Multispecific T cell linker composition and method of using the same

Abstract

A method and composition for manufacturing and using a therapeutic agent comprising a multispecific molecule, used for immunotherapy of cancer or infection.

Description

Technology Field

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 592,013 filed on October 20, 2023 and U.S. Provisional Application No. 63 / 698,816 filed on September 25, 2024, the entirety of which is incorporated herein by reference. Background Technology

[0003] T cell receptors (TCRs) are a crucial element in the adaptive immune system's recognition and elimination of 'non-self' intracellular antigens. It is estimated that over 70% of tumor-specific targets are processed intracellularly and presented as peptides on the cell surface in a state bound to the major histocompatibility complex (MHC); when referring to human MHC, the major histocompatibility complex is known as human leukocyte antigen (HLA). While these peptide HLA complexes (pHLA) cannot be accessed by antibody targeting mechanisms, including CAR T cells, antibody-drug conjugates, and bispecific T cell linkages, T cells recognize them via TCRs. Additionally, these pHLAs may be recognized by allogeneic T cells containing the same HLA allele through TCR-peptide / HLA interactions. Therefore, TCR-based therapies have become a promising tool for eliminating cells expressing mutated tumor-specific or tumor-associated peptides derived from intracellular proteins and presented bound to MHC on the surface of tumor cells. However, the weak affinity and low stability of naturally selected TCRs present some difficulties in their therapeutic use. Consequently, the most widely used TCR-based therapies involve the adoptive transfer of proliferated antigen-specific T cells or genetically modified T cells expressing artificial antigen-specific TCRs [specific peptide affinity-enhanced receptors (SPEARs)]. These therapies are complicated by the need to manufacture therapeutic T cells on a patient-specific basis.

[0004] In one embodiment, a composition comprising a recombinant nucleic acid for expression in mammalian cells, wherein the recombinant nucleic acid comprises a sequence encoding a multispecific molecule, the multispecific molecule comprising (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; The present invention provides a composition comprising an engineered T cell receptor (TCR) construct comprising: (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the T cell conjugate comprises an anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGIYSDSSDFIGNL (SEQ ID NO. 713) or GIYSDSSDFIGNL (SEQ ID NO. 707).

[0005] In another embodiment, a composition comprising a recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a TCR alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain, wherein the peptide of the MHC-peptide complex comprises a RAS peptide sequence; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the T cell conjugate comprises an anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGIYSDSSDFIGNL (SEQ No. 713) or GIYSDSSDFIGNL (SEQ No. 707). In some embodiments, the multispecific molecule comprises a post-translational modification. In some embodiments, the multispecific molecule comprises a mammalian glycosylation signature. In some embodiments, the multispecific molecule is soluble. In some embodiments, the multispecific molecule lacks a transmembrane domain. In some embodiments, the multispecific molecule consists of two polypeptides, namely a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and the second polypeptide comprise one or more disulfide bonds between the two polypeptides. In some embodiments, the multispecific molecule comprises the first polypeptide and the second polypeptide, wherein the first polypeptide comprises a TRAV domain and the second polypeptide comprises a TRBV domain. In some embodiments, the peptide of the MHC-peptide complex is a cancer antigen-derived peptide. In some embodiments, the peptide of the MHC-peptide complex is a mutant peptide, and the MHC of the MHC-peptide complex binds to the mutant peptide with a higher affinity compared to the corresponding wild-type peptide. In some embodiments, the peptide of the MHC-peptide complex does not contain the amino acid sequence YLEPGPVTA.In some embodiments, the MHC of the MHC-peptide complex comprises a Class I MHC. In some embodiments, the MHC of the MHC-peptide complex is a Class I MHC polypeptide. In some embodiments, the MHC of the MHC-peptide complex is encoded by an HLA that appears in less than 1% of the human population. In some embodiments, the MHC of the MHC-peptide complex has a peptide binding affinity greater than at least 50 nM. In some embodiments, one or more binding domains of the T cell linkage bind to an extracellular domain of an endogenous receptor expressed by a T cell. In some embodiments, said one or more binding domains of the T cell linkage bind to an extracellular domain of a receptor selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICAM-1, and CD8. In some embodiments, one or more binding domains of the T cell linkage bind to CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, one or more binding domains of the T cell linkage comprise an antibody domain or an antigen-binding fragment thereof. In some embodiments, the binding domain of the T cell linkage comprises scFv or sdAb. In some embodiments, one or more binding domains of the T cell linkage comprise VHH.

