;119(24):5697-5705; Torikai, Hiroki et al "HLA and TCR Knockout by Zinc Finger Nucleases: Toward "off-the-Shelf" Allogeneic T-Cell Therapy for CD19+ Malignancies...." Blood 116.21(2010):3766; Blood. 2018 Jan 18;131(3):311-322. doi:10.1182 / blood-2017-05-787598, and WO2016069283, which are incorporated by reference in their entireties.

[0354] The cells may be genetically modified to enhance cytokine secretion. Such modifications have been reported in Hsu C, Hughes MS, Zheng Z, Bray RB, Rosenberg SA, Morgan RA. Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine. J Immunol. 2005;175:7226-34; Quintarelli C, Vera JF, Savoldo B, Giordano Attianese GM, Pule M, Foster AE. Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes. Blood. 2007;110:2793-802; and Hsu C, Jones SA, Cohen CJ, Zheng Z, Kerstann K, Zhou J. Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene. Blood. 2007;109:5168-77.

[0355] Discrepancies between chemokine receptors on T cells and tumor-secreted chemokines have been shown to be a major factor in suboptimal trafficking of T cells into the tumor microenvironment. To improve therapeutic efficacy, cells can be genetically engineered to enhance chemokine recognition in the tumor microenvironment. Examples of such modifications are described in Moon et al., "Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor," Clin Cancer Res. 2011;17:4719-4730; and Craddock et al., "Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b," J Immunother. 2010:33780-788.

[0356] Cells can be genetically modified to enhance expression of costimulatory / enhancing receptors such as CD28 and 41BB.

[0357] Adverse effects of T cell therapy can include cytokine release syndrome and prolonged B cell depletion. Introducing a suicide / safety switch into recipient cells may improve the safety profile of cell-based therapies. Thus, cells may be genetically engineered to contain a suicide / safety switch. A suicide / safety switch can be a gene that sensitizes the expressed cell to an agent, such as a drug, causing the cell to die when the cell comes into contact with or comes into contact with the agent. An exemplary suicide / safety switch is described in Protein Cell. 2017 Aug;8(8):573-589. The suicide / safety switch can be HSV-TK. The suicide / safety switch can be cytosine deaminase, purine nucleoside phosphorylase, or nitroreductase. The suicide / safety switch can be RapaCIDe™, which is described in U.S. Patent Application Publication No. US20170166877A1. The suicide / safety switch system can be CD20 / rituximab. This is described in Haematologica. 2009 Sep;94(9):1316-1320. These references are incorporated by reference in their entirety.

[0358] The TCR or CAR can be introduced into recipient cells as split receptors that assemble only in the presence of a heterodimerizing small molecule. Such a system is described in Science. 2015 Oct 16;350(6258):aab4077 and U.S. Patent No. 9,587,020, which are incorporated by reference in their entireties.

[0359] In some embodiments, the cells comprise one or more nucleic acids, e.g., polynucleotides encoding a TCR or CAR disclosed herein, which polynucleotides have been introduced by genetic engineering to thereby express a recombinant or engineered TCR or CAR disclosed herein. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as one obtained from another organism or cell, e.g., not normally found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is a non-naturally occurring (e.g., not found in nature) nucleic acid, including, for example, one that comprises a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0360] Nucleic acids may include codon-optimized nucleotide sequences. Without being bound by any particular theory or mechanism, codon optimization of nucleotide sequences is believed to enhance the translation efficiency of mRNA transcripts. Codon-optimizing a nucleotide sequence may involve replacing a codon encoding the same amino acid with a naturally occurring codon, allowing translation to occur via tRNAs that are more readily available in cells, improving translation efficiency. Codon-optimizing a nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, improving translation efficiency.

[0361] A construct or vector may be used to introduce the TCR or CAR into recipient cells. Exemplary constructs are described herein. The polynucleotides encoding the α and β chains of the TCR or CAR may be in a single construct or separate constructs. The polynucleotides encoding the α and β chains may be operably linked to a promoter, e.g., a heterologous promoter. The heterologous promoter may be a strong promoter, e.g., EF1α, CMV, PGK1, Ubc, β-actin, CAG promoter, and the like. The heterologous promoter may be a weak promoter. The heterologous promoter may be an inducible promoter. Exemplary inducible promoters include, but are not limited to, TRE, NFAT, GAL4, LAC, and the like. Other exemplary inducible expression systems are described in U.S. Patent Nos. 5,514,578, 6,245,531, 7,091,038, and European Patent No. 0517805, which are incorporated by reference in their entireties.

[0362] Constructs for introducing a TCR or CAR into recipient cells may also include a polynucleotide encoding a signal peptide (signal peptide element). Signal peptides can facilitate surface transport of the introduced TCR or CAR. Examples include, but are not limited to, CD8 signal peptide and immunoglobulin signal peptide, specific examples of which include GM-CSF and IgGκ. Such signal peptides are described in Trends Biochem Sci. 2006 Oct;31(10):563-71, Epub 2006 Aug 21, and An, et al. "Construction of a New Anti-CD19 Chimeric Antigen Receptor and the Anti-Leukemia Function Study of the Transduced T Cells," Oncotarget 7.9(2016):10638-10649. PMC.Web. 16 Aug. 2018, which are incorporated herein by reference in their entirety.

[0363] In some cases, for example, when the α chain and the β chain are expressed from a single construct or open reading frame, or when a marker gene is included in the construct, the construct may include a ribosome skipping sequence. The ribosome skipping sequence may be a 2A peptide, such as a P2A or T2A peptide. Exemplary P2A and T2A peptides are described in Scientific Reports volume 7, Article number: 2193 (2017), which is incorporated by reference in its entirety. In some cases, a FURIN / PACE cleavage site is introduced upstream of the 2A element. FURIN / PACE cleavage sites are described, for example, at http: / / www.nuolan.net / substrates.html. The cleavage peptide may also be a factor Xa cleavage site. When the α chain and the β chain are expressed from a single construct or open reading frame, the construct may include an internal ribosome entry site (IRES).

