Antigen-binding proteins targeting common antigens

Abstract

The present disclosure provides HLA-peptide targets and antigen-binding proteins that bind to the HLA-peptide targets. Also disclosed are methods for identifying HLA-peptide targets and for identifying one or more antigen-binding proteins that bind to a given HLA-peptide target.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 611,403, filed on December 28, 2017, and U.S. Provisional Application No. 62 / 756,508, filed on November 6, 2018, each of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted via EFS - Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on December 28, 2018, has the name 41174WO_CRF_sequencelisting.txt, and is 25,492,888 bytes in size. Background of the Invention

[0005] To provide antigen - specific protection against pathogens, the immune system employs two types of adaptive immune responses, namely the humoral immune response and the cellular immune response, which specifically recognize pathogen antigens through B lymphocytes and T lymphocytes, respectively.

[0006] Since T lymphocytes are the antigen - specific effectors of cellular immunity, T lymphocytes play a central role in the human defense against diseases mediated by intracellular pathogens (such as viruses, intracellular bacteria, mycoplasmas, and intracellular parasites) and in the defense against cancer cells by directly lysing the affected cells. The specificity of the T lymphocyte response is conferred by the T - cell receptor (TCR) and is activated by its binding to (major histocompatibility complex) MHC molecules on the surface of the affected cells. The T - cell receptor is an antigen - specific receptor that is clonally distributed on individual T lymphocytes, and its antigen - specific repertoire is generated via a somatic gene rearrangement mechanism similar to that involved in generating the antibody repertoire. The T - cell receptor consists of a heterodimer of transmembrane molecules, the main types of which are composed of the α - β polypeptide dimer and a smaller subset of γ - δ polypeptide dimers. The T - lymphocyte receptor subunits include variable and constant regions similar to immunoglobulins in the extracellular domain, a short hinge region with cysteines that promote α and β chain pairing, a transmembrane, and a short cytoplasmic region. Signal transduction triggered by the TCR is indirectly mediated by CD3 - ζ, which is a related multi - subunit complex containing signal - transduction subunits.

[0007] T lymphocyte receptors generally do not recognize native antigens, but rather recognize complexes displayed on the cell surface, including antigen fragments processed intracellularly and associated with major histocompatibility complex (MHC) molecules used to present peptide antigens. The major histocompatibility complex genes are highly polymorphic in a species population, including multiple common alleles for each individual gene. In humans, the MHC is known as human leukocyte antigen (HLA).

[0008] Major histocompatibility complex class I molecules are expressed on the surface of nearly all nucleated cells in the body and are dimeric molecules consisting of a transmembrane heavy chain (containing a peptide antigen-binding groove) and a smaller extracellular chain called β2-microglobulin. The peptides presented by MHC class I molecules are derived from proteasome-degraded cytosolic proteins, where the proteasome is a multi-unit structure in the cytoplasm (Niedermann G., 2002. Curr Top Microbiol Immunol. 268:91-136; for the processing of bacterial antigens, see Wick M J and Ljunggren H G., 1999. Immunol Rev. 172:153-62) (Niedermann G., 2002. Curr Top Microbiol Immunol. 268:91-136; for the processing of bacterial antigens, see Wick M J and Ljunggren H G., 1999. Immunol Rev. 172:153-62). 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 class I assembly molecule, and then the resulting MHC / peptide complex is transported to the cell membrane, enabling the antigen to be presented to T lymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7; Yewdell J W and Bennink J R., 2001. Curr Opin Immunol. 13:13-8) (Yewdell J W., 2001. Trends Cell Biol. 11:294-7; Yewdell J W. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8). Alternatively, the cleaved peptides can be loaded onto MHC class I molecules in a TAP-independent manner and can also present proteins of extracellular origin by the method of cross-presentation. Thus, once the identity of the complex's structure (peptide sequence and MHC subtype) is determined, a given MHC / peptide complex presents a novel protein structure on the cell surface that can be targeted by a novel antigen-binding protein (e.g., an antibody or TCR).

[0009] Tumor cells can express antigens and display such antigens on the surface of the tumor cells. Such tumor-associated antigens can be used to develop novel immunotherapeutic reagents to specifically target 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. SUMMARY OF THE INVENTION

[0010] 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, wherein the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and wherein: the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY, the HLA class I molecule is HLA subtype A*02:01 and the HLA-restricted peptide comprises the sequence AIFPGAVPAA, the HLA class I molecule is HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence ASSLPTTMNY, or the HLA class I molecule is HLA subtype A*01:01 and the HLA-restricted peptide comprises the sequence HSEVGLPVY.

[0011] In some embodiments, the length of the HLA-restricted peptide is about 5-15 amino acids. In some embodiments, the length of the HLA-restricted peptide is about 8-12 amino acids. In some embodiments, the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY, the HLA class I molecule is HLA subtype A*02:01 and the HLA-restricted peptide consists of the sequence AIFPGAVPAA, 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.

[0012] In some embodiments, the ABP comprises an antibody or an antigen-binding fragment thereof.

[0013] In some embodiments of the ABP comprising an antibody or an antigen-binding fragment thereof, the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide comprises the sequence EVDPIGHVY. In some embodiments, the HLA class I molecule is HLA subtype B*35:01 and the HLA-restricted peptide consists of the sequence EVDPIGHVY.

[0014] In some embodiments, the ABP comprises a CDR-H3, the CDR-H3 comprising a sequence selected from: CARDGVRYYGMDVW, CARGVRGYDRSAGYW, CASHDYGDYGEYFQHW, CARVSWYCSSTSCGVNWFDPW, CAKVNWNDGPYFDYW, CATPTNSGYYGPYYYYGMDVW, CARDVMDVW, CAREGYGMDVW, CARDNGVGVDYW, CARGIADSGSYYGNGRDYYYGMDVW, CARGDYYFDYW, CARDGTRYYGMDVW, CARDVVANFDYW, CARGHSSGWYYYYGMDVW, CAKDLGSYGGYYW, CARSWFGGFNYHYYGMDVW, CARELPIGYGMDVW, and CARGGSYYYYGMDVW.

[0015] In some embodiments, the ABP comprises a CDR-L3, the CDR-L3 comprising a sequence selected from: CMQGLQTPITF, CMQALQTPPTF, CQQAISFPLTF, CQQANSFPLTF, CQQANSFPLTF, CQQSYSIPLTF, CQQTYMMPYTF, CQQSYITPWTF, CQQSYITPYTF, CQQYYTTPYTF, CQQSYSTPLTF, CMQALQTPLTF, CQQYGSWPRTF, CQQSYSTPVTF, CMQALQTPYTF, CQQANSFPFTF, CMQALQTPLTF, and CQQSYSTPLTF.

[0016] In some embodiments, the ABP comprises a CDR-H3 and a CDR-L3 from an scFv, the scFv being named 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 comprises all three heavy-chain CDRs and all three light-chain CDRs from an scFv, the scFv being named 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.

[0018] In some embodiments, the ABP comprises a VH sequence selected from: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGIINPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVRYYGMDVWGQGTTVTVSSAS, QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSHDINWVRQAPGQGLEWMGWMNPNSGDTGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGVRGYDRSAGYWGQGTLVIVSSAS, EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISYISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHDYGDYGEYFQHWGQGTLVTVSSAS, EVQLLQSGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSWYCSSTSCGVNWFDPWGQGTLVTVSSAS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVASISSSGGYINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVNWNDGPYFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFGVSWLRQAPGQGLEWMGGIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATPTNSGYYGPYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDVMDVWGQGTTVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGGTFSGYLVSWVRQAPGQGLEWMGWINPNSGGTNTAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYIFRNYPMHWVRQAPGQGLEWMGWINPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDNGVGVDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNPNIGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIADSGSYYGNGRDYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWINPNSGVTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWINPNSGDTKYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGTRYYGMDVWGQGTTVTVSS, EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSYISSSSSYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDVVANFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWMNPDSGSTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGHSSGWYYYYGMDVWGQGTTVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYSMHWVRQAPGKGLEWVSSITSFTNTMYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLGSYGGYYWGQGTLVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSWFGGFNYHYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARELPIGYGMDVWGQGTTVTVSS and QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGSYYYYGMDVWGQGTTVTVSS.,

[0019] In some embodiments, the ABP comprises a VL selected from: DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPITFGQGTRLEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQSTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYMMPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGTKVEIK,DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGQGTKLEIK, DIVMTQSPDSLAVSLGERATINCKTSQSVLYRPNNENYLAWYQQKPGQPPKLLIYQASIREPGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRFLNWYQQKPGKAPKLLIYGASRPQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK, EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYAASARASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSWPRTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGTKVEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCQASEDISNHLNWYQQKPGKAPKLLIYDALSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK,DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGQGTKVEIK and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.,

[0020] In some embodiments, the ABP comprises a VH sequence and a VL sequence from an scFv, the scFv being named 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.

[0021] In some embodiments, the ABP binds to any one or more of the amino acid positions 2-8 on the restricted peptide EVDPIGHVY.

[0022] In some embodiments of the ABP comprising an antibody or an antigen-binding fragment thereof, the HLA class I molecule is HLA subtype A*02:01, and the HLA-restricted peptide comprises the sequence AIFPGAVPAA. In some embodiments of the ABP comprising an antibody or an antigen-binding fragment thereof, and the HLA class I molecule is HLA subtype A*02:01, and the HLA-restricted peptide consists of the sequence AIFPGAVPAA.

[0023] In some embodiments, the ABP comprises a CDR-H3, the CDR-H3 comprising a sequence selected from: CARDDYGDYVAYFQHW, CARDLSYYYGMDVW, CARVYDFWSVLSGFDIW, CARVEQGYDIYYYYYMDVW, CARSYDYGDYLNFDYW, CARASGSGYYYYYGMDVW, CAASTWIQPFDYW, CASNGNYYGSGSYYNYW, CARAVYYDFWSGPFDYW, CAKGGIYYGSGSYPSW, CARGLYYMDVW, CARGLYGDYFLYYGMDVW, CARGLLGFGEFLTYGMDVW, CARDRDSSWTYYYYGMDVW, CARGLYGDYFLYYGMDVW, CARGDYYDSSGYYFPVYFDYW, and CAKDPFWSGHYYYYGMDVW.

[0024] In some embodiments, the ABP comprises a CDR-L3, the CDR-L3 comprising a sequence selected from: CQQNYNSVTF, CQQSYNTPWTF, CGQSYSTPPTF, CQQSYSAPYTF, CQQSYSIPPTF, CQQSYSAPYTF, CQQHNSYPPTF, CQQYSTYPITI, CQQANSFPWTF, CQQSHSTPQTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQTYSTPWTF, CQQYGSSPYTF, CQQSHSTPLTF, CQQANGFPLTF, and CQQSYSTPLTF.

[0025] In some embodiments, the ABP comprises a CDR-H3 and a CDR-L3 from an scFv, the scFv being named 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 comprises all three heavy chain CDRs and all three light chain CDRs from an scFv, the scFv being named 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.

[0027] In some embodiments, the ABP comprises a VH sequence selected from: QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGWINPNSGATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDDYGDYVAYFQHWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYPFIGQYLHWVRQAPGQGLEWMGIINPSGDSATYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDLSYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWMNPIGGGTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVYDFWSVLSGFDIWGQGTLVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVEQGYDIYYYYYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGWISTYSGHADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSYDYGDYLNFDYWGQGTLVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSSISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGYYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYFMHWVRQAPGQGLEWMGMVNPSGGSETFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAASTWIQPFDYWGQGTLVTVSS,EVQLLESGGGLVQPGGSLRLSCAASGFDFSIYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNGNYYGSGSYYNYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLTTYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVYYDFWSGPFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPYSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGGIYYGSGSYPSWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGVSWVRQAPGQGLEWMGWISPYSGNTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLYYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFSNMYLHWVRQAPGQGLEWMGWINPNTGDTNYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDVWGQGTKVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLLGFGEFLTYGMDVWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGVINPSGGSTTYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRDSSWTYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDVWGQGTTVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGGTFSSHAISWVRQAPGQGLEWMGVIIPSGGTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYDSSGYYFPVYFDYWGQGTLVTVSS and QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPFWSGHYYYYGMDVWGQGTTVTVSS.

[0028] In some embodiments, the ABP comprises a VL sequence selected from: DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPWTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQAISNSLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSTPPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGGGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGINSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNSYPPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPITIGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKLEIK,DIQMTQSPSSLSASVGDRVTITCRASQDVSTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPQTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQGTKLEIK, EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPLTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQNIYTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK, and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.,

[0029] In some embodiments, the ABP comprises the VH and VL sequences from an scFv, the scFv being designated as 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.

[0030] In some embodiments, the ABP binds to any one or more of amino acids 1-5 of the restricted peptide AIFPGAVPAA. In some embodiments, the ABP binds to one or both of amino acids 4 and 5 of the restricted peptide AIFPGAVPAA.

[0031] In some embodiments, the ABP binds to any one or more of amino acid positions 45-60 of HLA subtype A*02:01.

[0032] In some embodiments, the ABP binds to any one or more of amino acid positions 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 HLA subtype A*02:01.

[0033] In some embodiments of the ABP comprising an antibody or an antigen-binding fragment thereof, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments of the ABP comprising an antibody or an antigen-binding fragment thereof, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide consists of the sequence ASSLPTTMNY.

[0034] In some embodiments, the ABP comprises a CDR-H3, and the CDR-H3 comprises a sequence selected from the following: CARDQDTIFGVVITWFDPW, CARDKVYGDGFDPW, CAREDDSMDVW, CARDSSGLDPW, CARGVGNLDYW, CARDAHQYYDFWSGYYSGTYYYGMDVW, CAREQWPSYWYFDLW, CARDRGYSYGYFDYW, CARGSGDPNYYYYYGLDVW, CARDTGDHFDYW, CARAENGMDVW, CARDPGGYMDVW, CARDGDAFDIW, CARDMGDAFDIW, CAREEDGMDVW, CARDTGDHFDYW, CARGEYSSGFFFVGWFDLW, and CARETGDDAFDIW.

[0035] In some embodiments, the ABP comprises a CDR-L3, and the CDR-L3 comprises a sequence selected from the following: CQQYFTTPYTF, CQQAEAFPYTF, CQQSYSTPITF, CQQSYIIPYTF, CHQTYSTPLTF, CQQAYSFPWTF, CQQGYSTPLTF, CQQANSFPRTF, CQQANSLPYTF, CQQSYSTPFTF, CQQSYSTPFTF, CQQSYGVPTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQYYSYPWTF, CQQSYSTPFTF, CMQTLKTPLSF, and CQQSYSTPLTF.

[0036] In some embodiments, the ABP comprises a CDR-H3 and a CDR-L3 from an scFv, and the scFv is named 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.

[0037] In some embodiments, the ABP comprises all three heavy-chain CDRs and all three light-chain CDRs from an scFv, the scFv being designated as 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.

[0038] In some embodiments, the ABP comprises a VH sequence selected from: EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISARSGRTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDQDTIFGVVITWFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIHPGGGTTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDKVYGDGFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDDSMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSGLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNLDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGVTFSTSAISWVRQAPGQGLEWMGWISPYNGNTDYAQMLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAHQYYDFWSGYYSGTYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSNSIINWVRQAPGQGLEWMGWMNPNSGNTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREQWPSYWYFDLWGRGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSTHDINWVRQAPGQGLEWMGVINPSGGSAIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGYSYGYFDYWGQGTLVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGNTFIGYYVHWVRQAPGQGLEWVGIINPNGGSISYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSGDPNYYYYYGLDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGIIGPSDGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAENGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGIIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGGYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGDAFDIWGQGTMVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRISPSDGSTTYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMGDAFDIWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREEDGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVTVSS,QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNFAISWVRQAPGQGLEWMGGIIPIFDATNYAQKFQGRVTFTADESTSTAYMELSSLRSEDTAVYYCARGEYSSGFFFVGWFDLWGRGTQVTVSS and QVQLVQSGAEVKKPGASVKVSCKASGYNFTGYYMHWVRQAPGQGLEWMGIIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARETGDDAFDIWGQGTMVTVSS.,

[0039] In some embodiments, the ABP comprises a VL sequence selected from: DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFTTPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIFDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEAFPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIIPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQTYSTPLTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPWTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQNISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPLTFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQDISRYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPRTFGQGTKVEIK,DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSLPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYDASKLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPWTFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLKTPLSFGGGTKVEIK and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.

[0040] In some embodiments, the ABP comprises a VH sequence and a VL sequence from an scFv, and the scFv is named 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.

[0041] In some embodiments, the ABP binds to any one or more of amino acid positions 4, 6, and 7 of the restricted peptide ASSLPTTMNY.

[0042] In some embodiments, the ABP binds to any one or more of amino acid positions 49-56 of HLA subtype A*01:01.

[0043] 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, wherein the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 portion of the HLA class I molecule, and wherein the HLA-peptide target is selected from Table A.

[0044] In some embodiments, the length of the HLA-restricted peptide is between about 5-15 amino acids. In some embodiments, the length of the HLA-restricted peptide is between about 8-12 amino acids.

[0045] In some embodiments, the ABP comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding protein is linked to a scaffold, optionally, wherein the scaffold comprises serum albumin or Fc, optionally, wherein the Fc is human Fc and is of the 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, wherein the linker is a peptide linker, optionally, wherein 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, an F(ab′)2 fragment, a Fab′ fragment, a scFv fragment, a scFv-Fc fragment and / or a single-domain antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding protein comprises a 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, human or chimeric antibody. In some embodiments, the antigen-binding protein is multispecific, optionally bispecific. In some embodiments, the antigen-binding protein binds to more than one antigen or more than one epitope on a single antigen. In some embodiments, the antigen-binding protein comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgD, IgE and IgM. In some embodiments, the antigen-binding protein comprises a human IgG class and a heavy chain constant region of a subclass selected from IgG1, IgG4, IgG2 and IgG3. In some embodiments, the antigen-binding protein comprises one or more modifications that prolong the half-life. In some embodiments, the antigen-binding protein comprises a modified Fc, optionally, the modified Fc comprises one or more mutations that prolong the half-life, optionally, the one or more mutations that prolong the half-life are YTE.

[0046] 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 an antigen-binding portion thereof comprises a TCR variable region. In some embodiments, the TCR or an antigen-binding portion thereof comprises one or more TCR complementarity-determining regions (CDRs).

[0047] In some embodiments, the TCR comprises an α chain and a β chain. In some embodiments, the TCR comprises a γ chain and a δ chain.

[0048] In some embodiments, the antigen-binding protein is part of a chimeric antigen receptor (CAR) that comprises: an extracellular portion comprising the antigen-binding protein; and an intracellular signaling domain. In some embodiments, the antigen-binding protein comprises a 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 ζ chain of the CD3-ζ (CD3) chain.

[0049] In some embodiments, the ABP further comprises a transmembrane domain that links the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28.

[0050] In some embodiments, the ABP further comprises the intracellular signaling domain of a T cell co-stimulatory molecule. In some embodiments, the T cell co-stimulatory molecule is CD28, 4-1BB, OX-40, ICOS, or any combination thereof.

[0051] In some embodiments of an ABP that comprises a TCR or an antigen-binding portion thereof, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide consists of the sequence ASSLPTTMNY. In some embodiments, the ABP comprises a TCRα CDR3 sequence selected from Table 15. In some embodiments, the ABP comprises a TCRβ CDR3 sequence selected from Table 15. In some embodiments, the ABP comprises the αCDR3 and βCDR3 sequences from any one of TCR clone type ID#: 1-344. In some embodiments, the ABP comprises a TCRα variable (TRAV) amino acid sequence, a TCRα joining (TRAJ) amino acid sequence, a TCRβ variable (TRBV) amino acid sequence, a TCRβ diversity (TRBD) amino acid sequence, and a TCRβ joining (TRBJ) amino acid sequence, wherein each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences of any one of the TCR clone types selected from TCR clone type ID#: 1-344.

[0052] In some embodiments, the ABP comprises a TCRα constant (TRAC) amino acid sequence. In some embodiments, the ABP comprises a TCRβ constant (TRBC) amino acid sequence.

[0053] In some embodiments, the ABP comprises a TCRα VJ sequence. It has at least 95%, 96%, 97%, 98%, 99% or 100% identity with an αVJ sequence selected from Table 16. In some embodiments, the ABP comprises a TCRβ V(D)J sequence, which has at least 95%, 96%, 97%, 98%, 99% or 100% identity with a β V(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 has at least 95%, 96%, 97%, 98%, 99% or 100% identity with the corresponding TCRα VJ and TCRβ V(D)J amino acid sequences of any one of the TCR clones selected from TCR clone ID#: 1-344.

[0054] In some embodiments of the ABP of an antibody comprising a TCR or an antigen-binding portion thereof, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide comprises the sequence HSEVGLPVY. In some embodiments, the HLA class I molecule is HLA subtype A*01:01, and the HLA-restricted peptide consists of the sequence HSEVGLPVY.

[0055] In some embodiments, the ABP comprises a TCRα CDR3 sequence selected from Table 18. In some embodiments, the ABP comprises a TCRβ CDR3 sequence selected from Table 18. In some embodiments, the ABP comprises the αCDR3 and βCDR3 sequences from any one of TCR clones ID#: 345-447. In some embodiments, the ABP comprises a TCRα variable (TRAV) amino acid sequence, a TCRα joining (TRAJ) amino acid sequence, a TCRβ variable (TRBV) amino acid sequence, a TCRβ diversity (TRBD) amino acid sequence, and a TCRβ joining (TRBJ) amino acid sequence, wherein each of the TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences has at least 95%, 96%, 97%, 98%, 99% or 100% identity with the corresponding TRAV, TRAJ, TRBV, TRBD, and TRBJ amino acid sequences of any one of the TCR clones selected from TCR clone ID#: 345-447. In some embodiments, the ABP comprises a TCRα constant (TRAC) amino acid sequence. In some embodiments, the ABP comprises a TCRβ constant (TRBC) amino acid sequence.

[0056] In some embodiments, the ABP comprises a TCRα VJ sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an α VJ sequence selected from Table 19. In some embodiments, the ABP comprises a TCRβ V(D)J sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a β V(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 has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding TCRαVJ and TCRβ V(D)J amino acid sequences of any one of the TCR clones selected from TCR clone ID#: 345 - 447.

[0057] 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, wherein the HLA - restricted peptide is located in the peptide - binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and wherein the HLA class I molecule - peptide target is selected from Table A.

[0058] In some embodiments, the HLA class I molecule is HLA subtype B*35:01 and the HLA - restricted peptide comprises the sequence EVDPIGHVY, the HLA class I molecule is HLA subtype A*02:01 and the HLA - restricted peptide comprises the sequence AIFPGAVPAA, or the HLA class I molecule is HLA subtype A*01:01 and the HLA - restricted peptide comprises the sequence ASSLPTTMNY. In some embodiments, the HLA class I molecule is HLA subtype B*35:01 and the HLA - restricted peptide consists of the sequence EVDPIGHVY, 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.

[0059] In some embodiments, the length of the HLA - restricted peptide is between about 5 and 15 amino acids. In some embodiments, the length of the HLA - restricted peptide is between about 8 and 12 amino acids.

[0060] In some embodiments, the association of the 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 the HLA subtype with the α-subunit of the HLA subtype is demonstrated by conditional peptide exchange.

[0061] 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 an antibody labeled with a fluorescent dye.

[0062] Also provided herein are compositions comprising the HLA-peptide targets described herein bound to a solid support. In some embodiments, the solid support comprises beads, wells, membranes, tubes, columns, plates, agarose, magnetic beads, or fragments.

[0063] 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. In some embodiments, the first member is streptavidin and the second member is biotin.