[0006] In some embodiments, one or more binding domains of a T cell linkage comprise a first binding domain that binds to an extracellular domain of a first receptor expressed by a T cell and a second binding domain that binds to an extracellular domain of a second receptor expressed by a T cell. In some embodiments, the first binding domain of the T cell linkage that binds to an extracellular domain of a first receptor expressed by a T cell comprises a first VHH, and the second binding domain of the T cell linkage that binds to an extracellular domain of a second receptor expressed by a T cell comprises a second VHH. In one embodiment, the C-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or the C-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. In some embodiments, the N-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or the N-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. In some embodiments, the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by the T cell comprises scFv, and the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by the T cell comprises VHH. In some embodiments, the binding domain that binds to the extracellular domain of the first receptor expressed by the T cell and the second binding domain that binds to the extracellular domain of the second receptor expressed by the T cell are connected by a peptide linker.In some embodiments, the first binding domain binds to the extracellular domain of a first receptor expressed by a T cell, selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8, and the second binding domain binds to the extracellular domain of a second receptor expressed by a T cell, selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8. In some embodiments, the first receptor expressed by a T cell and the second receptor expressed by a T cell are different. In some embodiments, the first receptor expressed by a T cell is CD3, and the second receptor expressed by a T cell is CD2. In some embodiments, the first receptor expressed by a T cell is CD2, and the second receptor expressed by a T cell is CD3. In some embodiments, the first binding domain and the second binding domain of the T cell linkage are configured to bind to the same receptor expressed by a T cell. In some embodiments, the VH of the anti-CD3 binding domain comprises the HC CDR2 sequence of IYTNNIGST (SEQ No. 712) or CIYTNNIGSTWYASWAKG (SEQ No. 706). In some embodiments, the VH of the anti-CD3 binding domain comprises the HC CDR1 sequence of GFSFSNNYY (SEQ No. 711) or NNYYMC (SEQ No. 705). In some embodiments, the anti-CD3 binding domain comprises a light chain variable region [VL] comprising the light chain complementarity determining region 3 [LC CDR3] sequence of QQYNTIINVDRT (SEQ No. 713). In some embodiments, the VL of the anti-CD3 binding domain comprises the LC CDR2 sequence of KAS (SEQ No. 715) or KASTLAS (SEQ No. 709).In some embodiments, the VL of the anti-CD3 binding domain comprises the LC CDR1 sequence of QTIYNY (SEQ No. 714) or QASQTIYNYLA (SEQ No. 708). In some embodiments, the anti-CD3 binding domain comprises the HC CDR1 sequence of GFSFSNNYY (SEQ No. 711), the HC CDR2 sequence of IYTNNIGST (SEQ No. 712), the HC CDR3 sequence of ARGIYSDSSDFIGNL (SEQ No. 713), the LC CDR1 sequence of QTIYNY (SEQ No. 714), the LC CDR2 sequence of KAS (SEQ No. 715), and the LC CDR3 sequence of QQYNTIINVDRT (SEQ No. 716), wherein the CDR sequences are determined based on the IMGT CDR numbering scheme. In some embodiments, the anti-CD3 binding domain comprises the HC CDR1 sequence of NNYYMC (SEQ No. 705), the HC CDR2 sequence of CIYTNNIGSTWYASWAKG (SEQ No. 706), the HC CDR3 sequence of GIYSDSSDFIGNL (SEQ No. 707), the LC CDR1 sequence of QASQTIYNYLA (SEQ No. 708), the LC CDR2 sequence of KASTLAS (SEQ No. 709), and the LC CDR3 sequence of QQYNTIINVDRT (SEQ No. 710), wherein the CDR sequences are determined based on the Kabat CDR numbering scheme.

[0007] In some embodiments, the VH of the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS (Sequence No. 703).

[0008] In some embodiments, the VL of the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIK (Sequence No. 704).

[0009] In some embodiments, the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSAIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIK (Sequence No. 701).

[0010] In some embodiments, the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS (Sequence No. 702).

[0011] In some embodiments, the T cell linkage comprises an additional anti-CD3 binding domain. In some embodiments, the T cell linkage further comprises an anti-TRBC1 binding domain. In some embodiments, the T cell linkage further comprises an anti-CD2 binding domain.