[0364] The construct may further comprise one or more marker genes. Exemplary marker genes include, but are not limited to, GFP, luciferase, HA, and lacZ. The marker may be a selectable marker known to those skilled in the art, such as an antibiotic resistance marker, a heavy metal resistance marker, or a biocide resistance marker. The marker may be a complementing marker for use in an auxotrophic host. Exemplary complementing markers and auxotrophic hosts are described in Gene. 2001 Jan 24;263(1-2):159-69. Such markers may be expressed via fusion with an IRES, a frameshift sequence, a 2A peptide linker, a TCR, or a CAR, or may be expressed separately from a separate promoter.

[0365] Exemplary vectors or systems for introducing a TCR or CAR into recipient cells include, but are not limited to, adeno-associated virus, adenovirus, adenovirus plus modified vaccinia, Ankara virus (MVA), adenovirus plus retrovirus, adenovirus plus Sendai virus, adenovirus plus vaccinia virus, alphavirus (VEE) replicon vaccines, antisense oligonucleotides, Bifidobacterium longum, CRISPR-Cas9, Escherichia coli (E. coli), flavivirus, gene gun, herpesvirus, herpes simplex virus, Lactococcus lactis, electroporation, lentivirus, lipofection, Listeria monocytogenes, measles virus, modified vaccinia virus, and the like. Vaccinia Ankara virus (MVA), mRNA electroporation, naked / plasmid DNA, naked / plasmid DNA + adenovirus, naked / plasmid DNA + modified vaccinia Ankara virus (MVA), naked / plasmid DNA + RNA transfer, naked / plasmid DNA + vaccinia virus, naked / plasmid DNA + vesicular stomatitis virus, Newcastle disease virus, non-viral, PiggyBac™ (PB) transposon, nanoparticle-based system, poliovirus, poxvirus, poxvirus + vaccinia virus, retrovirus, RNA transfer, RNA transfer + naked / plasmid DNA, RNA virus, Saccharomyces cerevisiae cerevisiae, Salmonella typhimurium, Semliki Forest virus, Sendai virus, Shigella dysenteriae, Simian virus, siRNA, Sleeping Beauty transposon, Streptococcus mutans, vaccinia virus, Venezuelan equine encephalitis virus replicon, vesicular stomatitis virus, and Vibrio cholera.

[0366] In preferred embodiments, the TCR or CAR is introduced into recipient cells via adeno-associated virus (AAV), adenovirus, CRISPR-CAS9, herpesvirus, lentivirus, lipofection, mRNA electroporation, PiggyBac™ (PB) transposon, retrovirus, RNA transfer, or Sleeping Beauty transposon.

[0367] In some embodiments, the vector for introducing TCR or CAR into recipient cells is a viral vector.Exemplary viral vectors include adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, herpesvirus vectors, retroviral vectors, and the like.Such vectors are described herein.

[0368] Exemplary embodiments of TCR constructs for introducing a TCR or CAR into recipient cells are shown in Figure 2. In some embodiments, a TCR construct includes, in a 5'-3' direction, a polynucleotide sequence: a promoter sequence, a signal peptide sequence, a TCR beta variable (TCRβv) sequence, a TCR beta constant (TCRβc) sequence, a truncated peptide (e.g., P2A), a signal peptide sequence, a TCR alpha variable (TCRαv) sequence, and a TCR alpha constant (TCRαc) sequence. In some embodiments, the TCRβc and TCRαc sequences of the construct include one or more mouse regions, e.g., the complete mouse constant sequence or a human-to-mouse amino acid exchange as described herein. In some embodiments, In some embodiments, the construct further comprises, 3' to the TCRαc sequence, a cleavage peptide sequence (e.g., T2A), followed by a reporter gene. In one embodiment, the construct comprises, in the 5'-3' direction, a polynucleotide sequence: a promoter sequence, a signal peptide sequence, a TCRβ variable (TCRβv) sequence, a TCRβ constant (TCRβc) sequence comprising one or more mouse regions, a cleavage peptide (e.g., P2A), a signal peptide sequence, a TCRα variable (TCRαv) sequence, and a TCRα constant (TCRαc) sequence comprising one or more mouse regions, a cleavage peptide (such as T2A), and a reporter gene.

[0369] An exemplary construct backbone sequence for cloning TCRs into expression systems for therapeutic development is depicted in FIG.

[0370] An exemplary construct sequence for cloning the identified A*0201_LLASSILCA-specific TCR into an expression system for therapeutic development purposes is depicted in FIG. 4.

[0371] An exemplary construct sequence for cloning the identified A*0101_EVDPIGHLY-specific TCR into an expression system for therapeutic development purposes is depicted in FIG. 5.

[0372] Nucleotides, Vectors, Host Cells, and Related Methods Also provided are isolated nucleic acids encoding HLA-PEPTIDE ABPs, vectors containing the nucleic acids, and host cells containing the vectors and nucleic acids, as well as recombinant techniques for producing the ABPs.

[0373] Nucleic acids can be recombinant. Recombinant nucleic acids can be constructed outside of living cells by joining natural or synthetic nucleic acid segments to a nucleic acid molecule capable of replicating within a living cell, or the product of that replication. For purposes herein, replication can be in vitro or in vivo.

[0374] For recombinant production of an ABP, the nucleic acid(s) encoding the ABP may be isolated and inserted into a replicable vector for replication (i.e., amplification of the DNA) or expression. In some embodiments, the nucleic acid may be generated by homologous recombination, as described, for example, in U.S. Patent No. 5,204,244, which is incorporated by reference in its entirety.

[0375] Many different vectors are known in the art. Vector components typically include one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. This is described, for example, in U.S. Patent No. 5,534,615, which is incorporated by reference in its entirety.

[0376] Exemplary vectors or constructs suitable for expressing ABPs (e.g., TCRs, CARs, antibodies, or antigen-binding fragments thereof) include, for example, 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 AGT10, AGT1 1, AZapII (Stratagene), AEMBL4, and ANM1 149 are also suitable for expressing the ABPs disclosed herein.