[0064] Also provided herein is a reaction mixture comprising an isolated and purified α-subunit of the HLA subtype from the HLA-peptide target as described in Table A; the isolated and purified β2-microglobulin subunit of the HLA subtype; the isolated and purified restricted peptide from the HLA-peptide target as described in Table A; and a reaction buffer.

[0065] Also provided herein is a reaction mixture comprising the isolated HLA-peptide targets described herein and a plurality of T cells isolated from a human subject. In some embodiments, the T cells are CD8+ T cells.

[0066] Also provided herein are isolated polynucleotides comprising a first nucleic acid sequence encoding an HLA-restricted peptide described herein 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 wherein the encoded peptide and the encoded HLA subtype form an HLA / peptide complex described herein.

[0067] The present disclosure also provides a kit for expressing the stable HLA-peptide targets described herein, which comprises a first construct comprising a first nucleic acid sequence encoding an HLA-restricted peptide described herein operably linked to a promoter; and instructions for 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 described herein. In some embodiments, one or both of the first construct and the second construct are lentiviral vector constructs.

[0068] The present disclosure also provides a host cell comprising a heterologous HLA-peptide target described herein. The present disclosure also provides a host cell that expresses an HLA subtype defined by any one of the targets in Table A. The present disclosure also provides a host cell that comprises a polynucleotide encoding an HLA-restricted peptide as described in Table A, e.g., a polynucleotide encoding an HLA-restricted peptide described herein.

[0069] In some embodiments, the host cell does not comprise an endogenous MHC. In some embodiments, the host cell comprises an exogenous HLA. In some embodiments, the host cell is a K562 or A375 cell.

[0070] In some embodiments, the host cell is a cultured cell from a tumor cell line. In some embodiments, the tumor cell line expresses the HLA subtype defined by any one of the targets in Table A. In some embodiments, the tumor cell line expresses the gene target and HLA subtype defined by any one of the targets in Table A. For example, the tumor cell line may express the gene ABCB5 and the HLA subtype HLA-C*16:01 defined by Target #1 in 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 the known expression of the gene target of any one of the targets listed in Table A. The selection may be based on the known expression of the HLA subtype of any one of the targets listed in Table A. The selection may be based on the known expression of the gene target and HLA subtype of any one of the targets listed in Table A. An exemplary catalog of tumor cell lines includes, for example, the American Type Culture Collection (ATCC), which 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.” Onco immunology 3.8 (2014): e954893. PMC. Web. October 8, 2018 (which is hereby 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.

[0071] The present invention also provides a cell culture system comprising the host cell defined herein and a cell culture medium. In some embodiments, the host cell expresses the HLA subtype defined by any one of the targets in Table A, and wherein the cell culture medium comprises a restricted peptide defined by the target in Table A. In some embodiments, the host cell is a K562 cell comprising an exogenous HLA, wherein the exogenous HLA is the HLA subtype defined by any one of the targets in Table A, and wherein the cell culture medium comprises a restricted peptide defined by the target in Table A.

[0072] In some embodiments of the ABP, the antigen-binding protein binds to the HLA-peptide target through the contact points with the class I HLA molecule of the HLA-peptide target and the contact points with the HLA-restricted peptide of the HLA-peptide target. In some embodiments of the ABP, the binding of the ABP to the amino acid positions on the restricted peptide or HLA subtype, or the contact points or residues that directly or indirectly affect the binding of the HLA-peptide target to the ABP, are determined by position scanning, hydrogen-deuterium exchange, or protein crystallography.

[0073] In some embodiments, the ABP can be used as a drug. In some embodiments, the ABP can be used to treat cancer, optionally, wherein the cancer expresses or is expected to express the HLA-peptide target. In some embodiments, the ABP can be used to treat cancer, wherein the cancer is selected from solid tumors and hematological malignancies.

[0074] Also provided herein is an ABP that is a conservatively modified variant of the ABP described herein. Also provided herein is an antigen-binding protein (ABP) that competes with the antigen-binding protein described herein for binding. Also provided herein is an antigen-binding protein (ABP) that binds to the same HLA-peptide epitope as the antigen-binding protein described herein.

[0075] Also provided herein are engineered cells expressing a receptor comprising the antigen-binding protein described herein. In some embodiments of the engineered cells, the engineered cells are T cells, optionally cytotoxic T cells (CTLs). In some embodiments, the antigen-binding protein is expressed by a heterologous promoter.

[0076] Also provided herein is an isolated polynucleotide or polynucleotide set encoding the antigen-binding protein or an antigen-binding portion thereof described herein.

[0077] Also provided herein is an isolated polynucleotide or polynucleotide set encoding the HLA / peptide target described herein.

[0078] Also provided herein is a vector or vector set comprising the polynucleotide or polynucleotide set described herein.

[0079] Also provided herein is a host cell comprising the polynucleotide or polynucleotide set described herein or the vector or vector set described herein, optionally, wherein the host cell is CHO or HEK293, or optionally, wherein the host cell is a T cell.

[0080] Also provided herein is a method for producing an antigen-binding protein, which comprises: expressing the antigen-binding protein with a host cell as described herein and isolating the expressed antigen-binding protein.

[0081] The present invention also provides a pharmaceutical composition comprising the antigen-binding protein described herein and a pharmaceutically acceptable excipient. The present invention also provides a method for treating cancer in a subject, comprising: administering to the subject an effective amount of the antigen-binding protein described herein or the pharmaceutical composition described herein, optionally, wherein the cancer is selected from solid tumors and hematological tumors. In some embodiments, the cancer expresses or is predicted to express an HLA-peptide target.

[0082] The present invention also provides a kit comprising the antigen-binding protein described herein or the pharmaceutical composition described herein and instructions for use.

[0083] The present invention also provides a composition comprising at least one HLA-peptide target described herein and an adjuvant.

[0084] The present invention also provides a composition comprising at least one HLA-peptide target described herein and a pharmaceutically acceptable excipient.

[0085] The present invention also provides a composition comprising an amino acid sequence, the amino acid sequence comprising a polypeptide of at least one HLA-peptide target disclosed in Table A, optionally, the amino acid sequence consisting essentially of or consisting of the polypeptide.

[0086] The present invention also provides a virus comprising the isolated polynucleotide or polynucleotide set described herein. In some embodiments, the virus is a filamentous phage.

[0087] The present invention also provides a yeast cell comprising the isolated polynucleotide or polynucleotide set described herein.

[0088] The present invention also provides a method for identifying the antigen-binding protein described herein, comprising: providing at least one HLA-peptide target listed in Table A; and binding the at least one target 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 different antigen-binding proteins. In some embodiments, the phage display library is substantially free of antigen-binding proteins of HLA that non-specifically bind to the HLA-peptide target.

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

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

[0092] In some embodiments, the method further comprises: contacting the antigen-binding protein with one or more peptide-HLA complexes different from the HLA-peptide target, thereby determining whether the antigen-binding protein selectively binds to the HLA-peptide target, optionally, wherein the selectivity is determined by measuring the binding affinity of the antigen-binding protein for the soluble target HLA-peptide complex relative to a soluble HLA-peptide complex different from the target complex; optionally, wherein the selectivity is determined by measuring the binding affinity of the antigen-binding protein for the target HLA-peptide complex expressed on the surface of one or more cells relative to an HLA-peptide complex different from the target complex expressed on the surface of the one or more cells.

[0093] Also provided herein are methods of identifying the antigen-binding proteins described herein, which comprise: obtaining at least one HLA-peptide target listed in Table A; administering the HLA-peptide target to a subject, optionally in combination with an adjuvant; and isolating the antigen-binding protein from the subject.

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

[0095] In some embodiments, the method further comprises: 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, optionally, wherein the selectivity is determined by measuring the binding affinity of the antigen-binding protein for the soluble target HLA-peptide complex relative to a soluble HLA-peptide complex different from the target complex, optionally, wherein the selectivity is determined by measuring the binding affinity of the antigen-binding protein for the target HLA-peptide complex expressed on the surface of one or more cells relative to an HLA-peptide complex different from the target complex expressed on the surface of the one or more cells.

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

[0097] In some embodiments, isolating an antigen-binding protein comprises: isolating B cells from a subject expressing the antigen-binding protein, and optionally, directly cloning the sequence encoding the antigen-binding protein from the isolated B cells. In some embodiments, the method further comprises generating hybridomas 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 by Epstein-Barr virus (EBV) transformation. In some embodiments, the method further comprises generating a library comprising the antigen-binding protein of the B cells, optionally wherein the library is a phage display library or a yeast display library.

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

[0099] Also provided herein are methods of identifying an antigen-binding protein described herein, which comprise: obtaining a cell comprising the antigen-binding protein; contacting the cell with an HLA multimer comprising at least one HLA-peptide target listed in Table A; and identifying the antigen-binding protein by binding between the HLA multimer and the antigen-binding protein.

[0100] Also provided herein are methods of identifying an antigen-binding protein described herein, which comprise: 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 the 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 isolation to isolate the cells. In some embodiments, the method further comprises sequencing the antigen-binding protein.

[0101] Also provided herein are methods of identifying an antigen-binding protein described herein, which comprise: providing at least one HLA-peptide target listed in Table A; and identifying the antigen-binding protein using the target. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The following description and drawings facilitate a better understanding of these and other features, aspects, and advantages of the present invention, wherein:

[0103] Figure 1Shows the general structure of human leukocyte antigen (HLA) class I molecules. Personal work by user atropos235, published via en.wikipedia, CC BY 2.5, URL: https: / / commons.wikimedia.org / w / index.php?curid=1805424

[0104] Figure 2 Depicts exemplary construct elements for cloning TCRs into an expression system for therapeutic development.

[0105] Figure 3 Shows the target and microtiter negative control designs for the HLA-peptide target "G5".

[0106] Figure 4 Shows the target and microtiter negative control designs for the HLA-peptide targets "G8" and "G10".

[0107] Figure 5 A and 5B show the HLA stability results for the G5 rescreen "microtiter" and the G5 target.

[0108] Figure 6 A-6E show the HLA stability results for the G5 "complete" pool rescreen peptides.

[0109] Figure 7 A and Figure 7 B show the HLA stability results for the rescreen peptides and the G8 target.

[0110] Figure 8 A and Figure 8 B show the HLA stability results for the G10 rescreen "microtiter" and the G10 target.

[0111] Figure 9 A-9D show the HLA stability results for additional G8 and G10 "complete" pool rescreen peptides.

[0112] Figure 10 A-10C show the phage supernatant ELISA results indicating the progressive enrichment of G5-, G8- and G10-binding phages with successive rounds of panning.

[0113] Figure 11 Shows a flow chart describing the antibody selection process, including the standard and expected applications in scFv, Fab and IgG formats.

[0114] Figure 12A, 12B, and 12C depict the results of biolayer interferometry (BLI) of Fab clones G5-P7A05 to the HLA peptide target B*35:01-EVDPIGHVY, Fab clones R3G8-P2C10 and G8-P1C11 to the HLA peptide target A*02:01-AIFPGAVPAA, and Fab clone R3G10-P1B07 to the HLA peptide target A*01:01-ASSLPTTMNY.

[0115] Figure 13 Shows the general experimental design of the position scanning experiment.

[0116] Figure 14 A shows the stability results of G5 position variants - HLA.

[0117] Figure 14 B shows the binding affinity of Fab clone G5-P7A05 to G5 position variant - HLA.

[0118] Figure 15 A shows the stability results of G8 position variant HLAs.

[0119] Figure 15 B shows the binding affinity of Fab clone G8-P2C10 to G8 position variant HLA.

[0120] Figure 16 A shows the stability results of G10 position variant - HLA.

[0121] Figure 16 B shows the binding affinity of Fab clone G10-P1B07 to G10 position variant HLA.

[0122] Figure 17 A, Figure 17 B, and Figure 17 C show representative examples of antibodies that bind to G5-, G8-, or G10-presenting K562 cells as detected by flow cytometry.

[0123] Figure 18 A - 18C show histograms of K562 cells binding to the generated target-specific antibodies.

[0124] Figure 19 A - 19C show histograms of cell binding assays using tumor cell lines expressing HLA subtypes and selected HLA-peptide targets of the target gene.

[0125] Figure 20A and 20B show the number of target-specific T cells (A) and the number of target-specific unique TCR clonotypes (B) from the tested donors.

[0126] Figure 21 A shows an exemplary heatmap of scFv G8-P1H08 that visualizes the entire HLA portion of the HLA-peptide target G8 using an integrated perturbation view. Figure 21 B shows an example of HDX data from scFv G8-P1H08 plotted on the crystal structure PDB5bs0.

[0127] Figure 22 A shows a heatmap of the HLA α1 helix of all ABPs tested against the HLA-peptide target G8 (HLA-A*02:01_AIFPGAVPAA). Figure 22 B shows a heatmap of the HLA α2 helix of all ABPs tested against the HLA-peptide target G8 (HLA-A*02:01_AIFPGAVPAA). Figure 22 C shows the resulting heatmap of the restricted peptide AIFPGAVPAA of all tested ABPs.

[0128] Figure 23 A shows an exemplary heatmap of scFv R3G10-P2G11 that visualizes the entire HLA portion of the HLA-peptide target G10 using an integrated perturbation view.

[0129] Figure 23 B shows an example of HDX data from scFv R3G10-P2G11 plotted on the crystal structure PDB5bs0.

[0130] Figure 24 A shows the resulting heatmap of the HLA α1 helix of all ABPs tested against the HLA-peptide target G10 (HLA-A*01:01_ASSLPTTMNY). Figure 24 B shows the resulting heatmap of the HLA α2 helix of all ABPs tested against the HLA-peptide target G10 (HLA-A*01:01_ASSLPTTMNY). Figure 24 C shows the resulting heatmap of the restricted peptide ASSLPTTMNY of all tested ABPs.

[0131] Figure 25 Depicts exemplary spectral data of the peptide EVDPIGHVY. The figure contains peptide fragmentation information as well as information related to patient samples, including HLA type.

[0132] Figure 26 Depicts exemplary spectral data of the peptide AIFPGAVPAA. The figure contains peptide fragmentation information as well as information related to patient samples, including HLA type.

[0133] Figure 27Depicts exemplary spectral data of the peptide ASSLPTTMNY. The figure contains peptide fragmentation information as well as information related to patient samples, including HLA type.

[0134] Figure 28 A and 28B depict size-exclusion chromatography fractions (A) and SDS-PAGE analysis of the chromatography fractions under reducing conditions (B).

[0135] Figure 29 Depicts a micrograph of an exemplary crystal of a complex containing the Fab clone G8-P1C11 and the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0136] Figure 30 Depicts the overall structure of the complex formed by the binding of the Fab clone G8-P1C11 to the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0137] Figure 31 Depicts the fine electron density region of the crystal structure of the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8"), and the depicted region corresponds to the restricted peptide AIFPGAVPAA.

[0138] Figure 32 Depicts a LigPlot of the interaction between HLA and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0139] Figure 33 Depicts a diagram of the interacting residues between the Fab VH and VL chains and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0140] Figure 34 Depicts a LigPlot of the interaction between the restricted peptide chain and the Fab chain. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0141] Figure 35 Depicts a LigPlot of the interaction between the Fab VH chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0142] Figure 36 A LigPlot depicting the interaction between the Fab VL chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0143] Figure 37 An interface summary of a Pisa analysis depicting the interaction between HLA and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0144] Figure 38 A Pisa analysis of the residues involved in the interaction between HLA and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0145] Figure 39 A Pisa analysis depicting the residues involved in the interaction between the Fab VH chain and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0146] Figure 40 A Pisa analysis of the residues involved in the interaction between the Fab VL chain and the restricted peptide. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0147] Figure 41 An interface summary of a Pisa analysis depicting the interaction between the Fab VH chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0148] Figure 42 A Pisa analysis of the residues involved in the interaction between the Fab VH chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0149] Figure 43 An interface summary of a Pisa analysis depicting the interaction between the Fab VL chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0150] Figure 44 Pisa analysis of the interaction residues between the Fab VL chain and HLA. The crystal structure corresponds to the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0151] Figure 45 A depicts an exemplary heat map of the HLA portion of the G8 HLA-peptide complex when incubated with the scFv clone G8-P1C11, which is made globally visible using integrated perturbation.

[0152] Figure 45 B depicts an example of HDX data of scFv G8-P1C11 plotted on the crystal structure of the Fab clone G8-P1C11 complexed with the HLA-peptide target A*02:01_AIFPGAVPAA ("G8").

[0153] Figure 46 Depicts the binding affinity of the Fab clone G8-P1C11 for G8 position variants of HLA.

[0154] Figure 47 Shows a histogram of K562 cells bound to G8-P1C11, a target-specific antibody against the HLA-peptide target A*02:01_AIFPGAVPAA ("G8"). DETAILED DESCRIPTION

[0155] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and such definitions included herein are not necessarily to be construed as indicating a difference from the commonly understood meaning in the art. The methods and procedures described or referenced herein are methods and procedures that are generally readily understandable by those of ordinary skill in the art and are typically applied using conventional methodologies, such as, for example, the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Appropriately, unless otherwise indicated, procedures regarding the use of commercially available kits and reagents are generally performed according to the protocols and conditions defined by the manufacturer.

[0156] Unless the context otherwise clearly indicates, the singular forms “a,” “an,” and “the” as used herein include plural referents. Unless otherwise clearly indicated, the terms “comprising,” “such as,” and the like are intended to convey a meaning of including without limitation.

[0157] Unless otherwise specifically indicated, the term “comprising” as used herein also specifically encompasses the implementation examples “consisting of the recited elements” and “consisting essentially of the recited elements.” For example, a multispecific ABP “comprising a bispecific antibody” includes a multispecific ABP “consisting of a bispecific antibody” and a multispecific ABP “consisting essentially of a bispecific antibody.”

[0158] The term “about” refers to and encompasses the indicated value and ranges greater than and less than the value. In certain embodiments, the term “about” means the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term “about” means the specified value ± one standard deviation of the value.

[0159] The term “immunoglobulin” refers to a class of structurally related proteins that typically comprise two pairs of polypeptide chains: a pair of light chains (L) and a pair of heavy chains (H). In a “complete immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology, 7th Edition, Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region typically comprises three domains, abbreviated as C H1 , C H2 , and C H3 . Each light chain typically comprises a light chain variable region (V L ) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated as C L .

[0160] The term “antigen-binding protein” or “ABP” as used herein is used in its broadest sense and includes certain types of molecules that comprise one or more antigen-binding domains that specifically bind to an antigen or epitope.

[0161] 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. The ABP specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, ABP fragments, and multispecific antibodies. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, 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, the CAR includes the ABP. As provided herein, an “HLA-peptide ABP”, “anti-HLA-peptide ABP”, or “HLA-peptide specific ABP” is an ABP that specifically binds to the antigen HLA-peptide. The ABP comprises a protein containing one or more antigen-binding domains that specifically bind to an antigen or epitope through variable regions, such as variable regions derived from B cells (e.g., antibodies) or T cells (e.g., TCRs).

[0162] As used herein, the term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, which fragments include fragment antigen-binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (V H ) regions capable of specifically binding an antigen, single-chain antibody fragments (including single-chain variable fragments (scFv)), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses immunoglobulin forms modified by genetic engineering and / or other means, such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and conjugated antibodies, multispecific antibodies (e.g., bispecific antibodies), diabodies, triabodies, and tetra-bodies, tandem bivalent scFv, tandem trivalent scFv. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0163] As used herein, a “variable region” refers to a variable nucleotide sequence produced by a recombination event, e.g., which may include V, J, and / or D regions of an immunoglobulin or T cell receptor (TCR) sequence from a B cell or T cell (such as an activated T cell or activated B cell).

[0164] The term "antigen-binding domain" refers to the part of an ABP that is capable of specifically binding an antigen or epitope. An example of an antigen-binding domain is an antigen-binding domain formed by the antibody V H -V L dimer. Another example of an antigen-binding domain is an antigen-binding domain formed by diversifying certain loops of the tenth fibronectin type III domain from Adnectin. An antigen-binding domain can sequentially include antibody CDR1, 2, and 3 from the heavy chain; and sequentially include antibody CDR1, 2, and 3 from the light chain. An antigen-binding domain can include TCR CDRs, e.g., αCDR1, αCDR2, αCDR3, βCDR1, βCDR2, and βCDR3. TCR CDRs are described herein.

[0165] The V H region and V L region of an antibody can be further subdivided into hypervariable regions ("hypervariable regions (HVRs)"; also referred to as "complementary determining regions" (CDRs)), which are interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V L usually contains three antibody CDRs and four FRs, which are arranged in the following order (from the N-terminus to the C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Antibody CDRs participate in antigen binding and affect antigen specificity and the binding affinity of the ABP. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991) Public HealthService, National Institutes of Health, Bethesda, MD, which is incorporated herein by reference in its entirety.

[0166] Based on the sequence of the constant domain of vertebrates, the light chains from any vertebrate can be divided into two types, called kappa (κ) and lambda (λ), respectively.

[0167] The heavy chain of any vertebrate can be classified into one of the following five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also referred to as α, δ, ε, γ, and μ, respectively. Based on differences in sequence and function, the IgG and IgA classes are further divided into subclasses. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0168] One of many known numbering schemes can be used by those skilled in the art to determine the amino acid sequence boundaries of antibody CDRs. The known numbering schemes include the numbering schemes described in the following references: ((Kabat et al., supra) the “Kabat” numbering scheme); ((A1-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the “Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the “Contact” numbering scheme); Lefran et al., Dev. Comp. Immunol., 2003, 27:55-77 (the “IMGT” numbering scheme) and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 (the “AHo” numbering scheme); each of which is incorporated herein by reference in its entirety.

[0169] Table 20 provides the positions of antibody CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 identified by the Kabat and Chothia schemes. For CDR-H1, residue numbers are provided using both the Kabat and Chothia numbering schemes.

[0170] For example, ABP numbering software such as Abnum (which is available at www.bioinf.org.uk / abs / abnum / ) can be used to assign antibody CDRs. Abnum is available from the website www.bioinf.org.uk / abs / abnum / and is described in (Abhinandan and Martin, Immunology, 2008, 45:3832-3839) (which is incorporated herein by reference in its entirety).

[0171]

[0172] *When numbering using the Kabat numbering method, the C-terminus of CDR-H1 varies between H32 and H34 depending on the length of the CDR.

[0173] When referring to residues in the ABP heavy chain constant region (e.g., as reported by Kabat et al., supra), the “EU numbering scheme” is typically used. Unless otherwise specified, the EU numbering scheme is used to refer to residues in the ABP heavy chain constant region described herein.

[0174] As used herein, the terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably and refer to an antibody having a structure substantially similar to that of a naturally occurring antibody and having a heavy chain that includes an Fc region. For example, when referring to an IgG molecule, a "full-length antibody" is an antibody that includes two heavy chains and two light chains.

[0175] One of ordinary skill in the art may use any of a number of known numbering schemes to determine the amino acid sequence boundaries of TCR CDRs, including but not limited to the IMGT unique numbering described in the following references: 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. Immunol Genet., 15, 1-7 (1998); Lefranc and Lefranc, The T Cell Receptor FactsBook; and M.-P. Lefranc / Developmental and Comparative Immunology 27(2003)55-77; all of which are incorporated by reference.

[0176] An "ABP fragment" includes a portion of a full-length ABP, such as the antigen-binding or variable region of a full-length ABP. ABP fragments include, for example, Fv fragments, Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv((sFv)) fragments, and scFv-Fc fragments. ABP fragments include antibody fragments. Antibody fragments may include Fv fragments, Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv((sFv)) fragments, scFv-Fc fragments, and TCR fragments.

[0177] An "Fv" fragment includes a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain.

[0178] In addition to the heavy chain variable domain and the light chain variable domain, a "Fab" fragment also includes the constant domain of the light chain and the first constant domain ((CH1)) of the heavy chain. A Fab fragment, for example, can be produced by recombinant methods or by pepsin digestion of a full-length ABP.