[0012] In some embodiments, the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARYYDDHYCLDY (SEQ No. 720), ARYYDDHYSLDY (SEQ No. 721), YYDDHYCLDY (SEQ No. 675), or YYDDHYSLDY (SEQ No. 717). In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR2 sequence of INPSRGYT (SEQ No. 719) or YINPSRGYTNYNQKFKD (SEQ No. 674).

[0013] In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR1 sequence of GYTFTRYT (SEQ No. 718) or RYTMH (SEQ No. 673). In some embodiments, the additional anti-CD3 binding domain comprises a light chain variable region [VL] comprising the light chain complementarity determining region 3 [LC CDR3] sequence of QQWSSNPLT (SEQ No. 678). In some embodiments, the VL of the additional anti-CD3 binding domain comprises the LC CDR1 sequence of SSVSY (SEQ No. 722) or RASSSVSYMN (SEQ No. 676). In some embodiments, additional anti-CD3 binding domains include the HC CDR1 sequence of GYTFTRYT(IMGT) (SEQ No. 718), the HC CDR2 sequence of INPSRGYT(IMGT) (SEQ No. 719), the HC CDR3 sequence of ARYYDDHYCLDY(IMGT) (SEQ No. 720) or ARYYDDHYSLDY (SEQ No. 721), the LC CDR1 sequence of SSVSY (SEQ No. 722), the LC CDR2 sequence of DTS (SEQ No. 723), and the LC CDR3 sequence of QQWSSNPLT (SEQ No. 724).

[0014] In one embodiment, additional anti-CD3 binding domains include the HC CDR1 sequence of RYTMH (SEQ No. 673), the HC CDR2 sequence of YINPSRGYTNYNQKFKD (SEQ No. 674), the HC CDR3 sequence of YYDDHYCLDY (SEQ No. 675) or YYDDHYSLDY (SEQ No. 717), the LC CDR1 sequence of RASSSVSYMN (SEQ No. 676), the LC CDR2 sequence of DTSKVAS (SEQ No. 677), and the LC CDR3 sequence of QQWSSNPLT (SEQ No. 678). In some embodiments, the VH of the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS (Sequence No. 671) or QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 726). In some embodiments, the VL of the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (Sequence No. 672).In some embodiments, the additional anti-CD3 binding domain is QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID No. 727) or It includes a sequence having at least 80% sequence identity with QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS (Sequence No. 728).In some embodiments, the additional anti-CD3 binding domain is QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID No. 725) or It includes a sequence having at least 80% sequence identity with QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 728).

[0015] In one embodiment, the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARSGYYGDSDWYFDV (SEQ No. 731) or SGYYGDSDWYFDV (SEQ No. 607). In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR2 sequence of INPYKGVS (SEQ No. 730) or LINPYKGVSTYNQKFKD (SEQ No. 608). In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR1 sequence of GYSFTGYT (SEQ No. 729) or GYTMN (SEQ No. 605). In some embodiments, the additional anti-CD3 binding domain comprises a light chain variable region [VL] comprising the light chain complementarity determining region 3 [LC CDR3] sequence of QQGNTLPWT (SEQ No. 610). In some embodiments, the VL of the additional anti-CD3 binding domain comprises the LC CDR2 sequence of YTS (SEQ No. 733) or YTSRLES (SEQ No. 609). In some embodiments, the VL of the additional anti-CD3 binding domain comprises the LC CDR1 sequence of QDIRNY (SEQ No. 732) or RASQDIRNYLN (SEQ No. 608). In some embodiments, additional anti-CD3 binding domains include the HC CDR1 sequence of GYSFTGYT (SEQ No. 729), the HC CDR2 sequence of INPYKGVS (SEQ No. 730), the HC CDR3 sequence of ARSGYYGDSDWYFDV (SEQ No. 731), the LC CDR1 sequence of QDIRNY (SEQ No. 732), the LC CDR2 sequence of YTS (SEQ No. 733), and the LC CDR3 sequence of QQGNTLPWT (SEQ No. 610).

[0016] In some embodiments, additional anti-CD3 binding domains include the HC CDR1 sequence of GYTMN (SEQ No. 605), the HC CDR2 sequence of LINPYKGVSTYNQKFKD (SEQ No. 606), the HC CDR3 sequence of SGYYGDSDWYFDV (SEQ No. 607), the LC CDR1 sequence of RASQDIRNYLN (SEQ No. 608), the LC CDR2 sequence of YTSRLES (SEQ No. 609), and the LC CDR3 sequence of QQGNTLPWT (SEQ No. 610).