[0377] Illustrative examples of suitable host cells are provided below. These host cells are not meant to be limiting, and any suitable host cell can be used to produce the ABPs provided herein.

[0378] Suitable host cells include prokaryotic (eg, bacterial), lower eukaryotic (eg, yeast), or higher eukaryotic (eg, mammalian) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive bacteria, for example, Escherichia (E. coli), Enterobacter, 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 E. coli B, E. coli X1776, and E. coli W3110 are also suitable.

[0379] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding HLA-PEPTIDE ABPs. Saccharomyces cerevisiae (i.e., common baker's yeast) is a frequently used lower eukaryotic host microorganism. However, there are many other genera, species, and strains that are available and useful, such as Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, and others. crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

[0380] 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.

[0381] The host cells used to produce HLA-PEPTIDE ABPs can be cultured in a variety of media. For example, commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing 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 aforementioned references is incorporated by reference in its entirety.

[0382] Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (usually defined as inorganic compounds) present at final concentrations in the micromolar range, and glucose or an equivalent energy source. Other necessary supplements may also be included at appropriate concentrations known to those of skill in the art.

[0383] Culture conditions of temperature, pH, and the like will be those previously used for the host cell selected for expression and will be apparent to one of skill in the art.

[0384] When using recombinant techniques, ABPs can be produced intracellularly in the periplasmic space or directly secreted into the medium. If ABPs are produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Procedures for isolating ABPs secreted into the periplasmic space of E. coli are described, for example, in Carter et al. (Bio / Technology, 1992, 10:163-167), the entire contents of which are incorporated by reference. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris may be removed by centrifugation.

[0385] In some embodiments, the ABP is produced in a cell-free system. In some embodiments, the cell-free system is an in vitro transcription and translation system, as described in Yin et al., mAbs, 2012, 4:217-225, which is incorporated by reference in its entirety. In some embodiments, the cell-free system utilizes a cell-free extract from a eukaryotic or prokaryotic cell. In some embodiments, the prokaryotic cell is E. coli. Cell-free expression of the ABP can be useful, for example, when the ABP accumulates intracellularly as insoluble aggregates or when the yield from periplasmic expression is low.

[0386] When ABPs are secreted into the medium, supernatants from such expression systems are typically first concentrated using commercially available protein concentration filters, such as Amicon® or Millipore® Pellcon® ultrafiltration units. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0387] ABP compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Affinity chromatography is a particularly useful purification technique. The suitability of Protein A as an affinity ligand varies depending on the type and isotype of immunoglobulin Fc domain present in the ABP. Protein A may be used to purify ABPs containing human γ1, γ2, or γ4 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 human γ3 (Guss et al., EMBO J., 1986, 5:1567-1575, incorporated by reference in its entirety).

[0388] The substrate to which the affinity ligand is attached is most often agarose, although other substrates may be used. Mechanically stable substrates such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. ABP is C H3 If the domain is present, BakerBond ABX® resin is useful for purification.

[0389] Other protein purification techniques, such as fractionation on ion exchange columns, 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.

[0390] After the preliminary purification step(s), the mixture containing the ABP of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography (typically performed at low salt concentrations, e.g., about 0 to about 0.25 M salt) using an elution buffer at a pH of about 2.5 to about 4.5.

[0391] Method for producing HLA-PEPTIDE ABP Preparation of HLA-PEPTIDE antigens The HLA-PEPTIDE antigen used to isolate or generate the ABPs provided herein can be an intact HLA-PEPTIDE or a fragment of HLA-PEPTIDE. The HLA-PEPTIDE antigen can be, for example, in the form of an isolated protein or a protein expressed on the surface of a cell.

[0392] In some embodiments, the HLA-PEPTIDE antigen is a non-naturally occurring variant of HLA-PEPTIDE, such as an HLA-PEPTIDE protein having an amino acid sequence or post-translational modification that does not occur in nature.

[0393] In some embodiments, the HLA-PEPTIDE antigen is truncated, for example, by removal of an intracellular or transmembrane sequence, or a signal sequence. In some embodiments, the HLA-PEPTIDE antigen is fused at its C-terminus to a human IgG1 Fc domain or a polyhistidine tag.

[0394] Methods for identifying ABPs ABPs that bind to HLA-PEPTIDEs can be identified using any method known in the art, for example, phage display or immunization of the subject.

[0395] One method for identifying an antigen binding protein comprises providing at least one HLA-PEPTIDE target and allowing the at least one target to bind to the antigen binding protein, thereby identifying the antigen binding protein. The antigen binding protein may be present in a phage display library comprising a plurality of individual antigen binding proteins.

[0396] In some embodiments, the library is a phage display library. The phage display library can be developed to be substantially free of antigen binding proteins that nonspecifically bind to the HLA of the HLA-PEPTIDE target. The antigen binding proteins can be present in a yeast display library that includes a plurality of individual antigen binding proteins. The yeast display library can be developed to be substantially free of antigen binding proteins that nonspecifically bind to the HLA of the HLA-PEPTIDE target.

[0397] In some embodiments, the library is a yeast display library.

[0398] In some embodiments, the library is a TCR display library. Exemplary TCR display libraries and methods for using such TCR display libraries are described in WO98 / 39482; WO01 / 62908; WO2004 / 044004; WO2005116646, WO2014018863, WO2015136072, WO2017046198, and Helmut et al., (2000) PNAS 97(26)14578-14583, which are incorporated by reference in their entireties.

[0399] In some embodiments, the binding step is performed more than once, optionally at least three times, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0400] Additionally, the method may also include contacting the antigen binding protein with one or more peptide-HLA complexes that are different from the HLA-PEPTIDE target to determine whether the antigen binding protein selectively binds to the HLA-PEPTIDE target.