[0179] The "F(ab′)2" fragment contains two Fab fragments that are linked by a disulfide bond near the hinge region. The F(ab′)2 fragment can be produced, for example, by recombinant methods or by pepsin digestion of intact ABP. The F(ab′) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0180] The "single-chain Fv" or "sFv" or "scFv" fragment contains the VH domain and the VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). Any suitable linker can be used. In some embodiments, the linker is (GGGGS) n . In some embodiments, n = 1, 2, 3, 4, 5, or 6. See ABP from Escherichia coli. Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal ABPs Volume 113 (pp. 269 - 315). Springer-Verlag, New York, which is incorporated herein by reference in its entirety.

[0181] The "scFv-Fc" fragment contains an scFv bound to an Fc domain. For example, the Fc domain can be bound to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH), the Fc domain can follow VH or VL. Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain includes an IgG4 Fc domain.

[0182] The term "single-domain antibody" refers to a molecule in which one variable domain of an ABP specifically binds an antigen in the absence of the other variable domain. Single-domain ABPs and their fragments are described in: 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 herein by reference in its entirety. Single-domain ABPs are also referred to as sdAbs or nanobodies.

[0183] The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain, which interacts with certain proteins of the Fc receptor and the complement system in naturally occurring antibodies. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, which is incorporated herein by reference in its entirety. The Fc region can be a naturally occurring Fc region or a modified Fc region as described elsewhere in the art or in the present disclosure.

[0184] 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 binds to the antigen or epitope with specificity and affinity similar to that of an ABP. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins TM ), β-sandwich (e.g., iMab), lipocalin (e.g., ), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, protein A (e.g., ), ankyrin repeats (e.g., DARPins), γ-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and (LDLR-A module) e.g., Avimers). Additional information on alternative scaffolds is provided in the following references: 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 herein by reference in its entirety. An alternative scaffold is an ABP.

[0185] "Multi-specific ABP" is an ABP that contains two or more different antigen-binding domains that together specifically bind 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 on different antigens (e.g., different HLA-peptide molecules expressed by the same cell, or an HLA-peptide molecule and a non-HLA-peptide molecule). In some aspects, the multi-specific ABP binds two different epitopes (i.e., "bispecific ABP"). In some aspects, the multi-specific ABP binds three different epitopes (i.e., "trispecific ABP").

[0186] "Monospecific ABP" is an ABP that contains one or more binding sites that specifically bind a single epitope. For example, an example of a monospecific ABP is a naturally occurring IgG molecule, which, although bivalent (i.e., has two antigen-binding domains), recognizes the same epitope on both antigen-binding domains. The binding specificity can be present in any suitable valence.

[0187] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies contains antibodies that are substantially similar and bind the same epitope, except for variants that may typically arise during the production of monoclonal antibodies. There are usually only a small number of such variants. Monoclonal antibodies are typically obtained by methods that include selecting one antibody from a plurality of antibodies. For example, the selection method can be to select a unique clone from a plurality of clones, such as a collection 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.

[0188] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0189] The "humanized" form of a non-human antibody is a chimeric antibody that contains minimal sequences derived from the non-human antibody. A humanized antibody is typically a human antibody (the recipient antibody) in which the residues of one or more CDRs are replaced with the residues of one or more CDRs of a non-human antibody (the donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody with the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced with the corresponding framework region residues of the donor antibody. A humanized antibody can also contain residues not found in either the recipient antibody or the donor antibody. Such modifications can be made to further improve antibody function. For more 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 herein by reference in its entirety.

[0190] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an amino acid sequence of non-human origin that utilizes a human antibody library or human antibody-encoding sequences (e.g., obtained from a human source or designed de novo). A human antibody specifically excludes a humanized antibody.

[0191] "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 intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an ABP and an antigen or epitope). The affinity of a molecule X for its partner Y can be expressed as the dissociation equilibrium constant (KD). The kinetic elements related to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by conventional methods known in the art, including the methods described herein, such as surface plasmon resonance (SPR) technology (e.g., ) or biolayer interferometry (e.g., ).

[0192] Regarding the binding of an ABP to a target molecule, the terms "bind", "specifically bind", "bind specifically to", "specific for", "selectively bind", and "selective" with respect to a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen refer to binding that is significantly different from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, if the control molecule competitively inhibits the binding of the ABP to the target molecule, specific binding is indicated. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 50% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 40% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 30% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 20% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 10% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 1% less than its affinity for the HLA-peptide. In some aspects, the HLA-peptide ABP has an affinity for non-target molecules that is approximately 0.1% less than its affinity for the HLA-peptide.

[0193] As used herein, the term "k" d "(sec -1 ) refers to the dissociation rate constant for a particular ABP-antigen interaction. This value is also referred to as the koff value.

[0194] As used herein, the term "k" a "(M -1 ×sec -1 ) refers to the association rate constant for a particular ABP-antigen interaction. This value is also referred to as the kon value.

[0195] As used herein, the term "K" D "(M) refers to the dissociation equilibrium constant for a particular ABP-antigen interaction. K D = k d / k a . In some embodiments, the affinity of the ABP is described in terms of K D for the interaction between the ABP and its antigen. For clarity, as is known in the art, a smaller K D value indicates a higher affinity interaction, while a larger K DA value indicating a lower affinity interaction.

[0196] As used herein, the term "K" A "(M" -1 ) refers to the association equilibrium constant of a specific ABP-antigen interaction. K A = k a / k d .

[0197] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules, such as a therapeutic agent (e.g., a cytokine) or a diagnostic agent.

[0198] "Fc effector function" refers to the biological activities mediated by the Fc region of an ABP having an Fc region, which activities can vary by isotype. Examples of ABP effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cell cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP).

[0199] When used in the context of two or more ABPs, the terms "compete with" or "cross-compete with" mean that two or more ABPs compete for binding to an antigen (e.g., an HLA-peptide). In one exemplary assay, an HLA-peptide is coated on a surface and contacted with a first HLA-peptide ABP, and then a second HLA-peptide ABP is added. In another exemplary assay, a first HLA-peptide ABP is coated on a surface and contacted with an HLA-peptide, and then a second HLA-peptide ABP is added. If, in either assay, the presence of the first HLA-peptide ABP reduces the binding of the second HLA-peptide ABP, the ABPs compete with each other. The term "compete with" also encompasses combinations of ABPs in which one ABP reduces the binding of another ABP, but no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit each other's binding, regardless of their order of addition. In some embodiments, one 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%. One of ordinary skill in the art can select the concentration of the ABPs for a competition assay based on the affinity of the ABPs for the HLA-peptide and the valency of the ABPs. The assays described in this definition are illustrative, and one of ordinary skill in the art can use any suitable assay to determine whether ABPs compete with each other. Suitable assays are described in the following references: for example, "Immunoassay Methods" in Assay Guidance Manual [Internet], updated December 24, 2014, by Cox et al. (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.

[0200] The term "epitope" refers to a portion of an antigen that binds specifically to an ABP. Epitopes typically consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural properties as well as specific charge properties. The difference between conformational and non-conformational epitopes is that, in the presence of a denaturing solvent, binding to the former but not the latter may be lost. An epitope can contain amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. Known techniques for determining epitopes can be used to determine the epitope that binds to an ABP, such as, for example, testing the binding of an ABP to HLA-peptide variants with different point mutations or to chimeric HLA-peptide variants.

[0201] The "identity" percentage 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 reference sequence after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity. The alignment used to determine the percentage of amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0202] A "conservative substitution" or "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid that is chemically or functionally similar. Tables of conservative substitutions of similar amino acids are well known in the art. By way of example, in some embodiments, the amino acid groups provided in Tables 21-23 are considered to be conservative substitutions of one another.

[0203] Table 21. Selected amino acid groups that are considered to be conservative substitutions of one another in certain embodiments.

[0204]

[0205]

[0206] Table 22. Additional selected amino acid groups that are considered to be conservative substitutions of one another in certain embodiments.

[0207] Group 1 A, S, and T Group 2 D and E Group 3 N and Q Group 4 R and K Group 5 I, L, and M Group 6 F, Y, and W

[0208] Table 23. Further selected amino acid groups that are considered to be conservative substitutions of one another in certain embodiments.

[0209] Group A A and G Group B D and E Group C N and Q Group D R, K, and H Group E I, L, M, V Group F F, Y, and W Group G S and T Group H C and M

[0210] 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. An ABP produced by making one or more conservative substitutions of the amino acid residues of the parental ABP is referred to as a "conservatively modified variant".

[0211] The term "amino acid" refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (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).

[0212] The term "vector" as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of a nucleic acid operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0213] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced and its progeny. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "transfected cells"), each of which includes the primary transformed or transfected cell and its derived progeny. Such progeny may not be exactly identical in nucleic acid content to the parental cell and may contain mutations.

[0214] The term "treating" (and its variants such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or condition in a subject in need thereof. Treatments can be prophylactic and during the clinical pathological process. Desirable treatment outcomes include preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and relieving or improving the prognosis.

[0215] The term "therapeutically effective amount" or "effective amount" as used herein refers to the amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective in treating a disease or disorder.

[0216] 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 has a disease or condition that can be treated with the ABP provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0217] The term "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic or diagnostic product (e.g., a kit), which contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings for using such a therapeutic or diagnostic product.

[0218] The term "tumor" refers to the growth and proliferation of all neoplastic cells (whether malignant or benign), as well as all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive 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 certain aspects, the tumor is a solid tumor. In certain aspects, the tumor is a hematological malignancy.

[0219] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient(s) contained therein to effectively treat a subject and that does not contain additional components that are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0220] The terms "modulate" and "modulation" refer to reducing or inhibiting, or alternatively, activating or increasing the recited variable.

[0221] The terms "increase" and "activate" refer to an increase in the recited variable by 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 more.

[0222] The terms "reduce" and "inhibit" refer to a reduction in the recited variable by 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 more.

[0223] The term "agonize" refers to activating receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and agonizes a receptor.

[0224] The term "antagonize" refers to inhibiting receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0225] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can include, but are not limited to, coding or non-coding regions of genes or gene fragments, loci defined in terms of 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 include 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-galactosylinosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0226] Isolated HLA-peptide target

[0227] The major histocompatibility complex (MHC) is a complex of antigens encoded by a linked set of loci, which are collectively called H-2 in mice and HLA in humans. MHC antigens have two main classes, namely class I and class II, each of which includes a set of cell surface glycoproteins that play a role in determining tissue type and transplant compatibility. In the transplant response, cytotoxic T cells (CTLs) mainly respond to class I glycoproteins, while helper T cells mainly respond to class II glycoproteins.

[0228] Human major histocompatibility complex (MHC) class I molecules (which may be interchangeably referred to herein as HLA class I molecules) are expressed on the surface of almost all cells. The function of these molecules is to present peptides mainly from endogenously synthesized proteins to, for example, CD8+ T cells by interacting with the α-β T cell receptor. MHC class I molecules comprise a heterodimer consisting of an α chain of 46 kDa in size, which is non-covalently associated with a light chain β-2 microglobulin of 12 kDa in size. The α chain usually contains α1 and α2 domains, which form a groove for presenting HLA-restricted peptides, and an α3 transmembrane domain that interacts with the CD8 co-receptor of T cells. Figure 1 (Prior art) depicts the general structure of HLA class I molecules. Some TCRs can bind MHC class I independently of the CD8 co-receptor (see, for example, 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).

[0229] Class I MHC-restricted peptides (which may also be interchangeably referred to herein as HLA-restricted antigens, HLA-restricted peptides, MHC-restricted antigens, restricted peptides or peptides) generally bind to the heavy chain α1-α2 groove through about two or three anchor residues, which interact with the corresponding binding pockets in the MHC molecule. The β-2 microglobulin chain plays an important role in the intracellular transport, peptide binding and conformational 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 β-2 microglobulin results in protein maturation and export to the cell surface.

[0230] The binding of a given HLA subtype to an HLA-restricted peptide forms a complex with a unique and novel surface that can be specifically recognized by an ABP, such as, for example, a TCR on a T cell or an antibody or antigen-binding fragment thereof. The HLA complexed with the HLA-restricted peptide is referred to herein as an HLA-peptide or an HLA-peptide target. In some cases, the restricted peptide is located in 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 anchoring residues that interact with the corresponding binding pockets in the HLA molecule.

[0231] Accordingly, antigens comprising HLA-peptide targets are provided herein. The HLA-peptide target can comprise a specific HLA-restricted peptide having a defined amino acid sequence complexed with a specific HLA subtype.

[0232] The HLA-peptide targets identified herein can be used in tumor immunotherapy. In some embodiments, the HLA-peptide targets identified herein are present on the surface of tumor cells. The HLA-peptide targets identified herein can be expressed by tumor cells in a human subject. 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 common antigens that are typically expressed in a population of human subjects with cancer.

[0233] The HLA-peptide targets discovered in this article may have prevalence rates in individual tumor types. The prevalence rates in individual tumor types may be 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% or 100%. The prevalence rates in individual tumor types can be from approximately 0.1% to 100%, 0.2 to 50%, 0.5 to 25% or 1 to 10%.

[0234] Preferably, HLA-peptide targets are generally not expressed in most normal tissues. For example, in some cases, HLA-peptide targets may not be expressed in the tissues in the Genotype-Tissue Expression (GTEx) project, or in some cases, may be expressed only in immune-privileged tissues or non-essential tissues. Exemplary immune-privileged tissues or non-essential tissues include the testis, minor salivary glands, endocervix, and thyroid. In some cases, if the median expression of the gene from which the restricted peptide is derived is less than 0.5 RPKM (reads per kilobase per million reads) in GTEx samples, if the expression of the gene in GTEX samples does not exceed 10 RPKM, or if the gene expression is greater than or equal to 5 RPKM in no more than two samples among all essential tissue samples, or any combination thereof, the HLA-peptide target may be considered not expressed in essential tissues or non-immune-privileged tissues.

[0235] Exemplary HLA class I subtypes of HLA-peptide targets

[0236] There are many MHC haplotypes in humans (which may be interchangeably referred to as MHC subtypes, HLA subtypes, MHC types, and HLA types in this text). By way of illustration only, exemplary HLA subtypes include: 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*05:01, 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 of their subtypes, including 4-digit, 6-digit, and 8-digit subtypes. As is known to those skilled in the art, there are allelic variants of the above HLA types, and the present invention encompasses all such allelic variants. 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.

[0237] HLA-restricted peptide

[0238] The HLA-restricted peptide (which may be interchangeably referred to herein as "restricted peptide") can be a peptide fragment of a tumor-specific gene (e.g., a cancer-specific gene). Preferably, the cancer-specific gene is expressed in a cancer sample. Genes abnormally expressed in a cancer sample can be identified through databases. By way of illustration only, exemplary databases include: The Cancer Genome Atlas (TCGA) Research Network: http: / / cancergenome.nih.gov / ; The International Cancer Genome Consortium: https: / / dcc.icgc.org / . In some embodiments, the cancer-specific gene has an observed expression of at least 10 RPKM in at least 5 samples from the TCGA database. The cancer-specific gene may have an observable bimodal distribution.

[0239] The cancer-specific gene may have an observed expression greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transcripts per million (TPM) in at least one TCGA tumor tissue. In a preferred embodiment, the cancer-specific gene has an observed expression greater than 100 TPM in at least one TCGA tumor tissue. In some cases, the cancer-specific gene has an observed bimodal expression distribution in TCGA samples. Without wishing to be bound by theory, this bimodal expression pattern is consistent with a biological model in which the amount of expression at baseline is minimal in all tumor samples, while the amount of expression is higher in a subset of tumors that have undergone epigenetic dysregulation.

[0240] Preferably, the cancer-specific gene is generally not expressed in most normal tissues. For example, in some cases, the cancer-specific gene may not be expressed in the tissues in the Genotype-Tissue Expression (GTEx) project, or in some cases, may be expressed in immune-privileged tissues or non-essential tissues. Exemplary immune-privileged tissues or non-essential tissues include the testis, minor salivary glands, endocervix, and thyroid. In some cases, if the median expression of the cancer-specific gene in GTEx samples is less than 0.5 RPKM (reads per kilobase per million reads), if the expression of the gene in GTEX samples does not exceed 10 RPKM, or if the gene expression is greater than or equal to 5 RPKM in no more than two samples among all essential tissue samples, or any combination thereof, the cancer-specific gene may be considered not expressed in essential tissues or non-immune-privileged tissues.

[0241] In some embodiments, by evaluating GTEx, cancer-specific genes meet the following criteria: (1) the GTEx median expression in the brain, heart, or lung is less than 0.1 transcripts per million (TPM), and no sample exceeds 5 TPM; (2) the GTEx median expression in other essential organs (excluding testis, thyroid, minor salivary glands) is below 2 TPM, and no sample exceeds 10 TPM.

[0242] In some embodiments, cancer-specific genes are generally unlikely to be expressed in immune cells. For example, they are not interferon family genes, not eye-related genes, not olfactory or taste receptor genes, and not genes related to the circadian rhythm cycle (e.g., not CLOCK, PERIOD, CRY genes).

[0243] The restricted peptide preferably can be present on the surface of the tumor.

[0244] The size of the restricted peptide 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 molecular residues, and any range derivable therefrom. In certain embodiments, the size of the restricted peptide is about 8, about 9, about 10, about 11, or about 12 amino molecular residues. The length of the restricted peptide can be about 5 to 15 amino acids, preferably about 7 to 12 amino acids, or more preferably about 8 to 11 amino acids.

[0245] Exemplary HLA-peptide targets

[0246] Exemplary HLA-peptide targets are shown in Table A. Each row in Table A shows the HLA allele of each complex and the corresponding HLA-restricted peptide sequence. The peptide sequence can consist of the corresponding sequence shown in each row of Table A. Alternatively, the peptide sequence can include the corresponding sequence shown in each row of Table A. Alternatively, the peptide sequence can consist essentially of the corresponding sequence shown in each row of Table A.

[0247] In some embodiments, the HLA-peptide target is the target shown in Table A.

[0248] In some embodiments, the HLA-restricted peptide is not a gene selected from WT1 or MART1.

[0249] HLA class I molecules that do not associate with restricted peptide ligands are generally unstable. Thus, the association of the restricted peptide with the α1 / α2 groove of the HLA molecule can stabilize the non-covalent association between the β2-microglobulin subunit and the α-subunit of the HLA subtype.

[0250] The stability of the non-covalent association between the β2-microglobulin subunit of an HLA subtype and the α-subunit of an HLA subtype can be determined using any suitable method. For example, this stability can be evaluated by dissolving insoluble aggregates of the HLA molecule in a high concentration of urea (e.g., about 8 M urea), and determining the ability of the HLA molecule to refold in the presence of a restricted peptide upon removal of the urea (e.g., by dialysis). Such a refolding method is described in, for example, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 3429-3433, April 1992, which is hereby incorporated by reference.

[0251] For other embodiments, conditional HLA class I ligands can be used to assess this stability. Conditional HLA class I ligands are typically designed as short, restricted peptides that can stabilize the association between the β2 and α subunits of HLA class I molecules by binding to the α1 / α2 groove of the HLA molecule and contain one or more amino acid modifications such that the restricted peptide will cleave once exposed to a conditional stimulus. Once the conditional ligand cleaves, the β2 and α-subunits of the HLA molecule dissociate, unless the conditional ligand is exchanged for 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 HLA peptide ligands or predicted high-affinity HLA peptide ligands. For HLA alleles for which structural information is available, the water accessibility of the side chains can also be used to select the positions at which to introduce amino acid modifications. Using conditional HLA ligands may be advantageous by allowing for the batch preparation of stable HLA-peptide complexes that can be used to interrogate the restricted peptides being tested in a high-throughput manner.Conditional HLA class I ligands and methods for their production are described, for example, in Proc Natl Acad Sci U S A. Mar 11, 2008; 105(10):3831 - 3836; Proc Natl Acad Sci U S A. Mar 11, 2008; 105(10):3825 - 3830; J Exp Med. May 7, 2018; 215(5):1493 - 1504; Choo, J.A.L. et al. Bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides. Angew Chem Int Ed Engl 53, 13390 - 13394 (2014); 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 Cell Characterisation. ChemBioChem 14, 123 - 131 (2012); Rodenko, B. et al. Class I Major Histocompatibility Complexes Loaded by a Periodate Trigger. J Am Chem Soc 131, 12305 - 12313 (2009); and Chang, C.X.L. et al. Conditional ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses in acute and chronic viral diseases. Eur J Immunol 43, 1109 - 1120 (2013). These references are incorporated by reference in their entirety.

[0252] Thus, in some embodiments, the ability of an HLA-restricted peptide, as described herein (e.g., as described in Table A), to stabilize the association of the β2- and α-subunits of an HLA molecule is evaluated by performing a conditional ligand-mediated exchange reaction and an HLA stability assay. HLA stability can be determined using any suitable method, including, for example, mass spectrometry, immunoassays (e.g., ELISA), size exclusion chromatography, and HLA multimer staining followed by flow cytometry assessment of T cells.

[0253] Other exemplary methods for assessing the stability of the non-covalent association between the β2-microglobulin subunit of an HLA subtype and the α-subunit of an HLA subtype include peptide exchange using a dipeptide. Peptide exchange using a dipeptide is described in, for example, Proc Natl Acad Sci U S A. September 17, 2013; 110(38):15383-8); Proc Natl Acad Sci US A. January 6, 2015; 112(1):202-7), which are incorporated by reference.

[0254] Useful antigens comprising an HLA-peptide target are provided herein. The HLA-peptide target can comprise a specific HLA-restricted peptide having a defined amino acid sequence complexed with a specific HLA subtype allele.

[0255] The HLA-peptide target can be isolated and / or in substantially pure form. For example, the HLA-peptide target can be isolated from its natural environment or can be produced by technical methods. In some cases, the HLA-peptide target is provided in a form that is substantially free of other peptides or proteins.

[0256] The HLA-peptide target can exist in soluble form and, optionally, can be a recombinant HLA-peptide target complex. One of ordinary skill in the art can use any suitable method to produce and purify the recombinant HLA-peptide target. Suitable methods include, for example, using an Escherichia coli expression system, insect cells, etc. Other methods include synthetic production, such as using a cell-free system. WO2017089756 describes exemplary suitable cell-free systems, which are incorporated by reference in their entirety.

[0257] Compositions comprising an HLA-peptide target are also provided herein.

[0258] In some cases, the composition comprises an HLA-peptide target bound to a solid support. Exemplary solid supports include, but are not limited to, beads, wells, membranes, tubes, columns, plates, agarose gels, magnetic beads, and fragments. Exemplary solid carriers are described in, for example, Catalysts 2018, 8, 92; doi:10.3390 / catal8020092, which is hereby incorporated by reference in its entirety.

[0259] The HLA-peptide target can be attached to a solid support by any suitable method known in the art. In some cases, the HLA-peptide target is covalently attached to the solid support.

[0260] In some cases, the HLA-peptide target is attached to the solid support by an affinity binding pair. An affinity binding pair generally involves specific interactions between two molecules. A ligand that has an affinity for its binding partner molecule can be covalently attached to the solid support and thus serves as a bait for immobilizing a common affinity binding pair, which includes, for example, streptavidin and biotin, avidin and biotin; polyhistidine tags having metal ions (such as copper, nickel, zinc, and cobalt), etc.

[0261] The HLA-peptide target can comprise a detectable label.

[0262] A pharmaceutical composition comprising an HLA-peptide target.