[0017] In some embodiments, the VH of the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (Sequence No. 603). In some embodiments, the VL of the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (Sequence No. 604). In some embodiments, the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGSAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID No. 602).In some embodiments, the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (Sequence No. 601).

[0018] In some embodiments, the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] containing the heavy chain complementarity determining region 3 [HC CDR3] sequence of AAKIRPYIFKIAGQYDY (SEQ No. 736) or KIRPYIFKIAGQYDY (SEQ No. 618). In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR2 sequence of IVWSDGNT (SEQ No. 735) or AIVWSDGNTYYEDFVKG (SEQ No. 617). In some embodiments, the VH of the additional anti-CD3 binding domain comprises the HC CDR1 sequence of GRTYRGYS (SEQ No. 734) or GYSMA (SEQ No. 616). In some embodiments, the additional anti-CD3 binding domain is a VHH domain. In some embodiments, the additional anti-CD3 binding domain comprises the HC CDR1 sequence of GRTYRGYS (SEQ No. 734), the HC CDR2 sequence of IVWSDGNT (SEQ No. 735), and the HC CDR3 sequence of AAKIRPYIFKIAGQYDY (SEQ No. 736). In some embodiments, the additional anti-CD3 binding domain comprises the HC CDR1 sequence of GYSMA (SEQ No. 616), the HC CDR2 sequence of AIVWSDGNTYYEDFVKG (SEQ No. 617), and the HC CDR3 sequence of KIRPYIFKIAGQYDY (SEQ No. 618). In some embodiments, the VH of the additional anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS (Sequence No. 615).

[0019] In one embodiment, the T cell linkage further comprises an anti-TRBC1 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGAGYNFDGAYRFEDF (SEQ No. 742) or GAGYNFDGAYRFEDF (SEQ No. 739). In some embodiments, the VH of the anti-TRBC1 binding domain comprises the HC CDR2 sequence of INPYNDDI (SEQ No. 741) or FINPYNDDIQSNERFRG (SEQ No. 738). In some embodiments, the VH of the anti-TRBC1 binding domain comprises the HC CDR1 sequence of GYTFTGYV (SEQ No. 740) or GYVMH (SEQ No. 737). In some embodiments, the anti-TRBC1 binding domain comprises a light chain variable region [VL] comprising the sequence of light chain complementarity determining region 3 [LC CDR3] of SQSTHVPYT (SEQ No. 745). In some embodiments, the VL of the anti-TRBC1 binding domain comprises the sequence of LC CDR2 of RVS (SEQ No. 747) or RVSNRFP (SEQ No. 744). In some embodiments, the VL of the anti-TRBC1 binding domain comprises the sequence of LC CDR1 of QRLVHSNGNTY (SEQ No. 746) or RSSQRLVHSNGNTYLH (SEQ No. 743).

[0020] In some embodiments, the anti-TRBC1 binding domain comprises the HC CDR1 sequence of GYTFTGYV (SEQ No. 740), the HC CDR2 sequence of INPYNDDI (SEQ No. 741), the HC CDR3 sequence of ARGAGYNFDGAYRFEDF (SEQ No. 742), the LC CDR1 sequence of QRLVHSNGNTY (SEQ No. 746), the LC CDR2 sequence of RVS (SEQ No. 747), and the LC CDR3 sequence of SQSTHVPYT (SEQ No. 745). In one embodiment, the anti-TRBC1 binding domain comprises the HC CDR1 sequence of GYVMH (SEQ No. 737), the HC CDR2 sequence of FINPYNDDIQSNERFRG (SEQ No. 738), the HC CDR3 sequence of GAGYNFDGAYRFEDF (SEQ No. 739), the LC CDR1 sequence of RSSQRLVHSNGNTYLH (SEQ No. 743), the LC CDR2 sequence of RVSNRFP (SEQ No. 744), and the LC CDR3 sequence of SQSTHVPYT (SEQ No. 745).

[0021] In one embodiment, the anti-TRBC1 binding domain (VH) comprises a sequence having at least 80% sequence identity with the sequence EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (Sequence No. 625). In one embodiment, the VL of the anti-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with the sequence DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (Sequence No. 626). In some embodiments, the anti-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (SEQ ID No. 624).