[0401] Another method for identifying an antigen-binding protein can include obtaining at least one HLA-PEPTIDE target, administering the HLA-PEPTIDE target, optionally in combination with an adjuvant, to a subject (e.g., a mouse, rabbit, or llama), and isolating an 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. This method can also include contacting the antigen-binding protein with one or more peptide-HLA complexes different from the HLA-PEPTIDE target to determine whether the antigen-binding protein selectively binds to the HLA-PEPTIDE target. The identified antigen-binding protein can be humanized.

[0402] In some embodiments, isolating the antigen-binding protein comprises isolating B cells from a subject that express the antigen-binding protein. The B cells may be used to generate hybridomas. The B cells may also be used to clone one or more of the CDRs. The B cells may also be immortalized, for example, by using EBV transformation. The sequence encoding the antigen-binding protein may be cloned from the immortalized B cells or directly from B cells isolated from an immunized subject. Libraries comprising antigen-binding proteins from B cells may also be generated, where the library is phage-displayed or yeast-displayed.

[0403] Another method for identifying an antigen binding protein may include obtaining a cell containing the antigen binding protein, contacting the cell with an HLA multimer comprising at least one HLA-PEPTIDE target, and identifying the antigen binding protein via binding between the HLA multimer and the antigen binding protein.

[0404] The cells can be, for example, T cells, optionally cytotoxic T lymphocytes (CTLs), or, for example, natural killer (NK) cells. The method can further include isolating the cells, optionally using flow cytometry, magnetic separation, or single cell separation. The method can further include sequencing the antigen-binding protein.

[0405] Another method for identifying an antigen binding protein may include obtaining one or more cells containing 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 by selecting one or more cells activated by interaction with at least one HLA-PEPTIDE target.

[0406] The cells may be, for example, T cells, optionally CTLs, or NK cells. The method may further comprise isolating the cells, optionally using flow cytometry, magnetic separation, or single cell separation. The method may further comprise sequencing the antigen-binding protein.

[0407] How to make monoclonal ABP Monoclonal ABPs can be obtained, for example, using hybridoma techniques (first described by Kohler et al., Nature, 1975, 256:495-497, which is incorporated by reference in its entirety) and / or recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567, which is incorporated by reference in its entirety). Monoclonal ABPs can also be obtained, for example, using phage or yeast-based libraries. Sources: e.g., U.S. Pat. Nos. 8,258,082 and 8,691,730, each of which is incorporated by reference in its entirety.

[0408] In the hybridoma method, a mouse or other suitable host animal is immunized to elicit lymphocytes that produce or are capable of producing an ABP that specifically binds to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Source: Goding JW, Monoclonal ABPs: Principles and Practice 3 rd ed. (1986) Academic Press, San Diego, Calif., which is incorporated by reference in its entirety.

[0409] Hybridoma cells are seeded and grown in a suitable culture medium containing 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 phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0410] Useful myeloma cells are those that fuse efficiently, support stable, high-level production of ABP by the selected ABP-producing cells, and are sensitive to culture conditions (e.g., with or without HAT medium). Among these preferred myeloma cell lines, particularly prominent are mouse 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 have also been described for the production of human monoclonal ABPs. See, e.g., Kozbor, J. Immunol., 1984, 133:3001, incorporated by reference in its entirety.

[0411] After hybridoma cells producing ABPs with the desired specificity, affinity, and / or biological activity are identified, selected clones may be subcloned by limiting dilution and grown by standard methods. See Goding, supra. Suitable media for this use include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0412] DNA encoding the monoclonal ABP can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the monoclonal ABP). Therefore, hybridoma cells are believed to serve as a useful source of DNA encoding an ABP with desired properties. Once isolated, the DNA may be placed into an expression vector. The monoclonal ABP is then produced by transfecting this DNA into host cells (e.g., 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 the ABP).

[0413] Method for producing chimeric ABP Exemplary methods for producing chimeric ABPs are described, for example, in U.S. Patent 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, chimeric ABPs are produced by combining non-human variable regions (e.g., variable regions derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) with human constant regions using recombinant techniques.

[0414] Method for producing humanized ABP Humanized ABPs can be generated by replacing most or all of the structural portions of a non-human monoclonal ABP with the corresponding human ABP sequences, resulting in hybrid molecules in which only the antigen-specific variable, or CDRs, are composed of non-human sequences. Methods for obtaining humanized ABPs include, for example, those described in Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 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.

[0415] Method for producing human ABP Human ABPs can be produced by various techniques known in the art, for example, using transgenic animals (e.g., humanized mice). Sources: For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 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 obtained from phage display libraries (see, e.g., Hoogenboom et al., J. Mol. Biol., 1991, 227:381-388; Marks et al., J. Mol. Biol., 1991, 222:581-597, and U.S. Patent Nos. 5,565,332 and 5,573,905, each of which is incorporated by reference in its entirety). Human ABPs can also be produced 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 can also be derived from yeast-based libraries (see, e.g., U.S. Patent No. 8,691,730, each of which is incorporated by reference in its entirety).

[0416] Method for producing ABP fragments The ABP fragments provided herein can be produced by any suitable method, including the exemplary methods described herein or known in the art. Suitable methods include recombinant techniques and proteolytic digestion of the entire ABP. Illustrative methods for producing ABP fragments are described, for example, in Hudson et al., Nat. Med., 2003, 9:129-134, which is incorporated by reference in its entirety. Methods for producing scFvABPs are described, for example, in Plukthun, 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. Patent Nos. 5,571,894 and 5,587,458, each of which is incorporated by reference in its entirety.