[0263] Compositions comprising HLA-peptide targets can be pharmaceutical compositions. Such compositions can comprise multiple HLA-peptide targets. Exemplary pharmaceutical compositions are described herein. The compositions may be capable of eliciting an immune response. The compositions can comprise an adjuvant. Suitable adjuvants include but are not limited to: 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virus particles and other virus-like particles, YF-17D, aflibercept, R848, β-glucan, Pam3Cys, Aquila QS21 stimulator derived from saponins (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunoadjuvants specific for dendritic cells (e.g., MF59) and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison A C; Dev Biol Stand. 1998;92:3-11). Cytokines can also be used. Several cytokines have directly affected the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerated the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, incorporated herein by reference in its entirety) and act as immunoadjuvants (e.g., IL-12) (Gabrilovich DI et al. J Immunother Emphasis Tumor Immunol. 1996(6):414-418). The surface expression of HLA and the intracellular processing of proteins into peptides for presentation on HLA can also be enhanced by interferon-γ (IFN-γ).See, for example, York IA, Goldberg AL, MoXY, Rock KL. Proteolysis and class I major histocompatibility complex antigen presentation. Immunol Rev. 1999;172:49-66; and Rock KL, Goldberg AL. Degradation of cell proteins and the generation of MHC class I-presented peptides. Ann Rev Immunol. 1999;17:12.739-779, which are incorporated herein by reference in their entirety.

[0264] HLA-peptide ABP

[0265] Also provided herein are ABPs that specifically bind to the HLA-peptide targets described herein.

[0266] The HLA-peptide targets can be expressed on the surface of any suitable target cell, including tumor cells.

[0267] The ABP can specifically bind 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, and wherein the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule.

[0268] In some aspects, the ABP does not bind HLA class I in the absence of the HLA-restricted peptide. In some aspects, the ABP does not bind the HLA-restricted peptide in the absence of human MHC class I. In some aspects, the ABP binds to tumor cells that present human MHC class I complexed with an HLA-restricted peptide, optionally, wherein the HLA-restricted peptide is a tumor antigen that characterizes cancer.

[0269] The ABP can bind to each part of the HLA-peptide complex (i.e., HLA and the peptide representing each part of the complex), which, when bound together, form a new target and protein surface for interaction with and binding by the ABP, which is different from the surfaces presented by the peptide alone or by the HLA subtype alone. Generally, in the absence of each part of the HLA-peptide complex, there is no new target and protein surface formed by the binding of HLA to the peptide.

[0270] ABP is capable of specifically binding to a complex comprising HLA and an HLA-restricted peptide (HLA-peptide), e.g., derived from a tumor. In some aspects, in the absence of an HLA-restricted peptide derived from a tumor, ABP does not bind HLA. In some aspects, in the absence of HLA, ABP does not bind an HLA-restricted peptide derived from a tumor. In some aspects, when the HLA-restricted peptide is naturally present on a cell (such as a tumor cell), ABP binds to the complex comprising HLA and the HLA-restricted peptide.

[0271] In some embodiments, the ABP provided herein modulates the binding of an HLA-peptide to one or more ligands of the HLA-peptide.

[0272] ABP can specifically bind to any one of the HLA-peptide targets shown in Table A. In some embodiments, the HLA-restricted peptide is not a gene selected from WT1 or MART1.

[0273] In more specific embodiments, ABP specifically binds to an HLA-peptide target selected from any one of the following: HLA subtype B*35:01 complexed with an HLA-restricted peptide comprising the sequence EVDPIGHVY, HLA subtype HLA-A*02:01 complexed with an HLA-restricted peptide comprising the sequence AIFPGAVPAA, and HLA subtype A*01:01 complexed with an HLA-restricted peptide comprising the sequence ASSLPTTMNY.

[0274] In more specific embodiments, ABP specifically binds to an HLA-peptide target selected from any one of 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.

[0275] In some embodiments, ABP specifically binds to an HLA-peptide target selected from any one of HLA subtype B*35:01 complexed with an HLA-restricted peptide consisting of the sequence EVDPIGHVY, HLA subtype A*02:01 complexed with an HLA-restricted peptide consisting of the sequence AIFPGAVPAA, and HLA subtype A*01:01 complexed with an HLA-restricted peptide consisting of the sequence ASSLPTTMNY.

[0276] In some embodiments, ABP is an ABP that competes with the illustrative ABP provided herein. In some aspects, the ABP that competes with the illustrative ABP provided herein and the illustrative ABP provided herein bind the same epitope.

[0277] In some embodiments, the ABP described herein is referred to herein as a "variant". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method 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 for obtaining ABP. In some embodiments, the variant is derived from any of the sequences provided herein, wherein one or more conservative amino acid substitutions are made. In some embodiments, the variant is derived from any of the sequences provided herein, wherein one or more non-conservative amino acid substitutions are made. Conservative amino acid substitutions are described herein. Exemplary non-conservative amino acid substitutions include those described in J Immunol. May 1, 2008; 180(9):6116-31, which is incorporated herein by reference in its entirety. In preferred embodiments, the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. In more preferred embodiments, the non-conservative amino acid substitutions enhance the biological activity of the functional variant, such that the biological activity of the functional variant is enhanced compared to the parental ABP.

[0278] ABP comprising an antibody or antigen-binding fragment thereof

[0279] The ABP can comprise an antibody or an antigen-binding fragment thereof.

[0280] In some embodiments, the ABP provided herein comprises a light chain. In some aspects, the light chain is a κ light chain. In certain aspects, the light chain is a λ light chain.

[0281] In some embodiments, the ABP provided herein comprises a heavy chain. In some aspects, the heavy chain is IgA. In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG1. In some aspects, the heavy chain is IgG2. In some aspects, the heavy chain is IgG3. In some aspects, the heavy chain is IgG4. In some aspects, the heavy chain is IgA1. In some aspects, the heavy chain is IgA2.

[0282] 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 aspects, the ABP fragment is an Fv fragment. In some aspects, the ABP fragment is a Fab fragment. In some aspects, the ABP fragment is an F(ab′)2 fragment. In some aspects, the ABP fragment is a Fab′ fragment. In some aspects, the ABP fragment is a scFv (sFv) fragment. In some aspects, the ABP fragment is a scFv-Fc fragment. In some aspects, the ABP fragment is a fragment of a single-domain ABP.

[0283] In some embodiments, the ABP fragment provided herein is derived from the illustrative ABP provided herein. In some embodiments, the ABP fragment provided herein is not derived from the illustrative ABP provided herein, but can be isolated de novo, for example, according to the methods provided herein for obtaining ABP fragments.

[0284] In some embodiments, the ABP fragment provided herein retains 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 fragment provided herein retains the ability to prevent the interaction of an HLA-peptide with one or more of its ligands, as described herein.

[0285] In some embodiments, the ABP provided herein is a monoclonal ABP. In some embodiments, the ABP provided herein is a polyclonal ABP.

[0286] In some embodiments, the ABP provided herein includes 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 includes 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 includes 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.

[0287] In some embodiments, the ABP provided herein comprises an alternative scaffold. In some embodiments, the ABP provided herein consists of an alternative scaffold. In some embodiments, the ABP provided herein consists essentially of an alternative scaffold. Any suitable alternative scaffold can be used. In some aspects, the alternative scaffold is selected from: AdnectinTM, iMab, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, DARPin, Affilin, Tetranectin, Fynomer, and Avimer.

[0288] Also disclosed herein are isolated humanized, human, or chimeric ABPs that compete with the ABPs disclosed herein for binding to HLA-peptides.

[0289] Also disclosed herein are isolated humanized, human, or chimeric ABPs that bind to the HLA-peptide epitopes bound by the ABPs described herein.

[0290] In some aspects, the ABP comprises a human Fc region that comprises at least one modification that reduces binding to human Fc receptors.

[0291] It is known that when expressed in cells, ABPs are post-translationally modified. Examples of post-translational modifications include: cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase; modification of glutamine or glutamate at the N-terminus of the heavy and light chains to pyroglutamate by pyroglutamyl methylation, glycosylation, oxidation, deamidation; and glycation, which is known to occur in various ABPs (see Journal of Pharmaceutical Sciences, 2008, Vol. 97, pp. 2426-2447, which is incorporated herein by reference in its entirety). In some embodiments, the ABP is a post-translationally modified ABP or an antigen-binding fragment thereof. Examples of post-translationally modified ABPs or antigen-binding fragments thereof include: ABPs or antigen-binding fragments thereof with pyroglutamyl methylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications due to N-terminal pyroglutamyl methylation and C-terminal lysine deletion have no effect on the activity of the ABP or its fragments (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39, which is incorporated herein by reference in its entirety).

[0292] Monospecific and multispecific HLA-peptide ABP

[0293] In some embodiments, the ABP provided herein is a monospecific ABP.

[0294] In some embodiments, the ABP provided herein is a multispecific ABP.

[0295] In some embodiments, the multispecific ABP provided herein binds more than one antigen. In some embodiments, the multispecific ABP binds 2 antigens. In some embodiments, the multispecific ABP binds 3 antigens. In some embodiments, the multispecific ABP binds 4 antigens. In some embodiments, the multispecific ABP binds 5 antigens.

[0296] In some embodiments, the multispecific ABP provided herein binds more than one epitope on an HLA-peptide antigen. In some embodiments, the multispecific ABP binds 2 epitopes on an HLA-peptide antigen. In some embodiments, the multispecific ABP binds 3 epitopes on an HLA-peptide antigen.

[0297] Many multispecific ABP constructs are known in the art, and the ABP provided herein can be provided in the form of any suitable multispecific construct.

[0298] In some embodiments, the multispecific ABP comprises an immunoglobulin that comprises at least two different heavy chain variable regions, each heavy chain variable region paired with a common light chain variable region (i.e., a "common light chain ABP"). The common light chain variable region forms a different antigen-binding domain with each of the two different heavy chain variable regions. See Merchant et al., Nature Biotechnol., 1998, 16:677-681, which is incorporated herein by reference in its entirety.

[0299] In some embodiments, the multispecific ABP comprises an immunoglobulin that comprises an ABP or a fragment thereof that binds to one or more of the N- or C-terminus of the heavy or light chain of the immunoglobulin. See Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163, which is incorporated herein by reference in its entirety. In some aspects, such ABP includes a tetravalent bispecific ABP.

[0300] In some embodiments, the multispecific ABP includes a hybrid immunoglobulin that comprises at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello Nature, 1983, 305:537-540); and (Staerz and Bevan, Proc. Natl. Acad. Sci. USA, 1986, 83:1453-1457), each of which is incorporated herein by reference in its entirety.

[0301] In some embodiments, the multispecific ABP comprises immunoglobulin chains with variable sequences to reduce the formation of by-products that are not multispecific. In some aspects, the ABP comprises one or more "knobs-into-holes" modifications as described in U.S. Patent No. 5,731,168, which is incorporated herein by reference in its entirety.

[0302] In some embodiments, the multispecific ABP comprises immunoglobulin chains having one or more electrostatic modifications to facilitate Fc heteropolymer assembly. See WO 2009 / 089004, which is incorporated herein by reference in its entirety.

[0303] In some embodiments, the multispecific ABP comprises bispecific single-chain molecules. 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 herein by reference.

[0304] In some embodiments, the multispecific ABP comprises a heavy-chain variable domain and a light-chain variable domain linked via a polypeptide linker, wherein the linker length is selected to promote the assembly of the multispecific ABP with the desired multispecificity. For example, when the heavy-chain variable domain and the light-chain variable domain are linked by a polypeptide linker having a size of more than 12 amino acid residues, monospecific scFvs are generally formed. See U.S. Patent Nos. 4,946,778 and 5,132,405, both of which are incorporated herein by reference. In some embodiments, reducing the polypeptide linker length to less than 12 amino acid residues prevents the pairing of the heavy-chain and light-chain variable domains on the same polypeptide chain, thereby enabling the heavy-chain and light-chain variable domains from one chain to pair with the complementary domains on another chain. Thus, the resulting ABP has multispecificity, with the specificity of each binding site being assigned by more than one polypeptide chain. Polypeptide chains comprising heavy-chain and light-chain variable domains linked by a linker of 3 to 12 amino acid residues predominantly form dimers (referred to as diabodies). Linkers having 0 to 2 amino acid residues, i.e., trimers (referred to as triabodies) and tetramers (referred to as tetra-bodies), are advantageous. However, in addition to the linker length, the exact type of oligomerization appears to depend on the amino acid residue composition and the order of the variable domains in each polypeptide chain (e.g., VH-linker-VL versus VL-linker-VH). One skilled in the art can select an appropriate linker length based on the desired multispecificity.

[0305] Fc region and variants

[0306] In certain embodiments, the ABP provided herein comprises an Fc region. The Fc region can be wild-type or a variant thereof. In certain embodiments, the ABP provided herein comprises an Fc region having one or more amino acid substitutions, insertions, or deletions compared to a naturally occurring Fc region. In some aspects, such substitutions, insertions, or deletions result in an ABP having altered stability, glycosylation, or other characteristics. In some aspects, such substitutions, insertions, or deletions result in a glycosylated ABP.

[0307] A "variant Fc region" or "engineered Fc region" comprises an amino acid sequence that differs from a native sequence Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the native sequence Fc region or the Fc region of the parental polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or with the Fc region of the parental polypeptide, and most preferably has at least about 90% homology therewith, and more preferably has at least about 95% homology therewith.

[0308] The term "Fc region-containing ABP" refers to an ABP that comprises an Fc region. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region can be removed, e.g., during purification of the ABP or by recombinant engineering of the nucleic acid encoding the ABP. Thus, an ABP having an Fc region can include an ABP that contains or does not contain K447.

[0309] In some aspects, the Fc region of the ABP provided herein is modified to produce an ABP having an altered affinity for an Fc receptor or to produce a more immunologically inert ABP. In some embodiments, the ABP variant provided herein has some but not all effector functions. For example, such an ABP can be useful when the half-life of the ABP is important in vivo, but when certain effector functions (e.g., complement activation and ADCC) are unnecessary or detrimental.

[0310] In some embodiments, the Fc region of the ABP provided herein is a human IgG4 Fc region that comprises one or more mutations, S228P and L235E, that stabilize the hinge. See Aalberse et al., Immunology, 2002, 105:9-19, which is incorporated herein by reference in its entirety. In some embodiments, the IgG4 Fc region comprises one or more of the following mutations: E233P, F234V, and L235A. See Armour et al., Mol. Immunol., 2003, 40:585-593, which is incorporated herein by reference in its entirety. In some embodiments, the IgG4 Fc region comprises a deletion at position G236.

[0311] In some embodiments, the Fc region of the ABP provided herein is a human IgG1 Fc region that includes one or more mutations that reduce Fc receptor binding. In some aspects, the one or more mutations occur 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 aspects, the ABP includes the PVA236 mutation. PVA236 refers to the substitution of the amino acid sequence ELLG from amino acid positions 233 to 236 of IgG1 or EFLG of IgG4 with PVA. See U.S. Patent No. 9,150,641, which is incorporated by reference in its entirety.

[0312] In some embodiments, the Fc region of the ABP provided herein is modified 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.

[0313] In some embodiments, the Fc region of the ABP provided herein is a human IgG2 Fc region that includes one or more mutations A330S and P331S.

[0314] In some embodiments, the Fc region of the ABP provided herein has an amino acid substitution at one or more of the following positions: 238, 265, 269, 270, 297, 327, and 329. See U.S. Patent No. 6,737,056, which is incorporated by reference in its entirety. Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so - called "DANA" Fc mutant, which has alanine substitutions at residues 265 and 297. See U.S. Patent No. 7,332,581, which is incorporated by reference in its entirety. In some embodiments, the ABP includes alanine at amino acid position 265. In some embodiments, the ABP includes alanine at amino acid position 297.

[0315] In certain embodiments, the ABP provided herein comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of positions 298, 333, and 334 in the Fc region. In some embodiments, the ABP provided herein comprises an Fc region having 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 herein by reference in its entirety.

[0316] In some embodiments, the ABP provided herein comprises one or more alterations that improve or reduce C1q binding and / or CDC. See 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 herein by reference in its entirety.

[0317] In some embodiments, the ABP provided herein comprises one or more alterations to increase the half - life. An ABP having an increased half - life and improved binding to the neonatal Fc receptor (FcRn) is described in, for example, Hinton et al., J. Immunol., 2006, 176:346 - 356; and U.S. Patent Publication No. 2005 / 0014934; each of which is incorporated herein by reference in its entirety. Such Fc variants comprise Fc variants substituted at one or more of the following Fc region residues of IgG: 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, and 434. In some embodiments, the ABP comprises one or more non - Fc modifications that prolong the half - life. Exemplary non - Fc modifications that prolong the half - life are described in, for example, US20170218078, which is hereby incorporated herein by reference.

[0318] In some embodiments, the ABP provided herein comprises one or more Fc region variants, as 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 herein by reference in its entirety.

[0319] Antibody specific for B*35:01_EVDPIGHVY (HLA-peptide target "G5")

[0320] In some aspects, the present disclosure provides an ABP that comprises an antibody or an antigen-binding fragment thereof that specifically binds to an HLA-peptide target, wherein the HLA class I molecule of the HLA-peptide target is HLA subtype B*35:01, and the HLA-restricted peptide of the HLA-peptide target comprises the sequence EVDPIGHVY (“G5”) or consists of or consists essentially of the same.

[0321] CDR

[0322] The ABP specific for B*35:01_EVDPIGHVY may comprise one or more antibody complementarity-determining region (CDR) sequences. For example, it may comprise three heavy-chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light-chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0323] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-H3 sequence. The CDR-H3 sequence may be selected from CARDGVRYYGMDVW, CARGVRGYDRSAGYW, CASHDYGDYGEYFQHW, CARVSWYCSSTSCGVNWFDPW, CAKVNWNDGPYFDYW, CATPTNSGYYGPYYYYGMDVW, CARDVMDVW, CAREGYGMDVW, CARDNGVGVDYW, CARGIADSGSYYGNGRDYYYGMDVW, CARGDYYFDYW, CARDGTRYYGMDVW, CARDVVANFDYW, CARGHSSGWYYYYGMDVW, CAKDLGSYGGYYW, CARSWFGGFNYHYYGMDVW, CARELPIGYGMDVW, and CARGGSYYYYGMDVW.

[0324] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-L3 sequence. The CDR-L3 sequence may be selected from CMQGLQTPITF, CMQALQTPPTF, CQQAISFPLTF, CQQANSFPLTF, CQQANSFPLTF, CQQSYSIPLTF, CQQTYMMPYTF, CQQSYITPWTF, CQQSYITPYTF, CQQYYTTPYTF, CQQSYSTPLTF, CMQALQTPLTF, CQQYGSWPRTF, CQQSYSTPVTF, CMQALQTPYTF, CQQANSFPFTF, CMQALQTPLTF, and CQQSYSTPLTF.

[0325] An ABP specific for B*35:01_EVDPIGHVY may contain a specific heavy chain CDR3 (CDR-H3) sequence and a specific light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP contains CDR-H3 and CDR-L3 from an scFv named 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 the scFvs that specifically bind B*35:01_EVDPIGHVY are shown in Table 5. For clarity, each identified scFv is named by its clone name, and each row contains the CDR sequences of that specific clone name. For example, the one identified by the clone name G5_P7_E7 contains the heavy chain CDR3 sequence CARDGVRYYGMDVW and the light chain CDR3 sequence CMQGLQTPITF.

[0326] An ABP specific for B*35:01_EVDPIGHVY may contain all 6 CDRs from an scFv named 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.

[0327] VH

[0328] The ABP specific to B*35:01_EVDPIGHVY may contain a VH sequence. The VH sequence may be selected from QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGIINPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVRYYGMDVWGQGTTVTVSSAS, QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSHDINWVRQAPGQGLEWMGWMNPNSGDTGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGVRGYDRSAGYWGQGTLVIVSSAS, EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISYISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHDYGDYGEYFQHWGQGTLVTVSSAS, EVQLLQSGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSWYCSSTSCGVNWFDPWGQGTLVTVSSAS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVASISSSGGYINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVNWNDGPYFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFGVSWLRQAPGQGLEWMGGIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATPTNSGYYGPYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDVMDVWGQGTTVTVSS.QVQLVQSGAEVKKPGASVKVSCKASGGTFSGYLVSWVRQAPGQGLEWMGWINPNSGGTNTAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYIFRNYPMHWVRQAPGQGLEWMGWINPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDNGVGVDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNPNIGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIADSGSYYGNGRDYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWINPNSGVTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWINPNSGDTKYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGTRYYGMDVWGQGTTVTVSS, EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSYISSSSSYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDVVANFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWMNPDSGSTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGHSSGWYYYYGMDVWGQGTTVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYSMHWVRQAPGKGLEWVSSITSFTNTMYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLGSYGGYYWGQGTLVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSWFGGFNYHYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARELPIGYGMDVWGQGTTVTVSS, and QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGSYYYYGMDVWGQGTTVTVSS.,

[0329] VL

[0330] The ABP specific to B*35:01_EVDPIGHVY may contain a VL sequence. The VL sequence may be selected from DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPITFGQGTRLEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQSTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYMMPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGTKVEIK,DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGQGTKLEIK, DIVMTQSPDSLAVSLGERATINCKTSQSVLYRPNNENYLAWYQQKPGQPPKLLIYQASIREPGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTIPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRFLNWYQQKPGKAPKLLIYGASRPQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK, EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYAASARASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSWPRTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGTKVEIK, DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCQASEDISNHLNWYQQKPGKAPKLLIYDALSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK,DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGQGTKVEIK and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.,

[0331] VH-VL combination

[0332] ABPs specific for B*35:01_EVDPIGHVY may comprise a specific VH sequence and a specific VL sequence. In some embodiments, an ABP specific for B*35:01_EVDPIGHVY comprises the VH and VL sequences from an scFv named 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 the identified scFv hits that specifically bind B*35:01_EVDPIGHVY are shown in Table 4. For clarity, each identified scFv hit is named with a clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv hit identified by the clone name G5_P7_E7 comprises the VH sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGIINPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVRYYGMDVWGQGTIVTVSSAS and the VL sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPITFGQGTRLEIK.,

[0333] Antibody specific for A*02:01_AIFPGAVPAA (HLA-peptide target "G8")

[0334] In some aspects, the present disclosure provides an ABP that comprises an antibody or an antigen-binding fragment thereof that specifically binds to an HLA-peptide target, wherein the HLA class I molecule of the HLA-peptide target is HLA subtype A*02:01, and the HLA-restricted peptide of the HLA-peptide target comprises the sequence AIFPGAVPAA (“G8”), consists of, or consists essentially of, the same.

[0335] CDR

[0336] The ABP specific for A*02:01_AIFPGAVPAA may comprise one or more antibody complementarity-determining region (CDR) sequences, for example, may comprise three heavy-chain CDRs (CDR-H1, CDR-H2, CDR-H3) and three light-chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0337] The ABP specific for A*02:01_AIFPGAVPAA may comprise a CDR-H3 sequence. The CDR-H3 sequence may be selected from CARDDYGDYVAYFQHW, CARDLSYYYGMDVW, CARVYDFWSVLSGFDIW, CARVEQGYDIYYYYYMDVW, CARSYDYGDYLNFDYW, CARASGSGYYYYYGMDVW, CAASTWIQPFDYW, CASNGNYYGSGSYYNYW, CARAVYYDFWSGPFDYW, CAKGGIYYGSGSYPSW, CARGLYYMDVW, CARGLYGDYFLYYGMDVW, CARGLLGFGEFLTYGMDVW, CARDRDSSWTYYYYGMDVW, CARGLYGDYFLYYGMDVW, CARGDYYDSSGYYFPVYFDYW, and CAKDPFWSGHYYYYGMDVW.

[0338] The ABP specific for A*02:01_AIFPGAVPAA may comprise a CDR-L3 sequence. The CDR-L3 sequence may be selected from CQQNYNSVTF, CQQSYNTPWTF, CGQSYSTPPTF, CQQSYSAPYTF, CQQSYSIPPTF, CQQSYSAPYTF, CQQHNSYPPTF, CQQYSTYPITI, CQQANSFPWTF, CQQSHSTPQTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQTYSTPWTF, CQQYGSSPYTF, CQQSHSTPLTF, CQQANGFPLTF, and CQQSYSTPLTF.