[0417] Methods for fabricating alternative scaffolds The alternative scaffolds provided herein can be prepared by any suitable method, including the exemplary method described herein or known in the art.For example, the preparation method of Adnectins™ is described in Emanuel et al., mAbs, 2011, 3:38-48, which is incorporated by reference in its entirety.The preparation method of iMabs is described in U.S. Patent Application No. 2003 / 0215914, which is incorporated by reference in its entirety.The preparation method of Anticalin™ is described in Vogt and Skerra, Chem.Biochem., 2004, 5:191-199, which is incorporated by reference in its entirety. Methods for preparing Kunitz domains are described in Wagner et al., Biochem. & Biophys. Res. Comm., 1992, 186:118-1145, which is incorporated by reference in its entirety. Methods for preparing thioredoxin peptide aptamers are provided in Geyer and Brent, Meth. Enzymol., 2000, 328:171-208, which is incorporated by reference in its entirety. Methods for preparing affibodies are provided in Fernandez, Curr. Opinion in Biotech., 2004, 15:364-373, which is incorporated by reference in its entirety. Methods for preparing DARPins are provided in Zahnd et al., J. Mol. Biol., 2007, 369:1015-1028, which is incorporated by reference in its entirety. Methods for preparing affilin are provided in Ebersbach et al., J. Mol. Biol., 2007, 372:172-185, which is incorporated by reference in its entirety. Methods for preparing tetranectin are provided in Graversen et al., J. Biol. Chem., 2000, 275:37390-37396, which is incorporated by reference in its entirety.Methods for preparing Avimers are provided in Silverman et al., Nature Biotech., 2005, 23:1556-1561, which is incorporated by reference in its entirety. Methods for preparing Phinomers are provided in Silacci et al., J. Biol. Chem., 2014, 289:14392-14398, which is incorporated by reference in its entirety. Further information on alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005, 23:1257-1268, and Skerra, Current Opin. in Biotech., 2007, 18:295-304, which are incorporated by reference in their entirety.

[0418] Method for producing multispecific ABPs The multispecific ABPs provided herein can be produced by any suitable method, including the exemplary methods described herein or methods known in the art. A general method for producing light chain ABPs is described in Merchant et al., Nature Biotechnol., 1998, 16:677-681, which is incorporated by reference in its entirety. A method for producing tetravalent bispecific ABPs is described in Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163, which is incorporated by reference in its entirety. A method for producing hybrid immunoglobulins is described in 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. A method for producing immunoglobulins using knob-into-holes is described in U.S. Patent No. 5,731,168, which is incorporated by reference in its entirety. A method for producing immunoglobulins with electrostatic modifications is provided in WO2009 / 089004, which is incorporated by reference in its entirety. A method for making bispecific single-chain ABPs is described in 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. A method for making single-chain ABPs with variable linker lengths is described in U.S. Patent Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety. Methods for producing diabodies are described in Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90:6444-6448, which is incorporated by reference in its entirety.Methods for making triabodies and tetrabodies are described in Todorovska et al., J. Immunol. Methods, 2001, 248:47-66, which is incorporated by reference in its entirety. Methods for making trispecific F(ab')3 derivatives are described in Tutt et al., J. Immunol., 1991, 147:60-69, which is incorporated by reference in its entirety. Methods for making cross-linked ABPs are described in U.S. Patent No. 4,676,980; Brennan et al., Science, 1985, 229:81-83; Staerz, et al., Nature, 1985, 314:628-631; and EP 0453082, each of which is incorporated by reference in its entirety. A method for producing antigen-binding domains assembled by leucine zippers is described in Kostelny et al., J. Immunol., 1992, 148:1547-1553, which is incorporated by reference in its entirety. A method for producing ABPs by the DNL approach is described in U.S. Patent Nos. 7,521,056, 7,550,143, 7,534,866, and 7,527,787, which are incorporated by reference in their entirety. A method for producing hybrids of ABP molecules and non-ABP molecules is described in WO93 / 08829, which is incorporated by reference in its entirety, for example, with respect to such ABPs. A method for producing DAF ABPs is described in U.S. Patent Application No. 2008 / 0069820, which is incorporated by reference in its entirety. Methods for producing ABPs by reduction and oxidation are described in Carlring et al., PLoS One, 2011, 6:e22533, which is incorporated by reference in its entirety. Methods for producing DVD-Igs™ are described in U.S. Patent No. 7,612,181, which is incorporated by reference in its entirety. Methods for producing DARTs™ are described in Moore et al., Blood, 2011, 117:454-451, which is incorporated by reference in its entirety.Methods for making DuoBodies® are described in Labrijn et al., Proc. Natl. Acad. Sci. USA, 2013, 110:5145-5150; Gramer et al., mAbs, 2013, 5:962-972; and Labrijn et al., Nature Protocols, 2014, 9:2450-2463, each of which is incorporated by reference in its entirety. H3 A method for producing an ABP comprising an scFv fused to the C-terminus of an antibody is described in Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163, which is incorporated by reference in its entirety. A method for producing an ABP in which a Fab molecule is attached to the constant region of an immunoglobulin is described in Miller et al., J. Immunol., 2003, 170:4854-4861, which is incorporated by reference in its entirety. A method for producing a CovX body is described in Doppalapudi et al., Proc. Natl. Acad. Sci. USA, 2010, 107:22611-22616, which is incorporated by reference in its entirety. Methods for producing Fcab ABPs are described in Wozniak-Knopp et al., Protein Eng. Des. Sel., 2010, 23:289-297, which is incorporated by reference in its entirety. Methods for producing TandAb® ABPs are described in Kipriyanov et al., J. Mol. Biol., 1999, 293:41-56 and Zhukovsky et al., Blood, 2013, 122:5116, which are incorporated by reference in their entirety. Methods for producing TandemFabs are described in WO2015 / 103072, which is incorporated by reference in its entirety. Methods for producing Zybodies™ are described in LaFleur et al., mAbs, 2013, 5:208-218, which is incorporated by reference in its entirety.

[0419] Method for generating mutants Any suitable method can be used to introduce variability into the polynucleotide sequence(s) encoding the ABP. These methods include, for example, error-prone PCR, chain shuffling, and oligonucleotide-directed mutagenesis such as trinucleotide-directed mutagenesis (TRIM). In some embodiments, several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted for mutation.

[0420] The introduction of diversity into the variable regions and / or CDRs can be used to generate a secondary library. The secondary library is then screened to identify ABP variants with improved affinity. Construction of a secondary library and affinity maturation by reselection is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 2001, 178:1-37, which is incorporated by reference in its entirety.