[0339] An ABP specific for A*02:01_AIFPGAVPAA can 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 an scFv named 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 the identified scFv hits that specifically bind A*02:01_AIFPGAVPAA are shown in Table 7. For clarity, each identified scFv hit is named with a clone name, and each row contains the CDR sequences for that particular clone name. For example, the scFv hit identified by the clone name G8-P1A03 comprises the heavy chain CDR3 sequence CARDDYGDYVAYFQHW and the light chain CDR3 sequence CQQNYNSVTF.

[0340] An ABP specific for A*02:01_AIFPGAVPAA can comprise all 6 CDRs from an scFv named 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.

[0341] VH

[0342] ABPs specific for A*02:01_AIFPGAVPAA may contain VH sequences. The VH sequences may be selected from QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGWINPNSGATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDDYGDYVAYFQHWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYPFIGQYLHWVRQAPGQGLEWMGIINPSGDSATYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDLSYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWMNPIGGGTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVYDFWSVLSGFDIWGQGTLVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVEQGYDIYYYYYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGWISTYSGHADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSYDYGDYLNFDYWGQGTLVTVSS, EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSSISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGYYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYFMHWVRQAPGQGLEWMGMVNPSGGSETFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAASTWIQPFDYWGQGTLVTVSS,EVQLLESGGGLVQPGGSLRLSCAASGFDFSIYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNGNYYGSGSYYNYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLTTYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVYYDFWSGPFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPYSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGGIYYGSGSYPSWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGVSWVRQAPGQGLEWMGWISPYSGNTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLYYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFSNMYLHWVRQAPGQGLEWMGWINPNTGDTNYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDVWGQGTKVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLLGFGEFLTYGMDVWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGVINPSGGSTTYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRDSSWTYYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDVWGQGTTVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGGTFSSHAISWVRQAPGQGLEWMGVIIPSGGTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYDSSGYYFPVYFDYWGQGTLVTVSS and QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPFWSGHYYYYGMDVWGQGTTVTVSS.,

[0343] VL

[0344] The ABP specific to A*02:01_AIFPGAVPAA may contain a VL sequence. The VL sequence may be selected from DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPWTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQAISNSLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSTPPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGGGTKVDIK, DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGINSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNSYPPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPITIGQGTKVEIK,DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQDVSTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPQTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQGTKLEIK, EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPLTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQNIYTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK, and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.,

[0345] VH-VL combination

[0346] An ABP specific for A*02:01_AIFPGAVPAA may comprise a specific VH sequence and a specific VL sequence. In some embodiments, an ABP specific for A*02:01_AIFPGAVPAA may comprise the VH and VL sequences from an scFv named 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 the identified scFv hits that specifically bind A*02:01_AIFPGAVPAA are shown in Table 6. For clarity, each identified scFv hit is named by its clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv hit identified by the clone name G8-P1A03 comprises the VH sequence QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGWINPNSGATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDDYGDYVAYFQHWGQGTLVTVSS and the VL sequence DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTKLEIK.

[0347] Antibody specific for A*01:01_ASSLPTTMNY (HLA-peptide target "G10")

[0348]

[0349] CDR

[0350] ​The ABP specific to A*01:01_ASSLPTTMNY may comprise one or more antibody complementarity-determining region (CDR) sequences, for example, may comprise 3 heavy-chain CDRs (CDR-H1, CDR-H2, CDR-H3) and 3 light-chain CDRs (CDR-L1, CDR-L2, CDR-L3).

[0351] The ABP specific to A*01:01_ASSLPTTMNY may comprise a CDR-H3 sequence. The CDR-H3 sequence may be selected from CARDQDTIFGVVITWFDPW, CARDKVYGDGFDPW, CAREDDSMDVW, CARDSSGLDPW, CARGVGNLDYW, CARDAHQYYDFWSGYYSGTYYYGMDVW, CAREQWPSYWYFDLW, CARDRGYSYGYFDYW, CARGSGDPNYYYYYGLDVW, CARDTGDHFDYW, CARAENGMDVW, CARDPGGYMDVW, CARDGDAFDIW, CARDMGDAFDIW, CAREEDGMDVW, CARDTGDHFDYW, CARGEYSSGFFFVGWFDLW, and CARETGDDAFDIW.

[0352] The ABP specific to A*01:01_ASSLPTTMNY may comprise a CDR-L3 sequence. The CDR-L3 sequence may be selected from CQQYFTTPYTF, CQQAEAFPYTF, CQQSYSTPITF, CQQSYIIPYTF, CHQTYSTPLTF, CQQAYSFPWTF, CQQGYSTPLTF, CQQANSFPRTF, CQQANSLPYTF, CQQSYSTPFTF, CQQSYSTPFTF, CQQSYGVPTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQYYSYPWTF, CQQSYSTPFTF, CMQTLKTPLSF, and CQQSYSTPLTF.

[0353] 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 CDR-H3 and CDR-L3 from an scFv named R3G10-PLA07, 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 the identified scFv hits that specifically bind A*01:01_ASSLPTTMNY are shown in Table 9. For clarity, each identified scFv hit is named with a clone name, and each row contains the CDR sequences for that specific clone name. For example, the scFv hit identified by the clone name R3G10-P1A07 comprises the heavy chain CDR3 sequence CARDQDTIFGVVITWFDPW and the light chain CDR3 sequence CQQYFTTPYTF.

[0354] An ABP specific for A*01:01_ASSLPTTMNY may comprise all 6 CDRs from an scFv named 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.

[0355] VH

[0356] The ABP specific for A*01:01_ASSLPTTMNY may comprise a VH sequence. The VH sequence may be selected from EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISARSGRTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDQDTIFGVVITWFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIHPGGGTTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDKVYGDGFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDDSMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSGLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNLDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGVTFSTSAISWVRQAPGQGLEWMGWISPYNGNTDYAQMLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAHQYYDFWSGYYSGTYYYGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGGTFSNSIINWVRQAPGQGLEWMGWMNPNSGNTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREQWPSYWYFDLWGRGTLVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGGTFSTHDINWVRQAPGQGLEWMGVINPSGGSAIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGYSYGYFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGNTFIGYYVHWVRQAPGQGLEWVGIINPNGGSISYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSGDPNYYYYYGLDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGIIGPSDGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAENGMDVWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGIIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGGYMDVWGKGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGMIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGDAFDIWGQGTMVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRISPSDGSTTYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMGDAFDIWGQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREEDGMDVWGQGTTVTVSS,QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMGMIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNFAISWVRQAPGQGLEWMGGIIPIFDATNYAQKFQGRVTFTADESTSTAYMELSSLRSEDTAVYYCARGEYSSGFFFVGWFDLWGRGTQVTVSS, and QVQLVQSGAEVKKPGASVKVSCKASGYNFTGYYMHWVRQAPGQGLEWMGIIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARETGDDAFDIWGQGTMVTVSS.,

[0357] VL

[0358] ABP specific for A*01:01_ASSLPTTMNY may contain a VL sequence. The VL sequence may be selected from DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFTTPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIFDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEAFPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIIPYTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQTYSTPLTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPWTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQNISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPLTFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQDISRYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPRTFGQGTKVEIK,DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSLPYTFGQGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYDASKLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPTFGQGTKLEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, DIQMTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPWTFGQGTRLEIK, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLKTPLSFGGGTKVEIK and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK.

[0359] VH-VL combination

[0360] ABPs specific for A*01:01_ASSLPTTMNY can comprise a specific VH sequence and a specific VL sequence. In some embodiments, ABPs specific for A*01:01_ASSLPTTMNY can comprise the VH and VL sequences from scFvs named 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 VH and VL sequences of the identified scFvs that specifically bind A*01:01_ASSLPTTMNY are shown in Table 8. For clarity, each identified scFv hit is named by the clone name, and each row contains the VH and VL sequences for that particular clone name. For example, the scFv hit identified by the clone name R3G10-P1A07 comprises the VH sequence EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISARSGRTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDQDTIFGVVITWFDPWGQGTLVTVSS and the VL sequence DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFITPYTFGQGTKLEIK.

[0361] Receptor

[0362] In the provided ABP, for example, the HLA-peptide ABP is the receptor. The receptor can comprise an antigen receptor and other chimeric receptors that specifically bind to the HLA-peptide targets disclosed herein. The receptor can be a T cell receptor (TCR). The receptor can be a chimeric antigen receptor (CAR).

[0363] The TCR can be soluble or membrane-bound. There are functional non-TCR antigen receptors among the antigen receptors, such as chimeric antigen receptor (CAR). Also provided are cells expressing the receptor and their use in adoptive cell therapy, such as treating diseases and disorders associated with HLA-peptide expression, including cancer.

[0364] Exemplary antigen receptors (including CARs) and methods of engineering and introducing these receptors into cells include those described in, for example, International Patent Application Publications Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061; U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337; U.S. Patents 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. EP2537416, and / or those described in the following: Sadelain et al., Cancer Discov. April 2013; 3(4):388 - 398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5):633 - 39; Wu et al., Cancer, March 2012 18(2):160 - 75. In some aspects, the antigen receptor comprises the CAR described in U.S. Patent No. 7,446,190 and those CARs described in International Patent Application Publication No. WO / 2014055668 A1. Exemplary CARs include those CARs described in any of the above publications, such as WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, U.S. Patent No. 8,389,282, etc., where the antigen - binding portion (e.g., scFv) is replaced by an antibody (e.g., an antibody provided herein).

[0365] The chimeric receptor contains a chimeric antigen receptor (CAR). A chimeric receptor, such as a CAR, generally comprises an extracellular antigen-binding domain, which is contained in one of the provided anti-HLA-peptide ABP, for the anti-HLA-peptide ABP, comprising the anti-HLA-peptide ABP, such as an anti-HLA-peptide antibody. Thus, the extracellular portion of the chimeric receptor (e.g., CAR) typically comprises one or more HLA-peptide-ABP, such as one or more antigen-binding fragments, domains or portions, or one or more antibody variable domains, and / or antibody molecules (such as those described herein). In some embodiments, the CAR comprises a portion of an HLA-peptide-binding moiety or ABP (e.g., an antibody) molecule, such as the variable heavy (VH) chain region and / or variable light (VL) chain region of an antibody, e.g., an scFv antibody fragment.

[0366] TCR

[0367] In one aspect, the 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.

[0368] In most T cells, the TCR is a heterodimeric polypeptide having an alpha (α) chain and a beta (β) chain encoded by TRA and TRB, respectively. The α chain typically comprises an α variable region encoded by TRAV, an α joining region encoded by TRAJ, and an α constant region encoded by TRAC. The β chain typically comprises a β variable region encoded by TRBV, a β diversity region encoded by TRBD, a β joining region encoded by TRBJ, and a β constant region encoded by TRBC. The TCR-α chain is generated by VJ recombination, while the β chain receptor is generated by V(D)J recombination. Additional diversity of the TCR results from junctional diversity. Several bases can be deleted and several bases can be added (termed N and P nucleotides) at each junction. In most T cells, the TCR comprises a γ chain and a δ chain. The TCRγ chain is generated by VJ recombination, while the TCRδ 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 the TCR typically comprises six complementarity-determining regions (CDRs). The α chain contributes three CDRs: αCDR1, αCDR2, and αCDR3. The β chain also provides three CDRs: βCDR1, βCDR2, and βCDR3. αCDR3 and βCDR3 are the regions most affected by V(D)J recombination and are responsible for most of the variation in the TCR repertoire.

[0369] TCR can specifically recognize HLA-peptide targets, such as the HLA-peptide targets disclosed in Table A; thus, a TCR can be an ABP that specifically binds to an HLA-peptide. A TCR can be soluble, for example, similar to an antibody secreted by a B cell. A TCR can also be membrane-bound, for example, bound to a cell such as a T cell or a natural killer (NK) cell. Thus, TCRs can be used in the context corresponding to soluble antibodies and / or membrane-bound CARs.

[0370] Any TCR disclosed herein can comprise an alpha variable region, an alpha joining region, optionally an alpha constant region, a beta variable region, optionally a beta diversity region, a beta joining region, and optionally a beta constant region.

[0371] In some embodiments, the TCR or CAR is a recombinant TCR or CAR. A recombinant TCR or CAR can comprise any TCR identified herein, but comprises one or more modifications. Exemplary modifications are described herein, such as amino acid substitutions. The amino acid substitutions described herein can be referenced according to the IMGT nomenclature and the amino acid numbering on the website www.imgt.org.

[0372] A recombinant TCR or CAR can be a human TCR or CAR that comprises a complete human sequence, such as a native human sequence. A recombinant TCR or CAR can retain its native human variable domain sequence, but contains 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 can, for example, improve TCR assembly and expression in TCR gene therapy by driving preferential pairing of the exogenous TCR chains.

[0373] In some embodiments, the alpha and beta constant regions are modified by replacing the murine constant region sequences with the entire human constant region sequences. Such "humanized" TCRs and methods for their preparation are described in Cancer Res. September 1, 2006; 66(17):8878-86, which is incorporated herein by reference in its entirety.

[0374] In some embodiments, the α and β constant regions are modified by making one or more amino acid substitutions in the human TCRα constant (TRAC) region, the TCRβ constant (TRBC) region, or both the TRAC and TRAB regions, i.e., by substituting human residues with murine residues ((human→murine amino acid exchanges)). 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. 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.

[0375] In some embodiments, human TRAC contains an Asp substitution at residue 210, and human TRBC contains a Lys substitution at residue 134. Such substitutions can promote the formation of salt bridge bonds between the α and β chains and the formation of inter-TCR chain disulfide bonds. These targeted substitutions are described in J Immunol, June 1, 2010; 184(11):6232-6241, which is incorporated herein by reference in its entirety.

[0376] In some embodiments, the human TRAC region and the human TRBC region are modified to contain introduced cysteines, which can improve the preferential pairing of exogenous TCR chains by forming additional disulfide bonds. For example, human TRAC can contain a Cys substitution at residue 48, and human TRBC can contain a Cys substitution at residue 57, as described in the following references: Cancer Res., April 15, 2007; 67(8):3898-903 and Blood, March 15, 2007; 109(6):2331-8; which are incorporated herein by reference.

[0377] The recombinant TCR or CAR can contain other modifications to the α and β chains.

[0378] In some embodiments, the α and β chains are modified by linking the extracellular domains of the α and β chains to a full-length human CD3ζ ((CD3-zeta)) molecule. Such modifications are described in the following references: J Immunol, June 1, 2008, 180(11):7736-7746; Gene Ther., August 2000; 7(16):1369-77; and The Open Gene Therapy Journal, 2011, 4:11-22, which are incorporated herein by reference.

[0379] In some embodiments, the α-chain is modified by introducing hydrophobic amino acid substitutions in the transmembrane region of the α-chain, as described in J Immunol Jun 1, 2012; 188(11):5538-5546; Gene Ther. Aug 2000; 7(16):1369-77; and The Open Gene Therapy Journal, 2011, 4:11-22, which are hereby incorporated by reference in their entirety.

[0380] The α-chain or the β-chain can be modified by altering any one of the N-glycosylation sites in the amino acid sequence, as described in J ExpMed. Feb 16, 2009; 206(2):463-475, which is hereby incorporated by reference in its entirety.

[0381] The α-chain and the β-chain can each comprise a dimerization domain, such as a heterodimerization domain. It is known in the art that such a heterodomain can be a leucine zipper (5H3 domain) or a hydrophobic proline-rich reverse domain or other similar forms. In one example, the α-chain and the β-chain can be modified by introducing a 30-mer segment into the carboxyl terminus of the α and β extracellular domains, wherein the segments selectively associate to form a stable leucine zipper. Such modification is described in: PNAS Nov 22, 1994, 1994.91(24)11408-11412; https: / / doi.org / 10.1073 / pnas.91.24.11408, which is incorporated by reference in its entirety.

[0382] The TCRs identified herein can be modified to contain mutations that result in increased affinity or half-life, such as the mutations described in WO2012 / 013913, which is incorporated by reference in its entirety.

[0383] The recombinant TCR or CAR can be a single-chain TCR (scTCR). Such an scTCR can comprise an α-chain variable region sequence fused to the N-terminus of the extracellular sequence of the α-chain constant region of the TCR, a TCR β-chain variable region fused to the N-terminus of the extracellular sequence of the TCR β-chain constant region, 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 extracellular sequences of the constant regions of the α-segment and the β-segment 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-segment and the β-segment are oriented relative to each other substantially as in the native αβ T cell receptor. Exemplary scTCRs are described in U.S. Patent No. 7,569,664, which is incorporated by reference in its entirety.

[0384] In some cases, the variable regions of the scTCR can be covalently linked via a short peptide linker as described in Gene Therapy, Volume 7, pages 1369 - 1377 (2000). The short peptide linker may be a serine - rich or glycine - rich linker. For example, the linker can be (Gly4Ser)3 as described in Cancer Gene Therapy (2004) 11, 487 - 496 (which is incorporated by reference in its entirety).

[0385] The 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 fusion proteins are described in Cancer Res. (()) March 15, 2002; 62(6):1757 - 60. The TCR or its antigen - binding fragment can be fused to the Fc region of an antibody. Such fusion proteins are described in J Immunol, May 1, 2017, 198((1 Suppl))120.9.

[0386] In some embodiments, the recombinant receptor such as a TCR or CAR (such as its antibody portion) also includes a spacer, which can be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region, e.g., an IgG4 hinge region and / or CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer between the antigen recognition component (e.g., scFv) and the transmembrane domain. The length of the spacer can provide increased cellular responsiveness after antigen binding as compared to the absence of a spacer. In some instances, the length of the spacer is or about 12 amino acids, or its length does not exceed 12 amino acids. 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, and those including any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids or about 229 or fewer amino acids. Exemplary spacers include the IgG4 hinge alone, the IgG4 hinge linked to the CH2 and CH3 domains, or the 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 portion is IgD.

[0387] The antigen recognition domain of a receptor (such as a TCR or CAR) can be linked to one or more intracellular signaling components, such as in the case of a CAR, by mimicking the activated signaling components of an antigen receptor complex (such as a TCR complex), and / or by the signal of 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 is naturally associated with one of the domains in a receptor (e.g., a CAR) is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0388] In some embodiments, the transmembrane domain is natural or synthetic. If of natural origin, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. The transmembrane region comprises transmembrane regions (i.e., at least comprising those of) derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and / or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, there are triplets of phenylalanine, tryptophan, and valine at each end of the synthetic transmembrane domain. In some embodiments, connection is made through a linker, spacer, and / or transmembrane domain.

[0389] The intracellular signaling domain contains those that mimic or approximate the signals through the natural antigen receptor, the signals through the combination of such a receptor with a co-stimulatory receptor, and / or the signals through only the co-stimulatory receptor. In some embodiments, there are short oligonucleotide or polypeptide linkers, such as linkers between 2 and 10 amino acids in length, such as linkers containing glycine and serine, such as glycine-serine doublets, and form a connection between the transmembrane domain and the cytoplasmic signaling domain of the receptor.

[0390] The receptor, such as a TCR or CAR, can comprise at least one or more intracellular signaling components. In some embodiments, the receptor comprises the intracellular components of the TCR complex, such as the TCR CD3 chains that mediate T cell activation and cytotoxicity, such as the CD3ζ chain. Thus, in some aspects, 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 comprises 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 portions of one or more additional molecules, such as the Fc receptor-γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR comprises a chimeric molecule between CD3-ζ or the Fc receptor-γ and CD8, CD4, CD25, or CD16.

[0391] In some embodiments, once the TCR or CAR is ligated, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of normal effector functions or immune cell responses (e.g., engineered T cells expressing the receptor). For example, in some cases, the receptor induces functions of T cells, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, for example, if the intracellular signaling domain of an antigen receptor component transduces an effector function signal, a truncated portion of the intracellular signaling domain of the antigen receptor component or a costimulatory molecule is substituted for the intact immune-stimulatory chain. In some embodiments, one or more intracellular signaling domains comprise the cytoplasmic sequences of a T cell receptor (TCR), and in some aspects also include those of a coreceptor that synergizes with such a receptor in its native context to initiate signal transduction upon antigen receptor ligation, and / or any derivatives or variants of such molecules, and / or any synthetic sequences having the same function.

[0392] In the case of the native TCR, full activation generally requires not only signaling through the TCR but also costimulatory signals. Thus, in some embodiments, to facilitate full activation, components for generating secondary or costimulatory signals are also included in the receptor. In other embodiments, the receptor does not include components for generating costimulatory signals. In some aspects, additional receptors are expressed in the same cell and provide components for generating secondary or costimulatory signals.

[0393] In some aspects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the receptor comprises one or both of such signaling components.

[0394] In some aspects, the receptor comprises primary cytoplasmic signaling sequences that regulate primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are referred to as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include sequences derived from the TCR or CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in a CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0395] In some embodiments, the receptor comprises a signaling domain and / or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same receptor comprises both an activating component and a co-stimulatory component.

[0396] In some embodiments, the activating domain is contained within one receptor, while the co-stimulatory component is provided by another receptor that recognizes a different antigen. In some embodiments, the receptor comprises an activating or stimulatory receptor and a co-stimulatory receptor that are both expressed on the same cell (see WO2014 / 055668). In some aspects, the receptor targeting HLA-peptide is a stimulatory receptor or an activating receptor. In other aspects, it is a co-stimulatory receptor. In some embodiments, the cell further comprises an inhibitory receptor (e.g., iCAR, see Fedorov et al., Sci. Transl. Med., 5(215) (December 2013)), such as a receptor that recognizes an antigen other than the HLA-peptide, thereby reducing or inhibiting the activating signal transmitted through the receptor targeting the HLA-peptide by binding of the inhibitory receptor to its ligand, e.g., to reduce off-target effects.

[0397] In certain embodiments, the intracellular signaling domain comprises the CD28 transmembrane domain and signaling domain linked to the intracellular domain of CD3 (e.g., CD3-ζ). In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain linked to the CD3ζ intracellular domain.

[0398] In some embodiments, the receptor comprises one or more, e.g., two or more, co-stimulatory domains and an activating domain in the cytoplasmic portion, e.g., a major activating domain. Exemplary receptors comprise the intracellular components of CD3-ζ, CD28, and 4-1BB.

[0399] In some embodiments, the CAR or other antigen receptor, such as a TCR, further comprises a marker, such as a cell surface marker, which can be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated form of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker comprises all or part (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 linker sequence, such as a cleavable linker sequence or a ribosome skipping sequence, such as T2A. See WO2014031687. In some embodiments, introduction of a construct encoding a CAR and EGFRt separated by a T2A ribosome switch can express two proteins from the same construct such that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker and optional linker sequence can be any sequence disclosed in patent application publication No. WO2014031687. For example, the marker can be truncated EGFR ((tEGFR)), optionally linked to a linker sequence, such as a T2A ribosome skipping sequence.

[0400] In some embodiments, the marker is a molecule, such as a cell surface protein, that is not naturally present on T cells or on T cells or portions thereof in nature.

[0401] In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the immune system of the host into which the cell is adoptively transferred.

[0402] In some embodiments, in addition to serving as a marker for genetic engineering (e.g., for selecting successfully engineered cells), the marker has no therapeutic function and / or does not have an effect. In other embodiments, the marker can be a therapeutic molecule or otherwise play some desired role, such as a ligand for cells encountered in vivo, such as a co-stimulatory or immune checkpoint molecule, thereby enhancing and / or attenuating the response of the cell upon adoptive transfer and encounter with the ligand.

[0403] The TCR or CAR can include 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, α-aminodecanoic acid, homoserine, S-acetamidomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, ((3-phenylserine ((3-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyllysine, N,N′-dibenzyllysine, 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-tert-butylglycine.