[0421] Methods for manipulating cells with ABPs Also provided are methods, nucleic acids, compositions, and kits for expressing ABPs (e.g., receptors comprising antibodies, CARs, and TCRs) and for producing genetically engineered cells that express such ABPs. Genetic engineering generally involves introducing nucleic acids encoding the recombinant or engineered components into cells, such as by retroviral transduction, transfection, or transformation.

[0422] In some embodiments, gene transfer is achieved by first stimulating the cells with a combination of stimuli that induce a response such as proliferation, survival, and / or activation, e.g., as measured by expression of cytokines or activation markers, followed by transduction of the activated cells and expansion in culture to numbers sufficient for clinical use.

[0423] In some situations, overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) can be toxic to a subject. Thus, in some situations, engineered cells include gene segments that render the cells susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example, in some embodiments, cells are engineered so that they can be eliminated as a result of changes in the in vivo conditions of the patient to whom they are administered. A negatively selectable phenotype can be generated from the insertion of a gene that confers sensitivity to an administered agent, e.g., 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 phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0424] In some embodiments, cells are further designed to promote the expression of cytokines or other factors.Various methods for introducing genetically engineered components, such as antigen receptors, such as CAR, are well known, and can be used with the provided methods and compositions.Exemplary methods include the method for transferring the nucleic acid encoding receptor, including virus, such as retrovirus or lentivirus, transduction, transposon, and electroporation.

[0425] In some embodiments, recombinant nucleic acids are introduced into cells using recombinant infectious viral particles, such as vectors derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are introduced into T cells using retroviral vectors, such as recombinant lentiviral or 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 Ther Nucl Acids 2,e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11):550-557).

[0426] In some embodiments, the retroviral vector has a long terminal repeat (LTR), such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen follicular fungus virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from avian or mammalian cell sources. Retroviruses are typically amphotropic, meaning that they can infect host cells of several species, including humans. In one embodiment, the retroviral gag, pol, and / or env sequences are replaced with an expressed gene. Several exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (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).

[0427] Methods for lentiviral transduction are known. Exemplary methods are described, for example, in 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.

[0428] 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 introduced 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 for introducing and expressing genetic material into immune cells include calcium phosphate transfection (see, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated particle bombardment (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0429] Other approaches and vectors for introducing nucleic acids encoding recombinant products include those described in, for example, International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0430] Among the additional nucleic acids, e.g., genes for transfer, particularly prominent are those that improve the efficacy of therapy, such as by promoting the survival and / or function of transplanted cells; genes that provide genetic markers for cell selection and / or evaluation, e.g., genes for evaluating in vivo survival or localization; and genes that improve safety, e.g., by making cells susceptible to negative selection in vivo. Sources: Lupton SD et al., Mol. and Cell Biol., 11:6 (1991), and Riddell et al., Human Gene Therapy 3:319-338 (1992). See also publications: PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from the fusion of dominant positive and negative selectable markers. See, for example, Riddell et al., US Pat. No. 6,040,177, paragraphs 14-17.

[0431] Preparation of engineered cells In some embodiments, the preparation of engineered cells involves one or more culture and / or preparation steps. Cells for introducing an HLA-peptide-ABP, e.g., a TCR or CAR, can be isolated from a biological sample, e.g., a sample obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated has a disease or condition, or is in need of cell therapy, or is the subject to whom a cell therapy drug will be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, treated, and / or engineered.

[0432] Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples obtained by one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples (e.g., processed samples derived therefrom).

[0433] In some embodiments, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples of autologous and allogeneic origin in the context of cell therapy, e.g., adoptive cell therapy.

[0434] In some embodiments, the cells are derived from a cell line, such as a T cell line. The cells, in some embodiments, are obtained from a heterologous source, such as a mouse, rat, non-human primate, or pig.

[0435] In some embodiments, cell isolation involves one or more preparative and / or affinity-based cell separation steps. In some instances, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for components of interest, or lyse or remove cells sensitive to a particular reagent. In some instances, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, or resistance to a particular component.

[0436] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes (e.g., T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets), and in some embodiments, contains cells other than red blood cells and platelets.

[0437] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium in preparation 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, the wash step is performed using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor from Baxter) according to the manufacturer's instructions. In some aspects, the wash step is performed using tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, such as, for example, Ca++ / Mg++-free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0438] In some embodiments, the method involves preparing white blood cells from peripheral blood by a density-based cell separation method, for example, by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0439] In some embodiments, isolation methods involve separating different cell types based on the intracellular expression or presence of one or more specific molecules (e.g., surface markers such as surface proteins, intracellular markers, or nucleic acids). In some embodiments, any known method for separating based on such markers may be used. In some embodiments, separation is affinity- or immunoaffinity-based separation. For example, in some aspects, isolating involves separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers), e.g., by incubation with an antibody or binding partner that specifically binds to such markers, followed, typically by a washing step, and separating cells that are bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0440] Such separation steps can be based on positive selection, in which cells that bind to the reagent are retained for further use, and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some embodiments, negative selection is particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, such that separation is best performed based on markers expressed by cells other than the desired population.

[0441] Separation does not necessarily require the enrichment or removal of 100% of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (e.g., cells expressing a marker) refers to increasing the number or proportion of such cells, but does not necessarily require the complete elimination of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell (e.g., cells expressing a marker) refers to reducing the number or proportion of such cells, but does not necessarily require the complete removal of all such cells.

[0442] In some instances, multiple rounds of separation steps are performed, with the positively or negatively selected fraction from one step being subjected to a subsequent separation step (e.g., positive or negative selection). In some instances, a single separation step may deplete cells that simultaneously express multiple markers, for example, by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types may be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed in different cell types.

[0443] For example, in some embodiments, specific subpopulations of T cells, such as cells expressing positive or 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.

[0444] For example, CD3 / CD28 conjugated magnetic beads, such as DYNABEADS® M-450 CD3 / CD28 T cell expander, may be used to ensure selection of CD3+, CD28+ T cells.