[0404] In some cases, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some aspects, a first-generation CAR is a CAR that provides only CD3-chain-induced signals upon antigen binding; in some aspects, a second-generation CAR is a CAR that provides both signaling and co-stimulatory signals, such as a CAR that includes an intracellular signaling domain from a co-stimulatory receptor (such as CD28 or CD137); in some aspects, a third-generation CAR is a CAR that includes multiple co-stimulatory domains from different co-stimulatory receptors.

[0405] In some embodiments, the chimeric antigen receptor includes an extracellular portion that contains an antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion and an intracellular signaling domain, wherein the extracellular portion contains an antibody or fragment described herein. In some embodiments, the antibody or fragment includes an scFv or a single-domain VH antibody, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes the signaling domain of the ζ chain of CD3, i.e., the ζ(CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain that links the extracellular domain and the intracellular signaling domain.

[0406] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, such as any spacer described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell co-stimulatory molecule, such as an intracellular domain between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell co-stimulatory molecule is CD28 or 41BB.

[0407] In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some of the said embodiments, the receptor further comprises a spacer that contains 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.

[0408] In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (Accession No.: P10747.1).

[0409] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0410] In some embodiments, the intracellular signaling domain includes the intracellular co-stimulatory signaling domain of human CD28 or a functional variant or a portion thereof, such as its 41-amino acid domain and / or the domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular domain includes the intracellular co-stimulatory signaling domain of 4-1BB or a functional variant or a portion thereof, such as the 42-amino acid cytoplasmic domain of human 4-1BB (Accession No.: Q07011.1) or a functional variant or a portion thereof.

[0411] 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 isotype 3 of human CD3ζ (Accession No.: P20963.2), or the CD3ζ signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993.

[0412] In some aspects, the spacer contains only the hinge region of IgG, such as the hinge of only IgG4 or IgG1. In other embodiments, the spacer is an Ig hinge linked to the CH2 and / or CH3 domain, such as the IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked to the CH2 and CH3 domains, such as the IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked only to the CH3 domain, such as the IgG4 hinge. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.

[0413] For example, in some embodiments, the CAR comprises an antibody or a fragment thereof, such as any HLA-peptide antibody, comprising a single-chain antibody (sdAb((, e.g., comprising only the VH region)) and the scFv described herein; a spacer, such as any Ig-hinge containing a spacer; a CD28 transmembrane domain; a CD28 intracellular signaling domain; and a CD3ζ signaling domain. In some embodiments, the CAR comprises an antibody or fragment, such as any HLA-peptide antibody, comprising a sdAb and the scFv described herein; a spacer, such as any Ig-hinge containing a spacer; a CD28 transmembrane domain; a CD28 intracellular signaling domain; and a CD3ζ signaling domain.

[0414] Target-specific TCR against A*01:01_ASSLPTTMNY (SEQ ID NO:)[G10]

[0415] In some aspects, provided herein is an ABP comprising a TCR or an antigen-binding fragment thereof that specifically binds an HLA-peptide target, wherein the HLA class I molecule of the HLA-peptide target is the HLA subtype A*01:01, and the HLA-restricted peptide of the HLA-peptide target comprises the sequence ASSLPTTMNY (“G10”).

[0416] The TCR specific for A*01:01_ASSLPTTMNY may comprise an αCDR3 sequence. The αCDR3 sequence can be any of the αCDR3 sequences in Table 15. The α and βCDR3 sequences of the identified TCR clonotypes are shown in Table 15.

[0417] The TCR specific for A*01:01_ASSLPTTMNY may comprise a βCDR3 sequence. The βCDR3 sequence can be any of the βCDR3 sequences in Table 15.

[0418] TCRs specific for A*01:01_ASSLPTTMNY can comprise a specific αCDR3 sequence and a specific βCDR3 sequence. For example, TCRs specific for A*01:01_ASSLPTTMNY can comprise the αCDR3 sequence and the βCDR3 sequence from any one of the TCRs identified in Table 15. For clarity, each identified TCR is assigned a TCR ID number. For example, TCR ID#1 comprises the αCDR3 sequence CAGPGNTGKLIF and the βCDR3 sequence CASSNAGDQPQHF.

[0419] TCRs specific for A*01:01_ASSLPTTMNY can comprise TRAV, TRAJ, TRBV, optionally TRBD and TRBJ amino acid sequences, optionally a TRAC sequence and optionally a TRBC sequence. For example, TCRs specific for A*01:01_ASSLPTTMNY can 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 14. For clarity, each identified TCR is assigned a TCR ID number. For example, TCR ID#1 assigned to a TCR comprises the TRAV25 sequence, the TRAJ37 sequence, the TRAC sequence, the TRBV19 sequence, the TRBD1 sequence, the TRBJ1-5 sequence and the TRBC1 sequence.

[0420] TCRs specific for A*01:01_ASSLPTTMNY can comprise an αVJ sequence. The αVJ sequence can be any one of the αVJ sequences in Table 16.

[0421] TCRs specific for A*01:01_ASSLPTTMNY can comprise a βV(D)J sequence. The βV(D)J sequence can be any one of the βV(D)J sequences in Table 16.

[0422] TCRs 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. Table 16 shows the full-length αV(J) and βV(D)J sequences of the identified TCR clonotypes. For example, TCR ID#1 comprises the αV(J) sequence MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGPGNTGKLIFGQGTTLQVK and the βV(D)J sequence MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSNAGDQPQHFGDGTRLSIL.

[0423] Target-specific TCR against A*01:01_HSEVGLPVY

[0424] In some aspects, provided herein are ABP comprising a TCR or an antigen-binding fragment thereof that specifically binds an HLA-peptide target, wherein the HLA class I molecule of the HLA-peptide target is the HLA subtype A*01:01 and the HLA-restricted peptide of the HLA-peptide target comprises the sequence HSEVGLPVY.

[0425] A TCR specific for A*01:01_HSEVGLPVY may comprise an αCDR3 sequence. The αCDR3 sequence may be any one of the αCDR3 sequences in Table 18. The α and βCDR3 sequences of the identified TCR clonotypes are shown in Table 18.

[0426] A TCR specific for A*01:01_HSEVGLPVY may comprise a βCDR3 sequence. The βCDR3 sequence may be any one of the βCDR3 sequences in Table 18.

[0427] A TCR specific for A*01:01_HSEVGLPVY can comprise a specific αCDR3 sequence and a specific βCDR3 sequence. For example, a TCR specific for A*01:01_HSEVGLPVY can comprise the αCDR3 sequence and the βCDR3 sequence from any one of the TCRs identified in Table 18. For clarity, each identified TCR is assigned a TCR ID number. For example, TCR ID#345 comprises the αCDR3 sequence CAANPGDYKLSF and the βCDR3 sequence CASSSNYEQYF.

[0428] A TCR specific for A*01:01_HSEVGLPVY can comprise TRAV, TRAJ, TRBV, optional TRBD, and TRBJ amino acid sequences, an optional TRAC sequence, and an optional TRBC sequence. For example, a TCR specific for A*01:01_HSEVGLPVY can 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 is assigned a TCR ID number. For example, TCR ID#345 assigned to a TCR comprises the TRAV13-1 sequence, the TRAJ20 sequence, the TRAC sequence, the TRBV7-9 sequence, the TRBJ2-7 sequence, and the TRBC2 sequence.

[0429] A TCR specific for A*01:01_HSEVGLPVY can comprise an αVJ sequence. The αVJ sequence can be any one of the αVJ sequences in Table 19.

[0430] A TCR for A*01:01_HSEVGLPVY can comprise a βV(D)J sequence. The βV(D)J sequence can be any one of the βV(D)J sequences in Table 19.

[0431] TCRs specific for A*01:01_HSEVGLPVY can comprise an αVJ sequence and a βV(D)J sequence. For example, TCRs specific for A*01:01_HSEVGLPVY can 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 βV(D)J sequences of the identified TCR clonotypes are shown in Table 19. For example, TCR ID#345 comprises the αV(J) sequence MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAANPGDYKLSFGAGTTVTVR and the βV(D)J sequence MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSSNYEQYFGPGTRLTVT.

[0432] Engineered cell

[0433] Cells are also provided, such as cells containing an antigen receptor, e.g., an antigen receptor (e.g., a CAR or TCR) comprising an extracellular domain of an anti-HLA-peptide ABP as described herein. Populations of such cells and compositions containing such cells are also provided. In some embodiments, the composition or population is enriched for such cells, such as cells expressing HLA-peptide ABP that comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or greater than 99 percent of all cells in the composition, or a certain type of cell (such as a T cell or a CD8+ or CD4+ cell). In some embodiments, the composition comprises at least one cell containing an antigen receptor as disclosed herein. The composition includes pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). Therapeutic methods of administering the cells and compositions to a subject, e.g., a patient, are also provided.

[0434] Accordingly, genetically engineered cells expressing ABP comprising a receptor (e.g., TCR or CAR) are also provided. The cells are generally eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and the cells are cells of the immune system, such as cells of innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, generally 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). The cells are generally primary cells, such as cells isolated directly from a subject and / or cells isolated from a subject and cryopreserved. In some embodiments, the cells comprise one or more subsets of T cells or other cell types, such as total T cell populations, CD4+ cells, CD8+ cells, and their subsets, such as those defined by function, activation status, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. These methods include off-the-shelf methods. In some aspects, as in the prior art, the cells are multipotent and / or pluripotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject as described herein, preparing, processing, culturing, and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0435] Subtypes and subsets of T cells and / or CD4+ and / or CD8+ T cells include: naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MALT) cells, naturally occurring and adoptive 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, and δ / γ T cells.

[0436] 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.

[0437] Cells can be genetically modified to reduce expression or knockout endogenous TCR. Such modifications are described in the following references: Mol Ther Nucleic Acids. December 2012; 1(12): e63; Blood. August 11, 2011; 118(6): 1495-503; Blood. June 14, 2012; 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. January 18, 2018; 131(3): 311-322. doi: 10.1182 / blood-2017-05-787598; and WO2016069283, which are incorporated herein by reference in their entirety.

[0438] Cells can be genetically modified to promote cytokine secretion. Such modifications are described 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; Quintar elli 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.

[0439] It has been shown that the mismatch between chemokine receptors on T cells and chemokines secreted by tumors is responsible for the suboptimal trafficking of T cells into the tumor microenvironment. To improve the therapeutic effect, cells can be genetically modified to increase the recognition of chemokines 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;33:780-788.

[0440] Cells can be genetically modified to enhance the expression of co-stimulatory / enhancing receptors such as CD28 and 41BB.

[0441] Adverse reactions to T cell therapy can include cytokine release syndrome and persistent B cell depletion. Introducing a suicide / safety switch into the recipient cells can improve the safety of cell therapy. Thus, cells can be genetically modified to include a suicide / safety switch. A suicide / safety switch can be a gene that confers sensitivity to a reagent (e.g., a drug) on the cells expressing the gene and causes cell death when the cells come into contact with or are exposed to the reagent. Exemplary suicide / safety switches are described in Protein Cell. August 2017;8(8):573-589. A suicide / safety switch can be HSV-TK. A suicide / safety switch can be cytosine deaminase, purine nucleoside phosphorylase, or nitroreductase. A suicide / safety switch can be RapaCIDe TM , described in U.S. Patent Application Publication No. US20170166877A1. A suicide / safety switch system can be CD20 / rituximab, described in Haematologica. September 2009;94(9):1316-1320. These references are incorporated by reference in their entirety.

[0442] A TCR or CAR can be introduced into recipient cells as split receptors that assemble only in the presence of a heterodimerizing small molecule. Such systems are described in Science. October 16, 2015; 350(6258): aab4077 and U.S. Patent No. 9,587,020 (the literature and patent are hereby incorporated by reference in their entirety).

[0443] In some embodiments, the cell comprises one or more nucleic acids, e.g., a polynucleotide encoding a TCR or CAR disclosed herein, wherein the polynucleotide is introduced by genetic engineering to express a recombinant TCR or CAR or a genetically engineered TCR or CAR disclosed herein. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as a sample obtained from another organism or cell, e.g., not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as not found in nature, and comprises a chimeric combination of nucleic acids containing nucleic acids encoding various domains from multiple different cell types.

[0444] The nucleic acid may comprise a codon-optimized nucleotide sequence. Without being bound by a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing one codon encoding the same amino acid with a native codon, but it can be translated by a tRNA that is more readily available within the cell, thereby increasing the translation efficiency. Optimization of the nucleotide sequence can also reduce the mRNA secondary structure that would interfere with translation, thereby increasing the translation efficiency.

[0445] A TCR or CAR can be introduced into recipient cells using a construct or vector. Exemplary constructs are described herein. The polynucleotides encoding the α and β chains of the TCR or CAR can be present in a single construct or in separate constructs. The polynucleotides encoding the α and β chains can be operably linked to a promoter, e.g., a heterologous promoter. The heterologous promoter can be a strong promoter, such as EF1α, CMV, PGK1, Ubc, β-actin, CAG promoter, etc. The heterologous promoter can be a weak promoter. The heterologous promoter can be an inducible promoter. Exemplary inducible promoters include, but are not limited to, TRE, NFAT, GAL4, LAC, etc. 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 entirety.

[0446] Constructs for introducing TCR or CAR into recipient cells may also comprise a polynucleotide encoding a signal peptide (signal peptide element). The signal peptide may facilitate surface transport of the introduced TCR or CAR. Exemplary signal peptides include but are not limited to CD8 signal peptide, immunoglobulin signal peptide, specific examples of which include GM-CSF and IgGκ. Such signal peptides are described in Trends Biochem Sci. October 2006; 31(10): 563-71. Epub August 21, 2006; 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. August 16, 2018; they are hereby incorporated by reference.

[0447] In some cases, such as when expressing the α and β chains from a single construct or open reading frame, or when the construct contains a marker gene, the construct may comprise a ribosome skipping sequence. The ribosome skipping sequence may be a 2A peptide, such as P2A or T2A peptide. Exemplary P2A and T2A peptides are described in Scientific Reports Volume 7, Article number: 2193 (2017) (())(()), which is hereby incorporated by reference in its entirety. In some cases, a FURIN / PACE cleavage site is introduced upstream of the 2A element. For example, the FURIN / PACE cleavage site is described in http: / / www.nuolan.net / substrates.html. The cleavage peptide may also be a cleavage site for factor Xa. When expressing the α and β chains from a single construct or open reading frame, the construct may comprise an internal ribosome entry site ((IRES)).

[0448] The construct may also comprise one or more marker genes. Exemplary marker genes include but are not limited to GFP, luciferase, HA, lacZ. As known to those skilled in the art, the marker may be a selection marker, such as an antibiotic resistance marker, a heavy metal resistance marker or an anti-biocide marker. The marker may be a complementary marker for auxotrophic hosts. Exemplary complementary markers and auxotrophic hosts are described in Gene. January 24, 2001; 263(1-2): 159-69. Such markers may be expressed via an IRES, a frameshift sequence, a 2A peptide linker, a fusion with the TCR or CAR, or separately with a separate promoter.

[0449] Exemplary vectors or systems for introducing TCR or CAR into recipient cells include, but are not limited to: adeno-associated virus, adenovirus, adenovirus + modified vaccinia Ankara virus (MVA), adenovirus + retrovirus, adenovirus + Sendai virus, adenovirus + vaccinia virus, Venezuelan equine encephalitis virus (VEE) replicon vaccine, antisense oligonucleotide, Bifidobacterium longum, CRISPR-Cas9, Escherichia coli, flavivirus, gene gun, herpes virus, herpes simplex virus, Lactococcus lactis, electroporation, lentivirus, liposome transfection, Listeria monocytogenes, measles virus, modified 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 vector, PiggyBac TM (PB) transposon, nanoparticle-based system, poliovirus, poxvirus, poxvirus + vaccinia virus, retrovirus, RNA transfer, RNA transfer + naked / plasmid DNA, RNA virus, Saccharomyces 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 cholerae.

[0450] In a preferred embodiment, TCR or CAR is introduced into recipient cells by adeno-associated virus (AAV), adenovirus, CRISPR-CAS9, herpes virus, lentivirus, liposome transfection, mRNA electroporation, PiggyBac TM (PB) transposon, retrovirus, RNA transfer, or sleeping beauty transposon.

[0451] In some embodiments, the vector for introducing TCR or CAR into recipient cells is a viral vector. Exemplary viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, herpes virus vectors, retrovirus vectors, etc. Such vectors are described herein.

[0452] Exemplary embodiments of TCR constructs for introducing TCR or CAR into recipient cells are as Figure 2As shown. In some embodiments, the TCR construct comprises the following polynucleotide sequences in the 5′-3′ direction: a promoter sequence, a signal peptide sequence, a TCRβ variable (TCRβv) sequence, a TCRβ constant (TCRβc) sequence, a cleavage peptide (e.g., P2A), a signal peptide sequence, a TCRα variable (TCRαv) sequence, and a TCRα constant (TCRαc) sequence. In some embodiments, the TCRβc and TCRαc sequences of the construct comprise one or more murine regions, e.g., the full murine constant sequences or human→mouse amino acid exchanges described herein. In some embodiments, the construct further comprises, 3′ of the TCRαc sequence, a cleavage peptide sequence (e.g., T2A) and a reporter gene. In one embodiment, the construct comprises the following polynucleotide sequences in the 5′-3′ direction: a promoter sequence, a signal peptide sequence, a TCRβ variable ((TCRβv)) sequence, a TCRβ constant (TCRβc) sequence containing one or more murine regions, a cleavage peptide (e.g., P2A), a signal peptide sequence, a TCRα variable (TCRαv) sequence, and a TCRα constant (TCRαc) sequence, the TCRα constant (TCRαc) sequence containing one or more murine regions, a cleavage peptide (e.g., T2A), and a reporter gene.

[0453] Figure 3 Depicts an exemplary construct backbone sequence for cloning a TCR into an expression system for therapeutic development.

[0454] Figure 4 Depicts an exemplary construct sequence for cloning the identified A*0201-LLASSILCA-specific TCR into an expression system for therapeutic development.

[0455] Figure 5 Depicts an exemplary construct sequence for cloning the identified A*0101_EVDPIGHLY-specific TCR into an expression system for therapeutic development.

[0456] Nucleotides, vectors, host cells, and related methods

[0457] Also provided are an isolated nucleic acid encoding HLA-peptide ABP, a vector comprising the nucleic acid, a host cell comprising the vector and the nucleic acid, and a recombinant technique for producing the ABP.

[0458] The nucleic acid can be a recombinant nucleic acid. Recombinant nucleic acids can be constructed outside of living cells by ligating natural or synthetic nucleic acid fragments to a nucleic acid molecule or its replication product that can replicate in a living cell. For the purposes herein, replication can be in vitro replication or in vivo replication.

[0459] For recombinant production of ABP, the nucleic acid encoding ABP can be isolated and inserted into a replicable vector for further cloning (i.e., DNA amplification) or expression. In some aspects, the nucleic acid can be generated by homologous recombination, for example, as described in U.S. Patent No. 5,204,244, which is incorporated herein by reference in its entirety.

[0460] Many different vectors are known in the art. Vector components generally include one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, for example, as described in U.S. Patent No. 5,534,615, which is incorporated herein by reference in its entirety.

[0461] Exemplary vectors or constructs suitable for expressing ABP (e.g., TCR, CAR, antibody, or antigen-binding fragment thereof) include, for example, the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, La Jolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Phage vectors, such as AGT10, AGT11, AZapII (Stratagene), AEMBL4, and ANM1149, are also suitable for expressing the ABP described herein.

[0462] Illustrative examples of suitable host cells are provided below. These host cells are not limiting, and any suitable host cell can be used to produce the ABP provided herein.

[0463] Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescens), Shigella, Bacilli (B. subtilis and B.licheniformis), Pseudomonas (P. aeruginosa), and Streptomyces. A useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are also suitable.

[0464] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding HLA-peptide ABP. Saccharomyces cerevisiae or common baker's yeast is a commonly used lower eukaryotic host microorganism. However, there are many other available and useful genera, species, and strains, such as Schizosaccharomyces; Kluyveromyces (Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces bulgaricus, Kluyveromyces wickerhamii, Kluyveromyces waltii, Kluyveromyces drosophilarum, Kluyveromyces thermotolerans, and Kluyveromyces marxianus); Yarrowia; Pichia pastoris; Candida (Candida albicans); Trichoderma reesei; Neurospora crassa; Schwanniomyces (Schwanniomyces occidentalis); and filamentous fungi such as, for example, Penicillium, Torulopsis, and Aspergillus (Aspergillus nidulans and Aspergillus niger).

[0465] 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), etc.

[0466] Host cells for producing HLA-peptide ABP can be cultured in a variety of media. Commercially available media such as, for example, Ham F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Minimal Essential Medium (DMEM) are suitable for culturing host cells. Additionally, any medium 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 can be used, and each of the above references is incorporated by reference in its entirety.

[0467] Any of these media can 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, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other necessary supplements can also be included at appropriate concentrations known to those skilled in the art.

[0468] Culture conditions such as temperature, pH, etc., are those previously used with the host cells for expression and will be apparent to those of ordinary skill in the art.

[0469] When recombinant techniques are used, ABP can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If ABP is produced intracellularly, the first step is to remove particulate debris of host cells or cell lysate fragments by, for example, centrifugation or ultrafiltration. For example, Carter et al. (Bio / Technology, 1992, 10:163-167, incorporated by reference in its entirety) described a method for isolating ABP secreted into the periplasmic space of Escherichia coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation.

[0470] In some embodiments, the ABP is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system, as described in Yin et al. mAbs, 2012, 4:217 - 225, which is incorporated by reference in its entirety. In some aspects, the cell-free system utilizes cell-free extracts from eukaryotic or prokaryotic cells. In some aspects, the prokaryotic cell is Escherichia coli. Cell-free expression of ABP may be useful, for example, when the ABP accumulates in cells as insoluble aggregates or when the yield obtained from periplasmic expression is low.

[0471] In the case where the ABP is secreted into the culture medium, the supernatant from such expression systems is generally concentrated first using a commercially available protein concentration filter (e.g., or an ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0472] ABP compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, where affinity chromatography is a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the ABP. Protein A can be used to purify ABP containing human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth., 1983, 62:1 - 13, which is incorporated by reference in its entirety). Protein G can be used for all mouse isotypes and human γ3 (Guss et al., EMBO J., 1986, 5:1567 - 1575, which is incorporated by reference in its entirety).

[0473] The matrix to which the affinity ligand is attached is typically agarose, but other matrices can also be used. Mechanically stable matrices (such as controlled pore glass or poly(styrene - divinyl)benzene) have faster flow rates and shorter processing times than agarose. If the ABP contains a CH3 domain, resin can be used for purification.

[0474] Those skilled in the art can also use other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reverse - phase high - performance liquid chromatography (HPLC), silica gel chromatography, heparin chromatography, focused chromatography, SDS - PAGE, and ammonium sulfate precipitation, etc.

[0475] After any initial purification step, a mixture containing the target ABP and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and about 4.5, generally at a low salt concentration (e.g., about 0 to about 0.25 M salt).

[0476] Methods for preparing HLA-peptide ABP

[0477] Preparing HLA-peptide antigens

[0478] The HLA-peptide antigen for separating or generating the ABP described herein can be a full-length HLA-peptide or a fragment of an HLA-peptide. The HLA-peptide antigen can be, for example, in the form of an isolated protein or a protein expressed on the cell surface.

[0479] In some embodiments, the HLA-peptide antigen is a non-naturally occurring variant of an HLA-peptide, such as an HLA-peptide protein having an amino acid sequence or a post-translational modification not found in nature.

[0480] In some embodiments, the HLA-peptide antigen is truncated by removing, for example, intracellular sequences or transmembrane sequences or signal sequences. In some embodiments, the HLA-peptide antigen is fused at its C-terminus to a human IgG1 Fc domain or a polyhistidine tag.

[0481] Methods for identifying ABP

[0482] Any method known in the art, such as phage display or immunization of a subject, can be used to identify an ABP that binds to an HLA-peptide.

[0483] One method of identifying an antigen-binding protein includes: providing at least one HLA-peptide target; binding the at least one target to an antigen-binding protein, thereby identifying the antigen-binding protein. The antigen-binding protein can be present in a library containing a plurality of different antigen-binding proteins.

[0484] In some embodiments, the library is a phage display library. A phage display library can be developed such that it is substantially free of antigen-binding proteins of HLA that non-specifically bind to the HLA-peptide target. The antigen-binding protein can be present in a yeast display library containing a plurality of different antigen-binding proteins. A yeast display library can be developed such that it is substantially free of antigen-binding proteins that non-specifically bind to the HLA of the HLA-peptide target.

[0485] In some embodiments, the library is a yeast display library.

[0486] In some embodiments, the library is a TCR display library. Exemplary TCR display libraries and methods of their use are described in: WO 98 / 39482; WO 01 / 62908; WO 2004 / 044004; WO2005116646, WO2014018863, WO2015136072, and WO2017046198; and Helmut et al. ((2000)) PNAS 97(26)14578-14583, all of which are incorporated by reference in their entirety.

[0487] In some aspects, the binding step is performed more than once, optionally at least three times, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0488] Additionally, the method may further comprise: 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.

[0489] Another method of identifying an antigen-binding protein may comprise: 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 the antigen-binding protein from the subject. Isolating the antigen-binding protein may comprise: screening the serum of the subject to identify the antigen-binding protein. The method may further comprise: contacting the antigen-binding protein with one or more peptide-HLA complexes different from the HLA-peptide target, e.g., to determine whether the antigen-binding protein selectively binds to the HLA-peptide target. The identified antigen-binding protein may be humanized.

[0490] In some aspects, isolating the antigen-binding protein includes: isolating B cells from a subject expressing the antigen-binding protein. The B cells can be used to generate hybridomas. The B cells can also be used to clone one or more CDRs of the B cells. For example, immortalizing the B cells by EBV transformation. The sequence encoding the antigen-binding protein can be cloned from the immortalized B cells or can be directly cloned from B cells isolated from the immunized subject. A library containing the antigen-binding protein of the B cells can also be created, optionally, wherein the library is a phage display library or a yeast display library.

[0491] Another method of identifying an antigen-binding protein may comprise: obtaining a cell containing the antigen-binding protein; contacting the cell with an HLA multimer (e.g., a tetramer) containing at least one HLA-peptide target; and identifying the antigen-binding protein by the binding between the HLA multimer and the antigen-binding protein.

[0492] The cells can be, for example, T cells, optionally cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells. The method can further comprise: optionally using flow cytometry, magnetic separation or single cell isolation to isolate the cells. The method can further comprise sequencing the antigen-binding protein.

[0493] Another method of identifying an antigen-binding protein can comprise: obtaining one or more cells comprising the antigen-binding protein; activating the one or more cells with at least one HLA-peptide target presented on at least one antigen-presenting cell (APC); and identifying the antigen-binding protein by selecting the one or more cells that are activated by interacting with the at least one HLA-peptide target.

[0494] The cells can be, for example, T cells, optionally CTLs or NK cells. The method can further comprise: optionally using flow cytometry, magnetic separation or single cell isolation to isolate the cells. The method can further comprise sequencing the antigen-binding protein.

[0495] Methods for preparing monoclonal ABP

[0496] Monoclonal ABP can be obtained, for example, by the hybridoma method first described by Kohler et al., Nature, 1975, 256:495-497 (incorporated herein by reference in its entirety), and / or recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567, incorporated herein by reference in its entirety). Monoclonal ABP can also be obtained, for example, using phage or yeast libraries. See, e.g., U.S. Patent Nos. 8,258,082 and 8,691,730, both of which are incorporated herein by reference in their entirety.

[0497] 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 the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Then, the lymphocytes are fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. See Goding J.W., Monoclonal ABPs: Principles and Practice 3rd ed. ((1986) Academic Press, San Diego, CA), which is incorporated herein by reference in its entirety.

[0498] Hybridoma cells are inoculated into a suitable medium for growth, and the medium contains one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma usually contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0499] Useful myeloma cells are those that can fuse efficiently, support the stable and high-level production of ABP by the selected ABP-producing cells, and are sensitive to culture medium conditions (such as the presence or absence of HAT medium). Among them, preferred myeloma cell lines are murine myeloma cell lines, such as MOPC-21 and MC-11 murine tumor-derived ones (available from the Cell Distribution Center of the Salk Institute for Biological Studies in San Diego, California), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection in Rockville, Maryland). Human myelomas and human-mouse heteromyeloma cell lines have been described for the production of human monoclonal ABP. See, for example, Kozbor, J. Immunol., 1984, 133: 3001, which is incorporated herein by reference in its entirety.

[0500] After determining that the ABP produced by the hybridoma cells has the desired specificity, affinity, and / or biological activity, the selected clones can be subcloned by the limiting dilution method and grown by standard methods. See Goding, supra. Media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells can be grown as ascites tumors in animals.

[0501] The DNA encoding the monoclonal ABP can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal ABP). Thus, hybridoma cells can be used as a useful source of DNA encoding an ABP with desired properties. After isolation, the DNA can be placed in an expression vector and then transfected into host cells such as bacteria (e.g., Escherichia coli), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), COS cells, Chinese hamster ovary (CHO) cells, or other myeloma cells that do not produce ABP, thereby producing the monoclonal ABP.

[0502] Methods for preparing chimeric ABP

[0503] Exemplary methods for preparing chimeric ABP 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; which are hereby incorporated by reference in their entirety. In some embodiments, chimeric ABP is prepared by combining non-human variable regions (e.g., variable regions from mouse, rat, hamster, rabbit, or non-human primate (such as monkey) sources) and human constant regions by recombinant techniques.

[0504] Methods for preparing humanized ABP

[0505] Humanized ABP is generated by replacing most or all of the structural portions of a non-human monoclonal ABP with the corresponding human ABP sequence. As a result, a hybrid molecule is produced in which only the antigen-specific variable region or CDRs are composed of non-human sequences. Methods for obtaining humanized ABP include those described in the following documents: for example, Winter and Milstein, Nature, 1991, 349: 293-299; Rade et al., Proc. Nat. Acad. Sci. U.S.A., 1998, 95: 8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275: 36073-36078; Queen et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86: 10029-10033; and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; which are hereby incorporated by reference in their entirety.

[0506] Methods for preparing human ABP

[0507] Human ABP can be generated by a variety of techniques known in the art, for example, using genetically engineered animals (e.g., humanized mice). See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807; each of which is incorporated by reference in its entirety. Human ABP can also be derived 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 ABP 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 ABP can also be derived from yeast libraries (see, e.g., U.S. Patent No. 8,691,730, which is incorporated by reference in its entirety).

[0508] Methods for preparing ABP fragments

[0509] The ABP fragments provided herein can be prepared by any suitable method, including the illustrative methods described herein or methods known in the art. Suitable methods include recombinant techniques and proteolytic digestion of the whole ABP. Illustrative methods for preparing ABP fragments are described in, e.g., Hudson et al., Nat. Med., 2003, 9:129-134, which is incorporated by reference in its entirety. Methods for preparing scFvABP are described in, e.g., Plückthun's The Pharmacology of Monoclonal ABPs, Vol. 113, Rosenberg 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.

[0510] Methods for preparing alternative scaffolds

[0511] The alternative scaffolds provided herein can be prepared by any suitable method, including the illustrative methods described herein or methods known in the art. For example, Adnectins are described in Emanuel et al., mAbs, 2011, 3:38-48 (which is incorporated by reference in its entirety). TM The method of preparation. The method of preparation of iMab is described in U.S. Patent Publication No. 2003 / 0215914, which is incorporated by reference in its entirety. Vogt and Skerra, Chem. Biochem., 2004, 5:191-199 (which is incorporated by reference in its entirety) describe The method of preparation. The method of preparation of Kunitz domains is described in Wagner et al., Biochem. & Biophys. Res. Comm., 1992, 186:118-1145 (which is incorporated by reference in its entirety). The method of preparation of thioredoxin peptide aptamers is described in Geyer and Brent, Meth. Enzymol., 2000, 328:171-208 (which is incorporated by reference in its entirety). Fernandez, Curr. Opinion in Biotech., 2004, 15:364-373 (which is incorporated by reference in its entirety) provides a method for preparing affibodies. Zahnd et al., J. Mol. Biol., 2007, 369:1015-1028 (which is incorporated by reference in its entirety)) provides a method for preparing DARPins. (Ebersbach et al.), J. Mol. Biol., 2007, 372:172-185 (which is incorporated by reference in its entirety) provides a method for preparing Affilins. The method of preparation of Tetranectins is provided in Graversen et al., J. Biol. Chem., 2000, 275:37390-37396 (which is incorporated by reference in its entirety). The method of preparation of Avimers is provided in Silverman et al., Nature Biotech., 2005, 23:1556-1561 (which is incorporated by reference in its entirety). The method of preparation of Fynomers is provided in Silacci et al., J. Biol. Chem., 2014, 289:14392-14398 (which is incorporated by reference in its entirety). For more information on alternative scaffolds, see Binz et al., Nat. Biotechnol., 2005 23:1257-1268)); and Skerra, Current Opin. in Biotech., 2007 18:295-304, each of which is incorporated by reference in its entirety.

[0512] Methods for preparing multispecific ABP

[0513] The multispecific ABP provided herein can be prepared by any suitable method, including the exemplary methods described herein or methods known in the art. Methods for preparing common light chain ABP are described in Merchant et al., Nature Biotechnol., 1998, 16: 677-681 (which is incorporated by reference in its entirety). Methods for preparing tetravalent bispecific ABP are described in Coloma and Morrison, Nature Biotechnol., 1997, 15: 159-163 (which is incorporated by reference in its entirety). Methods for preparing hybrid immunoglobulins are 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). Methods for preparing immunoglobulins with knob-into-hole modifications are described in U.S. Patent No. 5,731,168 (which is incorporated by reference in its entirety). Methods for preparing immunoglobulins with electrostatic modifications are provided in WO 2009 / 089004 (which is incorporated by reference in its entirety). Methods for preparing bispecific single-chain ABP are 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). Methods for preparing single-chain ABP are described in U.S. Patent Nos. 4,946,778 and 5,132,405 (each of which is incorporated by reference in its entirety), wherein the linker length of the ABP can be varied. Methods for preparing 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 preparing triabodies and tetra-bodies are described in Todorovska et al. J. Immunol. Methods, 2001, 248: 47-66 (which is incorporated by reference in its entirety). Methods for preparing 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 preparing cross-linked ABP 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).The method for preparing antigen-binding domains assembled by leucine zippers was described in Kostelny et al., J. Immunol., 1992, 148: 1547-1553 (which is incorporated herein by reference in its entirety). The method for preparing ABP by the DNL method was described in U.S. Patent Nos. 7,521,056; 7,550,143; 7,534,866 and 7,527,787 (each of which is incorporated herein by reference in its entirety). The method for preparing hybrids of ABP and non-ABP molecules, such as the method for preparing such ABP, was described in WO 93 / 08829 (which is incorporated herein by reference). The method for preparing DAF ABP was described in U.S. Patent Publication No. 2008 / 0069820 (which is incorporated herein by reference). The method for preparing ABP by reduction and oxidation was described in Carlring et al., PLoS One, 2011, 6: e22533 (which is incorporated herein by reference)). The method for preparing DVD-IgsTM was described in U.S. Patent No. 7,612,181 (which is incorporated herein by reference). The method for preparing DARTsTM was described in Moore et al., Blood, 2011, 117: 454-451 (which is incorporated herein by reference). The method for preparing was 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 herein by reference. Coloma and Morrison, Nature Biotechnol., 1997, 15: 159-163 (which is incorporated herein by reference) described the method for preparing ABP, wherein the ABP comprises C of IgG H3scFv fused to the C-terminus of. The preparation method of ABP was described in Miler et al., J. Immunol., 2003, 170: 4854 - 4861 (which is incorporated herein by reference in its entirety), where the Fab molecule was linked to the constant region of the immunoglobulin. The preparation method of CovX - Bodies was described in Doppalapudi et al., Proc. Natl. Acad. Sci. USA, 2010, 107: 22611 - 22616 (which is incorporated herein by reference in its entirety). The preparation method of Fcab ABP was described in Wozniak - Knopp et al., Protein Eng. Des. Sel., 2010, 23: 289 - 297 (which is incorporated herein by reference in its entirety). The preparation method of ABP was described in Kipriyanov et al., J. Mol. Biol., 1999, 293: 41 - 56 and Zhukovsky et al., Blood, 2013, 122: 5116 (each of which is incorporated herein by reference in its entirety). The preparation method of tandem Fab was described in WO 2015 / 103072 (which is incorporated herein by reference in its entirety). The preparation method of Zybodies was described in LaFleur et al., mAbs, 2013, 5: 208 - 218 (which is incorporated herein by reference in its entirety). TM The preparation method.

[0514] Methods for preparing variants

[0515] Diversity can be introduced into the polynucleotide sequence encoding ABP by any suitable method, including error - prone PCR, strand shuffling, and oligonucleotide - directed mutagenesis, such as trinucleotide - directed mutagenesis (TRIM). In some aspects, several CDR residues (e.g., 4 to 6 residues at a time) are randomized. For example, alanine - scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding. In particular, CDR - H3 and CDR - L3 are often targeted for mutation.

[0516] The diversity introduced in the variable region and / or CDR can be used to generate a secondary library. Then, the secondary library is screened to identify ABP variants with improved affinity. For example, affinity maturation by construction and reselection of a secondary library was described in Hoogenboom et al., Methods in Molecular Biology, 2001, 178: 1 - 37 (which is incorporated herein by reference in its entirety).

[0517] Engineering methods for cells containing ABP

[0518] Also provided are methods, nucleic acids, compositions and kits for expressing ABP (including receptors comprising antibodies, CARs and TCRs) and for generating genetically engineered cells expressing such ABP. Genetic engineering generally involves, for example, introducing nucleic acids encoding recombinant or engineered components into cells by retroviral transduction, transfection or transformation.

[0519] In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining the cells with a stimulant that induces a response such as proliferation, survival and / or activation, e.g., as measured by the expression of a cytokine or an activation marker; then transducing the activated cells and expanding them in culture to a number sufficient for clinical use.

[0520] In some cases, overexpression of stimulatory factors (e.g., lymphokines or cytokines) may be toxic to the subject. Thus, in some cases, the engineered cells contain gene segments that render the cells amenable to negative selection in vivo, such as when administered in adoptive immunotherapy. For example, in some aspects, the cells are engineered such that they are eliminated due to changes in the in vivo condition of the patient to whom the cells are administered. The negative selection phenotype is created by the insertion of a gene that confers sensitivity to a reagent (e.g., a compound) administered. Negative selection genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers sensitivity to ganciclovir (Wigler et al., Cell II:223, 1977), the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase ((APRT)) gene, the bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992).

[0521] In some aspects, the cells are further engineered to promote the expression of cytokines or other factors. Various methods for introducing genetic engineering components, such as antigen receptors (e.g., CARs), are well known and can be used with the methods and compositions described. Exemplary methods include methods for transferring nucleic acids encoding the receptor, including methods by virus (e.g., retrovirus or lentivirus) transduction, transposons and electroporation.

[0522] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles that are, e.g., vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant lentiviral vectors or retroviral vectors, such as γ-retroviral vectors, are used to transfer recombinant nucleic acids into T cells (see, e.g., Koste et al., (2014) Gene Therapy Apr 3, 2014. 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. Nov 2011 29(11):550-557).

[0523] In some embodiments, retroviral vectors have long terminal repeats (LTRs), e.g., retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus comprises a retrovirus from any avian or mammalian cell source. Retroviruses are generally amphotropic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Numerous 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, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa et al (1991) Virology 180:849-852; Burns et al (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037 and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0524] Methods of lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al., 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.

[0525] In some embodiments, recombinant nucleic acids are transferred into T cells by electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3): e60298; Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437 and Roth et al. (2018) Nature 559: 405-409. In some embodiments, recombinant nucleic acids are transferred into T cells by inversion (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 in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microprojectile bombardment (Johnston, Nature, 346: 776-777 (1990); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987).

[0526] Other methods and vectors for transferring nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0527] Additional nucleic acids, e.g., genes for introduction are those such as to enhance therapeutic efficacy by promoting viability and / or function of the transferred cells; to provide gene markers for selection and / or evaluation of cells, such as to evaluate in vivo survival rate or localization; e.g., genes such as to facilitate negative selection of cells in vivo to enhance safety, as described in Lupton S.D. et al., Mol. and Cell Biol., 11:6 (1991) and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selection fusion genes obtained by fusing a dominant positive selection marker with a negative selection marker. See, e.g., Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0528] Preparing engineered cells

[0529] In some embodiments, the preparation of the engineered cells comprises one or more culturing and / or preparation steps. Cells for introduction of the HLA-peptide-ABP, e.g., TCR or CAR, can be isolated from a sample, such as a biological sample (isolated from or of subject origin) from a subject. In some embodiments, the subject from whom the cells are isolated is a subject having a disease or disorder or in need of or to be subjected to cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed, and / or engineered.

[0530] Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. The sample comprises tissues, fluids, and other samples taken directly from the subject, as well as samples produced by one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a processed sample. The biological sample includes but is not limited to body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.

[0531] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is a product of or derived from apheresis or leukapheresis. 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 organs, and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy), the sample includes samples of autologous and allogeneic origin.

[0532] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, these cells are obtained from xenogeneic sources, e.g., mouse, rat, non-human primate or pig.

[0533] In some embodiments, the isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some instances, the cells are washed, centrifuged and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich desired components, lyse or remove cells sensitive to a particular reagent. In some instances, the cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to a particular component.

[0534] In some instances, the cells are obtained from the circulating blood of a subject by, e.g., apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some aspects, contains cells other than red blood cells and platelets.

[0535] In some embodiments, the 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 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 by a semi-automatic "flow-through" centrifuge (e.g., the Cobe 2991 cell processor from Baxter) according to the manufacturer's instructions. In some aspects, the wash step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, e.g., PBS without Ca++ / Mg++. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in a medium.

[0536] In some embodiments, these methods include: density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0537] In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cells, and the specific molecules are, for example, surface markers, such as surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on affinity or immunological affinity. For example, in some aspects, the separation involves separating cells and cell populations based on the expression or expression level of one or more markers (usually cell surface markers) in the cells, such as by incubating with antibodies or binding partners that specifically bind to these markers; next, usually a washing step, and then separating the cells bound to the antibody or binding partner from the cells not bound to the antibody or binding partner.

[0538] This separation step can be carried out based on positive selection and / or negative selection, where, in positive selection, the cells bound to the reagent are retained for further use; in negative selection, the cells not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection may be particularly useful in the absence of antibodies available for specifically identifying cell types in a heterogeneous population, such that the separation is best carried out based on markers expressed by cells other than the desired population.

[0539] The separation does not need to result in 100% enrichment or depletion of a specific cell population or cells expressing a specific marker. For example, positive selection or enrichment of a specific type of cell (such as those expressing a marker) refers to increasing the number or percentage of such cells, but does not necessarily mean that cells not expressing the marker are completely absent. Similarly, negative selection, removal, or depletion of a specific type of cell (such as those expressing a marker) refers to reducing the number or percentage of such cells, but does not necessarily mean that all such cells are completely removed.

[0540] In some instances, multiple rounds of separation steps are carried out, where the fraction from a positive or negative selection in one step is subjected to another separation step, such as a subsequent positive or negative selection. In some instances, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each of which is specific for a marker targeted for negative selection. Similarly, negative selection of multiple cell types can be carried out simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types.

[0541] For example, in some aspects, specific subsets of T cells, such as cells that are positive or have a high expression level of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+, are isolated by positive or negative selection techniques.

[0542] For example, positive selection of CD3+, CD28+ T cells can be performed using CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS.RTM.M-450 CD3 / CD28 TCell Expander).

[0543] In some embodiments, isolation is performed by enriching a specific cell population by positive selection or depleting a specific cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed (marker+) or expressed at a relatively high level (marker high) on the cells being positively or negatively selected, respectively.

[0544] In some embodiments, T cells are isolated from a peripheral blood mononuclear cell (PBMC) sample by negative selection of markers expressed on non-T cells (such as B cells, monocytes or other white blood cells, such as CD14). In some aspects, CD4+ or CD8+ selection steps are used to isolate CD4+ helper cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be sorted into subsets in one step by positive or negative selection of markers expressed or expressed at a relatively high level on one or more naive, memory and / or effector T cell subsets.

[0545] In some embodiments, positive or negative selection based on surface antigens associated with respective subsets is used to further enrich or deplete naive cells, stem cells, central memory stem cells, effector memory stem cells and / or central memory stem cells in CD8+. In some embodiments, central memory T (TCM) cells are enriched to improve efficacy, such as improving long-term survival, expansion and / or engraftment survival after administration, and in some aspects, they are particularly robust in such subsets. See Terakura et al. (2012) Blood.1:72 - 82; Wang et al. (2012) J Immunother.35(9):689 - 701. In some embodiments, combining CD8+ T cells and CD4+ T cells enriched for TCM further improves efficacy.

[0546] In embodiments, memory T cells are present in the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. Peripheral blood mononuclear cells (PBMCs) can be enriched for or depleted of the CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0547] In some embodiments, enrichment of central memory T (TCM) cells is based on positive expression or high-level surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, a CD8+ population enriched in TCM cells is isolated by depleting cells expressing CD4, CD14, CD45RA and positively selecting or enriching cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting from the negative fraction of cells selected based on CD4 expression, which is negatively selected based on the expression of CD14 and CD45RA and positively selected based on the expression of CD62L. In some aspects, this selection is performed simultaneously, while in other aspects, it is performed sequentially in either order. In some aspects, the same CD4-expression-based selection steps used to prepare a CD8+ cell population or subset are also used to generate a CD4+ cell population or subset, such that the positive and negative fractions generated based on CD4 isolation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps.

[0548] In a particular example, selection of CD4+ cells is performed on a sample of PBMCs or other white blood cell sample, where both the negative and positive fractions are retained. Then, the negative fraction is negatively selected based on the expression of CD14 and CD45RA or ROR1, and the positive fraction is negatively selected based on the marker profile of central memory T cells (such as CD62L or CCR7), where the positive and negative selections are performed in either order.

[0549] CD4+ T helper cells are classified as naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0550] In one example, to enrich CD4+ cells by negative selection, a monoclonal antibody mixture (cocktail) typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, an antibody or binding partner is conjugated to a solid support or matrix (such as magnetic or paramagnetic beads) to separate cells for positive and / or negative selection. For example, in some embodiments, immuno-magnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Volume 58: Metastasis Research Protocols, Volume 2: Cell Behavior In Vitro and In Vivo, pages 17 - 25, edited by: S.A. Brooks and U. Schumacher Humana Press Inc., Totowa, N.J.).

[0551] In some aspects, a sample or cell composition to be separated is incubated with magnetizable or magnetoresponsive small substances, such as magnetoresponsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetoresponsive substance (e.g., particle) is typically directly or indirectly linked to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., surface marker) present in one or more cells or cell populations that need to be separated (e.g., need negative or positive selection).

[0552] In some embodiments, the magnetic particles or beads comprise a magnetoresponsive substance conjugated to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetoresponsive substances that can be used in magnetic separation methods. Suitable magnetic particles are those described in Molday's U.S. Patent No. 4,452,773 and European Patent Specification EP452342B, which are incorporated by reference. Other examples are colloidal-sized particles, such as those described in Owen's U.S. Patent No. 4,795,698 and Liberti et al.'s U.S. Patent No. 5,200,084.