[0445] In some embodiments, isolation is achieved by positive selection to enrich for a particular cell population, or negative selection to deplete a particular cell population. In some embodiments, one or more surface markers are selected that are expressed on positively or negatively selected cells (marker+) or at relatively high levels (marker+). 高 Positive or negative selection is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to the target protein.

[0446] In some embodiments, T cells are isolated from a peripheral blood mononuclear cell (PBMC) sample by negatively selecting for markers expressed on non-T cells (e.g., B cells, monocytes, or other white blood cells such as CD14). In some embodiments, a CD4+ or CD8+ selection step is used to separate CD4+ helper cells from CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positively or negatively selecting for markers expressed on, or at relatively high levels of, one or more naive, memory, and / or effector T cell subpopulations.

[0447] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to improve efficacy, e.g., long-term survival, expansion, and / or engraftment following administration, which in some embodiments is particularly robust for such subpopulations. Adapted from: 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 with CD4+ T cells further enhances efficacy.

[0448] In embodiments, memory T cells reside in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. For example, anti-CD8 and anti-CD62L antibodies may be used to enrich or deplete the CD62L-CD8+ and / or CD62L+CD8+ fractions of peripheral blood mononuclear cells (PBMCs).

[0449] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, a CD8+ population enriched for TCM cells is isolated by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed first from the negative fraction of cells selected based on CD4 expression (subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L). Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, e.g., retaining both the positive and negative fractions from the CD4-based separation for use in subsequent steps of the method, optionally followed by one or more additional positive or negative selection steps.

[0450] In a specific example, a sample of PBMCs or other white blood cell sample, retaining both negative and positive fractions, is subjected to selection of CD4+ cells. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on marker characteristics of central memory T cells (such as CD62L or CCR7). The order of positive and negative selection is arbitrary.

[0451] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations that possess cell surface antigens. CD4+ lymphocytes can be obtained using 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-.

[0452] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are attached to a solid support or substrate, such as magnetic or paramagnetic beads, to allow for cell separation following positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (see review: Methods in Molecular Medicine, vol. 58; Metastasis Research Protocols, vol. 2; Cell Behavior In Vitro and In Vivo, pp. 17-25, Edited by: SA Brooks and U. Schumacher, Humana Press Inc., Totowa, NJ).

[0453] In some embodiments, a sample or composition of cells to be separated is incubated with a small, magnetizable or magnetically responsive material, e.g., magnetically responsive particles or microparticles such as paramagnetic beads (e.g., Dynabeads or MACS beads, etc.). The magnetically responsive material, e.g., particles, are generally attached directly or indirectly to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell(s) or population of cells desired to be separated, e.g., negatively or positively selected.

[0454] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material, such as an antibody or other binding partner, bound to a specific binding member. Many known magnetically responsive materials are used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and European Patent Specification EP 452342(B), which are incorporated herein by reference in their entireties. Other examples of colloidal-sized particles include those described in Owen, U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084.

[0455] Incubation is generally carried out under conditions in which molecules, such as antibodies or binding partners attached to the magnetic particles or beads, or secondary antibodies or other reagents that specifically bind to such antibodies or binding partners, will specifically bind to the cell surface, if present, on cells in the sample.

[0456] In some embodiments, the sample is placed in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are attracted to the magnet and separated from unlabeled cells. In positive selection, cells attracted to the magnet are retained, while in negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained for further processing or further separation steps.

[0457] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via a coating of a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (such as streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with a biotinylated primary or secondary antibody.

[0458] In some embodiments, the magnetically responsive particles remain attached to the cells for subsequent culturing, cultivation, and / or manipulation. In some aspects, the particles remain attached to the cells in preparation 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, for example, using competitive unlabeled antibodies, magnetizable particles, or antibodies bound to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0459] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetically activated cell sorting (MACS) systems allow for the high-purity selection of cells with attached magnetic particles. In certain embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached species are eluted. After this first elution step is complete, species that were trapped in the magnetic field and prevented from eluting are then released in some manner, allowing for their elution and recovery. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0460] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, thereby minimizing, for example, error, user handling, and / or contamination. In one example, the system is the system described in International Patent Application Publication No. WO2009 / 072003 or U.S. Patent Application Publication No. 20110003380(A1).

[0461] In some embodiments, the system or device performs one or more (e.g., all) of the separation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some embodiments, the system or device includes a computer and / or computer program in communication with the system or device, thereby allowing a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0462] In some embodiments, separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec), for example, to automate cell separation at clinical-scale levels in a closed, sterile system. Components include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer, in some embodiments, controls all components of the instrument and directs the system to repeatedly perform steps in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for the selected column. The peristaltic pump, in conjunction with the pinch valves, controls the flow rate of the entire tubing set. This ensures a controlled flow of buffer through the system and a continuous suspension of the cells.

[0463] In some embodiments, the CliniMACS system uses antibody-bound magnetizable particles provided in a sterile, non-pyrogenic solution. In some embodiments, cells are labeled with magnetic particles and then washed to remove excess particles. A cell preparation bag is then connected to a tubing set, which is then 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 is intended for single use. After initiating a separation program, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, while unlabeled cells are removed through a series of washing steps. In some embodiments, the cell populations used in the methods described herein are unlabeled and therefore not retained in the column. In some embodiments, the cell populations used in the methods described herein are labeled and therefore retained in the column. In some embodiments, the cell populations used in the methods described herein are eluted from the column after the magnetic field is removed and collected in a cell collection bag.

[0464] In certain embodiments, separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system, in some embodiments, is equipped with a cell processing unit, allowing for automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system may be equipped with an onboard camera and image recognition software to identify the optimal cell fractionation endpoint by identifying the macroscopic layers of the source cell product. For example, peripheral blood can be automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber, enabling cell culture protocols such as cell differentiation and proliferation, antigen challenge, and long-term cell culture. An input port may allow for the sterile removal and replenishment of media. Cells can be monitored using an integrated microscope. Sources: 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.