[0553] Incubation is generally carried out under conditions such that the antibody or binding partner or molecule, such as a secondary antibody or other reagent that specifically binds to an antibody or binding partner conjugated to such linked magnetic particles or beads, specifically binds to cell surface molecules (if present) on the cells in the sample.

[0554] In some aspects, the sample is placed in a magnetic field, and cells having magnetoresponsive or magnetizable particles attached thereto will be attracted by the magnet and separated from the unlabeled cells. For positive selection, the cells attracted by the magnet are retained; for negative selection, the non-attracted cells (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed in the same selection step, wherein the positive and negative fractions are retained and further processed or further separation steps are performed.

[0555] In certain embodiments, the magnetoresponsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are linked to cells by coating with a primary antibody that is specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0556] In some embodiments, the magnetoresponsive particles are left attached to the cells that need to be incubated, cultured, and / or engineered subsequently; in some aspects, the particles are left attached to the cells to be administered to a patient. In some embodiments, the magnetizable or magnetoresponsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, using a competitive unlabeled antibody, a magnetizable particle, or an antibody conjugated to a cleavable linker. In some embodiments, the magnetizable particles are biodegradable.

[0557] In some embodiments, the affinity-based selection is performed by magnetic-activated cell sorting ((MACS)) (Miltenyi Biotech, Auburn, Calif.). The magnetic-activated cell sorting ((MACS)) system is capable of highly pure selection of cells to which magnetized particles are attached. In certain embodiments, MACS operates in the following mode: after applying an external magnetic field, non-target and target substances are eluted sequentially. That is, the cells attached with magnetized particles are kept in place while the unconnected substances are eluted. Then, after the first elution step is completed, the substances that remain in the magnetic field and are prevented from being eluted are released in some way so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and eliminated from the heterogeneous cell population.

[0558] In certain embodiments, the isolation or separation is performed using a system, apparatus, or device that implements 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, for the purpose of, for example, minimizing errors, user manipulation, and / or contamination as much as possible. In one example, the system is the system described in International Patent Application Publication No. WO2009 / 072003 or U.S. Patent Application Publication No. US20110003380 A1.

[0559] In some embodiments, the system or device performs one or more (e.g., all) of the separation, processing, engineering, and formulation steps in an integrated or stand-alone system, and / or in an automated or programmable form. In some aspects, the system or device includes a computer and / or computer program that communicates with the system or device and allows a user to program, control, evaluate the results of, and / or adjust aspects of the processing, separation, engineering, and formulation steps.

[0560] In some aspects, the CliniMACS system (Miltenyi ) is used to perform the separation and / or other steps, for example, for the automated separation of cells at the clinical level in a closed and sterile system. The components can include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some aspects, the integrated computer controls all components of the instrument and instructs the system to perform repetitive operations in a standardized sequence. In some aspects, the magnetic separation unit includes a movable permanent magnet and a holder for selecting columns. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valves, ensures a controlled flow rate of buffer and continuous cell suspension through the system.

[0561] In some aspects, the CliniMACS system uses antibody-conjugated magnetizable particles provided in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a set of tubes, which in turn are connected to a buffer-containing bag and a cell collection bag. The set of tubes consists of pre-assembled sterile tubing (including a pre-column and a separation column) and is for single use only. After the separation program is initiated, the system automatically loads the cell sample onto the separation column. The labeled cells are retained in the column, while the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population used in the methods described herein is unlabeled and does not remain in the column. In some embodiments, the cell population used in the methods described herein is labeled and remains in the column. In some embodiments, after removal of the magnetic field, the cell population used in the methods described herein is eluted from the column and collected in the cell collection bag.

[0562] In certain embodiments, the CliniMACS Prodigy system (Miltenyi Biotec) is used for separation and / or other steps. In some aspects, the CliniMACS Prodigy system is equipped with a cell processing unit that allows automated washing and centrifugal fractionation of cells. The CliniMACS Prodigy system may also include a built-in camera and image recognition software to determine the endpoint of optimal cell fractionation by discerning the visually distinguishable layers of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber for performing cell culture assays such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports may allow for sterile removal and replenishment of media, and the cells may be monitored using an integrated microscope. See, for example, 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.

[0563] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, wherein cells stained for multiple surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative fluorescence-activated cell sorting (FACS). In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a microelectromechanical system (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 Godi et al. (2008) J Biophoton. 1(5): 355-376). In both cases, the cells can be labeled with multiple markers in order to isolate highly pure, defined T cell subsets.

[0564] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate isolation by positive selection and / or negative selection. For example, the isolation can be based on binding to a fluorescently labeled antibody. In some instances, cell separation based on binding of an antibody or other binding partner specific for one or more cell surface markers is carried out in a fluid stream, such as by fluorescence-activated cell sorting (FACS) (which includes preparative (FACS) and / or microelectromechanical system (MEMS) chips), for example in combination with a flow cytometry detection system. This method allows for simultaneous positive and negative selection based on multiple markers.

[0565] In some embodiments, the preparation method includes the step of freezing (e.g., cryopreserving) the cells before or after separation, incubation, and / or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes from the cell population and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, for example after washing to remove plasma and platelets. Various known freezing solutions and parameters in some aspects can be employed. One example involves PBS containing 20% DMSO and 8% human serum albumin (HAS), or other suitable cell freezing media. Then, it is diluted 1:1 with media such that the final concentrations of DMSO and HSA are 10% and 4%, respectively. Other examples include CTL-Cryo TM ABC freezing media, etc. Then, the cells are generally frozen at a rate of 1 degree / minute to -80°C and stored in the vapor phase of a liquid nitrogen storage tank.

[0566] In some embodiments, the method includes cultivation, incubation, culture, and / or genetic engineering steps. For example, in some embodiments, methods are provided for incubating and / or engineering a cell population and a culture initiation composition.

[0567] Thus, in some embodiments, the cell population is incubated in the culture initiation composition. The incubation and / or engineering can be carried out in a culture vessel such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culturing or growing cells.

[0568] In some embodiments, the cells are incubated before and / or in conjunction with genetic engineering. The incubation step includes culture, cultivation, stimulation, activation, and / or proliferation. In some embodiments, the composition or cells are incubated in the presence of a stimulating condition or stimulant. Such conditions include those designed to achieve the following purposes: inducing proliferation, expansion, activation, and / or survival of cells in the population, mimicking antigen contact, and / or preparing the cells for genetic modification, such as introducing a recombinant antigen receptor.

[0569] The conditions can include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, reagents (e.g., nutrients), amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other substances designed to activate cells.

[0570] In some embodiments, the stimulating condition or reagent includes one or more reagents capable of activating the intracellular signaling domain of the TCR complex, such as a ligand. In some aspects, the reagent turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such a reagent can include antibodies, such as antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, which are bound, for example, to a solid support (such as beads and / or one or more cytokines). Optionally, the amplification method can further include the step of 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 stimulant includes IL-2 and / or IL-15, e.g., IL-2 at a concentration of at least about 10 units / mL.

[0571] In some aspects, incubation is carried out according to the techniques described in the following documents: such as U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1: 72-82 and / or Wang et al. (2012) J Immunother. 35(9): 689-701.

[0572] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture initiation composition (e.g., such that each T lymphocyte in the initial population to be expanded in the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells); and incubating the culture (e.g., incubating for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may include γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with γ-rays in the range of about 3000 to 3600 rads to prevent cell division. In some embodiments, the PBMC feeder cells are inactivated with Mytomicin C. In some aspects, the feeder cells are added to the culture medium before adding the T cell population.

[0573] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, generally at least about 30 degrees, and generally is or about 37 degrees Celsius. Optionally, the incubation may also include adding non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. The LCLs can be irradiated with γ-rays in the range of about 6000 to 10000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as the ratio of LCL feeder cells to initial T lymphocytes is at least about 10:1.

[0574] In embodiments, antigen-specific T cells are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen, such as antigen-specific CD4+ and / or CD8+ T cells. For example, an antigen-specific T cell line or clone against a cytomegalovirus antigen can be generated as follows: T cells are isolated from an infected subject and the cells are stimulated in vitro with the same antigen.

[0575] Assays

[0576] A variety of assays known in the art can be used to identify and characterize the HLA-peptide ABP described herein.

[0577] Binding, competition, and epitope mapping assays

[0578] Any suitable method can be used to evaluate the specific antigen-binding activity of the ABP provided herein, including methods using SPR, BLI, RIA, and MSD-SET (as described in other parts of the present disclosure). Additionally, antigen-binding activity can be evaluated by ELISA assays, using flow cytometry, and / or Western blot assays.

[0579] Assays for measuring competition between two ABPs or between an ABP and another molecule (e.g., one or more ligands of HLA-peptide, such as TCR) are described in other parts of the present disclosure and, for example, Harlow and Lane, ABPs: A Laboratory Manual Chapter 14, 1988, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is incorporated by reference in its entirety.

[0580] Assays for epitope mapping of the ABP binding provided herein are described in, for example, Morris "Epitope Mapping Protocols" in Methods in Molecular Biology Volume 66, 1996, Humana Press, Totowa, N.J., 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.

[0581] Effector function assays

[0582] The effector functions after ABP and / or cell therapy provided herein can be evaluated using a variety of in vitro and in vivo assays known in the art, including the methods described in the following references: Ravetch and Kinet, Annu. Rev. Immunol., 1991, 9:457-492; U.S. Patent Nos. 5,500,362, 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; WO 2006 / 029879; WO 2005 / 100402; Gazzano-Santoro et al., J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003, 101:1045-1052; Cragg et al., Blood, 2004, 103:2738-2743 and Petkova et al., Int’l. Immunol., 2006, 18:1759-1769; each of which is incorporated by reference in its entirety.

[0583] Pharmaceutical compositions

[0584] The ABP, cells or HLA-peptide targets provided herein can be formulated into any suitable pharmaceutical composition and administered by any applicable route of administration. Suitable routes of administration include, but are not limited to: intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, intranasal, parenteral, pulmonary and subcutaneous routes.

[0585] The pharmaceutical composition can contain one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used, and those of ordinary skill in the art can select suitable pharmaceutical excipients. Therefore, the pharmaceutical excipients provided below are merely exemplary and not restrictive. Additional pharmaceutical excipients include, for example, the pharmaceutical excipients described in Handbook of Pharmaceutical Excipients, Rowe et al. (eds.) 6th ed. (2009) (which is incorporated by reference in its entirety).

[0586] In some embodiments, the pharmaceutical composition comprises an antifoaming agent. Any suitable antifoaming agent can be used. In some aspects, the antifoaming agent is selected from alcohols, ethers, oils, waxes, silicones, surfactants, and combinations thereof. In some aspects, 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, silicoglycol, fluorosilicone, polyethylene glycol - polypropylene glycol copolymer, polydimethylsiloxane - silica, ether, octanol, decanol, sorbitan trioleate, ethanol, 2-ethylhexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.

[0587] In some embodiments, the pharmaceutical composition comprises a co-solvent. Illustrative examples of co-solvents include ethanol, poly((ethylene glycol)), butylene glycol, dimethylacetamide, glycerol, propylene glycol, and combinations thereof.

[0588] In some embodiments, the pharmaceutical composition comprises a buffering agent. Illustrative examples of buffering agents include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, sodium glutamate, and combinations thereof.

[0589] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, guar gum, and combinations thereof.

[0590] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include: d-α tocopherol, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, cetylpyridinium chloride, sodium docusate, glyceryl behenate, glyceryl monooleate, lauric acid, polyethylene glycol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, polyglycerol ester, sodium lauryl sulfate, sorbitan ester, vitamin E polyethylene((ethylene glycol)) succinate, and combinations thereof.

[0591] In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate((tribasic)), hydroxypropylmethyl cellulose, hydroxypropyl cellulose, magnesium oxide, and combinations thereof.

[0592] Other excipients that can be used with the pharmaceutical composition include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersants, solubilization enhancers, emulsifiers, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizers, solvents, stabilizers, sugars, and combinations thereof. Specific examples of these agents are described, for example, in Handbook of Pharmaceutical Excipients, 6th Edition (2009), Rowe et al. (editors) (which is incorporated herein by reference in its entirety).

[0593] In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is a salt solution, such as a sterile isotonic salt solution or a dextrose solution. In certain aspects, the solvent is water for injection.

[0594] In some embodiments, the pharmaceutical composition is in particulate form, such as microparticles or nanoparticles. The microparticles and nanoparticles can be formed from any suitable material, such as polymers or lipids. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymeric vesicles.

[0595] Since water can promote the degradation of some ABPs, anhydrous pharmaceutical compositions and dosage forms comprising ABPs are also provided herein.

[0596] The anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low-moisture ingredients under low-moisture or low-humidity conditions. If substantial contact with moisture and / or humidity is expected during manufacturing, packaging, and / or storage, the pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine can be anhydrous.

[0597] The anhydrous pharmaceutical compositions should be prepared and stored to maintain their anhydrous nature. Thus, the anhydrous compositions can be packaged using known materials that prevent contact with water, such that they are contained in suitable dispensing kits. Examples of suitable packaging include, but are not limited to, airtight foils, plastics, unit-dose containers (e.g., vials), blister packs, and strip packs.

[0598] In certain embodiments, the ABPs and / or cells provided herein are formulated into parenteral dosage forms. The parenteral dosage forms can be administered to a subject by a variety of routes, including but not limited to subcutaneous, intravenous (including infusion and bolus), intramuscular, and intraarterial. Since their route of administration typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are generally sterile or capable of being sterilized before being administered to the subject. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, dry (e.g., lyophilized) products to be dissolved or suspended in a pharmaceutically acceptable injectable vehicle, injectable suspensions, and emulsions.

[0599] Those skilled in the art are familiar with suitable vehicles for providing parenteral dosage forms. Examples include, but are not limited to: water for injection ((see USP)); aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-soluble vehicles such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and anhydrous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0600] Excipients may also be incorporated into the parenteral dosage form, which increase the solubility of one or more of the ABP and / or cells disclosed herein.

[0601] In some embodiments, the parenteral dosage form is lyophilized. Exemplary lyophilized formulations are described in, for example, U.S. Patent Nos. 6,267,958 and 6,171,586; and WO 2006 / 044908; which are hereby incorporated by reference in their entirety.

[0602] In human therapy, the physician will determine the dose that he or she deems most appropriate based on prophylactic or therapeutic treatment and according to age, body weight, medical condition, and other specific factors of the subject to be treated.

[0603] In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic ABP.

[0604] The amount of ABP, cell, or composition that is effective to prophylactically or therapeutically treat a disorder or one or more of its symptoms will vary with the nature and severity of the disorder or medical condition and the route of administration of the ABP and / or cell. The frequency and dose will also vary according to the specific factors of each subject, depending on the particular therapy ((e.g., therapeutic or prophylactic agent)) being administered, the severity of the disorder, disease, or medical condition, the route of administration, and the age, body, weight, response, and medical history of the subject. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0605] It will be readily appreciated by those of ordinary skill in the art that different therapeutically effective amounts may be applicable to different diseases and medical conditions. Similarly, the dosage and dosage frequency regimens provided herein also encompass doses that are sufficient to prevent, control, treat, or improve the disorder but are not sufficient to cause or are sufficient to mitigate the side effects associated with the ABP and / or cells provided herein. In addition, when multiple doses of the compositions provided herein are administered to a subject, not all doses need to be the same. For example, the dose administered to a subject may be increased to improve the prophylactic or therapeutic effect of the composition, or the dosage may be decreased to reduce one or more side effects that a particular subject is experiencing.

[0606] In certain embodiments, a subject may be treated or prophylactically treated with one or more loading doses of the ABP or composition provided herein, followed by one or more maintenance doses.

[0607] In certain embodiments, a dose of the ABP, cell, or composition provided herein is administered to achieve a steady-state concentration of the ABP and / or cell in the blood or serum of the subject. The steady-state concentration can be determined by measurement using techniques available to those of skill in the art, or can be determined based on the physical characteristics of the subject such as height, weight, and age.

[0608] As discussed in more detail elsewhere in the present disclosure, the ABP and / or cell provided herein may optionally be administered in combination with one or more additional agents for the prevention or treatment of a disease or disorder. The effective amount of such additional agent depends on the amount of ABP present in the formulation, the type of disorder or treatment, and other factors known in the art or described herein.

[0609] Therapeutic uses

[0610] For therapeutic use, the ABP and / or cell is administered to a mammal, typically a human, in a pharmaceutically acceptable dosage form (such as those known in the art and discussed above). For example, the ABP and / or cell may be administered intravenously to a human by bolus injection or continuous infusion over a period of time via a muscle, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intrathecal, or intratumoral route. The ABP may also be administered appropriately via a peritumoral, intralesional, or perilesional route to exert both local and systemic therapeutic effects. The intraperitoneal route, for example, may be particularly useful in the treatment of ovarian tumors.

[0611] The ABP and / or cell provided herein can be used to treat any HLA-peptide-related disease or disorder. In some embodiments, the disease or disorder is a disease or disorder that benefits from anti-HLA-peptide ABP and / or cell therapy. In some embodiments, the disease or disorder is a tumor. In some embodiments, the disease or disorder is a cell proliferative disorder. In some embodiments, the disease or disorder is cancer.

[0612] In some embodiments, the ABP and / or cell provided herein is used as a medicament. In some embodiments, the ABP and / or cell provided herein is used in the manufacture or preparation of a medicament. In some embodiments, the medicament is used to treat a disease or disorder that benefits from anti-HLA-peptide ABP and / or cell therapy. In some embodiments, the disease or disorder is a tumor. In some embodiments, the disease or disorder is a cell proliferative disorder. In some embodiments, the disease or disorder is cancer.

[0613] In some embodiments, provided herein are methods of administering an effective amount of an ABP and / or cell provided herein to a subject in need thereof to treat a disease or condition of the subject. In some aspects, the disease or condition is cancer.

[0614] In some embodiments, provided herein are methods of administering an effective amount of an ABP and / or cell provided herein to a subject in need thereof to treat a disease or condition of the subject, wherein the disease or condition is cancer, and the cancer is selected from solid tumors and hematological tumors.

[0615] In some embodiments, provided herein are methods of modulating an immune response in a subject in need thereof, the methods comprising: administering to the subject an effective amount of an ABP and / or cell or pharmaceutical composition disclosed herein.

[0616] Combination therapies

[0617] In some embodiments, the ABP and / or cell provided herein is administered in combination with at least one additional therapeutic agent. Any suitable additional therapeutic agent can be administered in combination with the ABP and / or cell provided herein. The additional therapeutic agent can be fused to the ABP. In some aspects, the additional therapeutic agent is selected from radiopharmaceuticals, cytotoxic agents, toxins, chemotherapeutic agents, cytostatic agents, antihormonal agents, EGFR inhibitors, immunomodulators, antiangiogenic agents, and combinations thereof. In some embodiments, the additional therapeutic agent is an ABP.

[0618] Diagnostic methods

[0619] Also provided are methods for predicting and / or detecting the presence of a given HLA-peptide on the cells of a subject. Such methods can be used, for example, to pr...

Claims

1. An isolated antibody or antigen-binding fragment thereof 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, wherein the HLA-restricted peptide is located in the peptide-binding groove of the α1 / α2 heterodimer portion of the HLA class I molecule, and wherein the HLA class I molecule is the HLA subtype B*35:01, and the HLA-restricted peptide comprises the sequence EVDPIGHVY, and wherein the antibody or antigen-binding fragment thereof comprises HCDR1 consisting of the sequence FSFSSYWMS, HCDR2 consisting of the sequence SYISGDSGYTNYA, HCDR3 consisting of the sequence CASHDYGDYGEYFQHW, LCDR1 consisting of the sequence QASQDISNYLN, LCDR2 consisting of the sequence AASSLQS, and LCDR3 consisting of the sequence CQQAISFPLTF.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH sequence that comprises the sequence EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISYISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHDYGDYGEYFQHWGQGTLVTVSSAS.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VL sequence that comprises the sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQSTKVEIK.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the VH sequence EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISYISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHDYGDYGEYFQHWGQGTLVTVSSAS and the VL sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQSTKVEIK.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is linked to a scaffold, and the scaffold is serum albumin or Fc.

6. The isolated antibody or antigen-binding fragment thereof according to claim 5, wherein the Fc is human Fc and is an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM isotype Fc.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is linked to a scaffold through a linker, and the scaffold is serum albumin or Fc.

8. The isolated antibody or antigen-binding fragment thereof according to claim 7, wherein the linker is a peptide linker.

9. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein the peptide linker is the hinge region of a human antibody.

10. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a scFv fragment, a scFv-Fc fragment, and / or a single-domain antibody or antigen-binding fragment thereof.

11. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a scFv fragment.

12. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

13. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized, human or chimeric antibody.

14. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

15. The isolated antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody or antigen-binding fragment thereof comprises a human IgG class and a heavy chain constant region of a subclass selected from IgG1, IgG4, IgG2 and IgG3.

16. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a modification for extending the half-life.

17. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc.

18. The isolated antibody or antigen-binding fragment thereof according to claim 17, wherein the modified Fc comprises one or more mutations for extending the half-life.

19. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is part of a chimeric antigen receptor (CAR) that comprises: an extracellular portion comprising the antibody or antigen-binding fragment thereof; and an intracellular signaling domain.

20. The isolated antibody or antigen-binding fragment thereof according to claim 19, wherein the antibody or antigen-binding fragment thereof comprises a scFv, and the intracellular signaling domain comprises an ITAM.

21. An isolated antibody or antigen-binding fragment thereof according to claim 19 or 20, wherein the intracellular signaling domain comprises the signaling domain of the ζ chain of the CD3-ζ (CD3) chain.

22. An isolated antibody or antigen-binding fragment thereof according to claim 19, wherein the antibody or antigen-binding fragment further comprises a transmembrane domain that links the extracellular domain and the intracellular signaling domain.

23. An isolated antibody or antigen-binding fragment thereof according to claim 22, wherein the transmembrane domain comprises the transmembrane portion of CD28.

24. An isolated antibody or antigen-binding fragment thereof according to claim 19, wherein the antibody or antigen-binding fragment further comprises the intracellular signaling domain of a T cell co-stimulatory molecule.

25. An isolated antibody or antigen-binding fragment thereof according to claim 24, wherein the T cell co-stimulatory molecule is CD28, 4-1BB, OX-40, ICOS, or any combination thereof.

26. An engineered cell that expresses a receptor comprising the antibody or antigen-binding fragment thereof according to claim 1.

27. The engineered cell according to claim 26, which is a T cell or a cytotoxic T cell (CTL).

28. The engineered cell according to claim 26, wherein the antibody or antigen-binding fragment thereof is expressed by a heterologous promoter.

29. An isolated polynucleotide or polynucleotide set that encodes the antibody or antigen-binding fragment thereof according to claim 1.

30. A vector or vector set that comprises the polynucleotide or polynucleotide set according to claim 29.

31. A host cell that comprises the polynucleotide or polynucleotide set according to claim 29, or the vector or vector set according to claim 30.

32. The host cell according to claim 31, wherein the host cell is CHO or HEK293.

33. The host cell according to claim 31, wherein the host cell is a T cell.

34. A method of generating an antibody or an antigen-binding fragment thereof, comprising: Expressing the antibody or antigen-binding fragment thereof using the host cell according to claim 31 or 32, and isolating the expressed antibody or antigen-binding fragment thereof.

35. A pharmaceutical composition that comprises the antibody or antigen-binding fragment thereof according to claim 1, and a pharmaceutically acceptable excipient.

36. A kit that includes the antibody or antigen-binding fragment thereof according to claim 1, or the pharmaceutical composition according to claim 35, and instructions for use.

37. A virus that comprises the isolated polynucleotide or polynucleotide set according to claim 29.

38. The virus according to claim 37, wherein the virus is a filamentous phage.

39. A yeast cell that comprises the isolated polynucleotide or polynucleotide set according to claim 29.

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