[0465] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, where cells stained for multiple cell surface markers are transported via a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative-scale fluorescence-activated cell sorting (FACS). In certain embodiments, the cell populations described herein are collected and enriched (or depleted) using a microelectromechanical systems (MEMS) chip 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. 1(5):355-376). In both cases, labeling cells with multiple markers allows for the isolation of well-defined T cell subsets with high purity.

[0466] In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some instances, cell separation based on binding of antibodies or other binding partners specific for one or more cell surface markers is achieved via fluid flow, e.g., using fluorescence-activated cell sorting (FACS), e.g., preparative-scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., flow cytometry detection systems. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0467] In some embodiments, the preparation method includes a freezing step (e.g., cryopreservation of cells before or after isolation, incubation, and / or manipulation). In some embodiments, the freezing and subsequent thawing steps remove granulocytes, and to some extent monocytes, from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after removing plasma and platelets in a washing step. Any of a variety of known freezing solutions and parameters may be used in some aspects. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA), which is then diluted 1:1 with medium to achieve final DMSO and HSA concentrations of 10% and 4%, respectively. Other examples include Cryostor®, CTL-Cryo™ ABC freezing medium, and the like. The cells are then frozen to -80°C at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0468] In some embodiments, provided methods include culturing, incubation, culturing, and / or genetic manipulation steps. For example, in some embodiments, methods are provided for incubating and / or manipulating depleted cell populations and culture starting compositions.

[0469] Thus, in some embodiments, the cell population is incubated in a culture starter composition. Incubation and / or manipulation may be performed in a culture vessel (e.g., a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for medium or cell culture).

[0470] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culturing, culturing, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of stimulatory agents. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prepare cells for genetic manipulation (e.g., introduction of a recombinant antigen receptor).

[0471] Conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, drugs (e.g., nutrients), amino acids, antibiotics, ions, and / or stimulatory factors (e.g., cytokines, chemokines, antigens, binding partners, fusion proteins), as well as recombinant soluble receptors and other agents designed to activate the cells.

[0472] In some embodiments, the stimulatory conditions or agents include one or more agents (e.g., a ligand that can activate the intracellular signaling domain of the TCR complex). In some aspects, the agent turns on (i.e., initiates) the TCR / CD3 intracellular signaling cascade in the T cell. Such agents can include antibodies, e.g., specific for TCR components and / or costimulatory receptors (e.g., anti-CD3, anti-CD28, solid supports such as beads), and / or those bound to one or more cytokines. Optionally, the expansion method can further comprise adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / ml.

[0473] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Pat. No. 6,040,177 (issued 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.

[0474] In some embodiments, T cells are expanded by adding culture starting composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population being expanded), and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some embodiments, PBMC feeder cells are inactivated with mitomycin C. In some aspects, the feeder cells are added to the medium before adding the population of T cells.

[0475] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25°C, typically at least about 30°C, typically at or about 37°C. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6000 to 10,000 rads. In some embodiments, the LCL feeder cells are provided in any suitable amount, e.g., at a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

[0476] 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 an antigen. For example, antigen-specific T cell lines or clones against a cytomegalovirus antigen can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0477] Assay A variety of assays known in the art may be used to identify and characterize the HLA-PEPTIDEABPs provided herein.

[0478] Binding, competition, and epitope mapping assays The specific antigen-binding activity of the ABPs provided herein can be assessed by any suitable method, including using SPR, BLI, RIA, and MSD-SET, as described elsewhere in this disclosure. Additionally, antigen-binding activity may be assessed using ELISA assays, flow cytometry, and / or Western blot assays.

[0479] Assays for measuring competition between two ABPs, or an ABP and another molecule (e.g., one or more ligands of an HLA-PEPTIDE, such as a TCR) are described elsewhere in this disclosure, e.g., Harlow and Lane, ABPs: A Laboratory Manual ch. 14, 1988, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, which is incorporated by reference in its entirety.

[0480] Assays for mapping epitopes to which the ABPs provided herein bind are described, for example, in Morris, "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66, 1996, Humana Press, Totowa, NJ, which is 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.

[0481] Effector function assays Effector function following treatment with the ABPs and / or cells provided herein can be assessed using a variety of in vitro and in vivo assays known in the art, including those described in Rev. Immunol., 1991, 9:457-492; U.S. Patent Nos. 5,500,362 and 5,821,337; Hellstrom et al., Proc. Nat'l Acad. Sci. USA, 1986, 83:7059-7063; Hellstrom et al., Proc. Nat'l Acad. Sci. USA, 1985, 82:1499-1502; Bruggemann et al., J. Exp. Med., 1987, 166:1351-1361; Clynes et al., Proc. Nat'l Acad. Sci. USA, 1998, 95:652-656; WO2006 / 029879; WO2005 / 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.

[0482] Pharmaceutical Composition The ABPs, cells, or HLA-PEPTIDE targets provided herein can be formulated into any suitable pharmaceutical composition and administered by any suitable route of administration, including, but not limited to, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

[0483] The pharmaceutical composition may contain one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used. Those skilled in the art can select suitable pharmaceutical excipients. Therefore, the pharmaceutical excipients provided below are intended to be illustrative and not limiting. Additional pharmaceutical additives include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009). This document is incorporated by reference in its entirety.

[0484] In some embodiments, the pharmaceutical composition includes an antifoaming agent. Any suitable antifoaming agent can be used. In some embodiments, the antifoaming agent is selected from alcohols, ethers, oils, waxes, silicones, surfactants, and combinations thereof. In some embodiments, the antifoaming agent is selected from mineral oil, vegetable oil, ethylene bisstearamide, paraffin wax, ester wax, fatty alcohol wax, long-chain fatty alcohol, fatty acid soap, fatty acid ester, silicone glycol, fluorosilicone, polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethylhexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.

[0485] In some embodiments, the pharmaceutical composition comprises a co-solvent. Illustrative examples of co-solvents include ethanol, poly(...

Claims

[Claim 1] The invention described herein.

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