[0022] In some embodiments, the anti-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (SEQ ID No. 748).

[0023] In one embodiment, the T cell linkage further comprises an anti-CD2 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of AAVRDYVGMPYYSGSAYEY (SEQ No. 749) or VRDYVGMPYYSGSAYEY (SEQ No. 750). In some embodiments, the VH of the anti-CD2 binding domain comprises the HC CDR2 sequence of IRPGTIP (SEQ No. 751) or AIRPGTIPYYSESVKG (SEQ No. 752). In some embodiments, the VH of the anti-CD2 binding domain comprises the HC CDR1 sequence of GLTFSSYG (SEQ No. 753) or SYGMA (SEQ No. 754). In some embodiments, the anti-CD2 binding domain is a VHH domain. In some embodiments, the anti-CD2 binding domain comprises the HC CDR1 sequence of GLTFSSYG (SEQ No. 753), the HC CDR2 sequence of IRPGTIP (SEQ No. 751), and the HC CDR3 sequence of AAVRDYVGMPYYSGSAYEY (SEQ No. 749).

[0024] In some embodiments, the anti-CD2 binding domain comprises the HC CDR1 sequence of SYGMA (SEQ No. 754), the HC CDR2 sequence of AIRPGTIPYYSESVKG (SEQ No. 752), and the HC CDR3 sequence of VRDYVGMPYYSGSAYEY (SEQ No. 750).

[0025] In some embodiments, the VH of the anti-CD2 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLVESGGGLVQAGGSLRLSCAASGLTFSSYGMAWFRRALGREREFVGAIRPGTIPYYSESVKGRFTVSKDNAKNTVSLQMNSLKPEDTAVYYCAAVRDYVGMPYYSGSAYEYWGQGTQVTVSS (Sequence No. 623).

[0026] In some embodiments, one or more binding domains of the T cell linkage are linked to the first polypeptide or the second polypeptide.

[0027] In some embodiments, the first polypeptide further comprises a dimeric domain fused to a TRAV domain, and the second polypeptide comprises a dimeric domain fused to a TRBV domain.

[0028] In some embodiments, the first polypeptide comprises a dimerization domain, a peptide linker, and a TRAV domain of the first polypeptide in the direction from the N-terminus to the C-terminus, and the second polypeptide comprises a dimerization domain, a peptide linker, and a TRBV domain of the second polypeptide in the direction from the N-terminus to the C-terminus.

[0029] In some embodiments, the dimerization domain of the first polypeptide comprises a TCR alpha constant (TRAC) domain or a part thereof, and the dimerization domain of the second polypeptide comprises a TCR beta constant (TRBC) domain or a part thereof.

[0030] In some embodiments, the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by one or more disulfide bridges.

[0031] In some embodiments, the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by a single disulfide bridge.

[0032] In some embodiments, the first polypeptide comprises a T cell linkage.

[0033] In some embodiments, the T cell linkage is connected to the dimerization domain of the first polypeptide.

[0034] In some embodiments, the first polypeptide comprises a T cell linker, a dimerization domain, and a TRAV domain from the N-terminus to the C-terminus.

[0035] In some embodiments, the first polypeptide comprises a TRAV domain and a T cell linkage from the N-terminus to the C-terminus.

[0036] In some embodiments, the second polypeptide comprises a T cell linkage.

[0037] In some embodiments, the second polypeptide comprises a T cell linker, a dimerization domain, and a TRBV domain from the N-terminus to the C-terminus.

[0038] In some embodiments, the second polypeptide comprises a TRBV domain and a T cell linkage from the N-terminus to the C-terminus.

[0039] In some embodiments, the T cell linker comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises scFv, a dimerization domain, and a TRAV domain from the N-terminus to the C-terminus.

[0040] In some embodiments, the T cell linker comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerizing domain, a TRAV domain, and scFv from the N-terminus to the C-terminus.

[0041] In some embodiments, the T cell linker comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises scFv, a dimerization domain, and a TRBV domain from the N-terminus to the C-terminus.

[0042] In some embodiments, the T cell linker comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerizing domain, a TRBV domain, and scFv from the N-terminus to the C-terminus.

[0043] In some embodiments, the T cell linker comprises VHH that binds to a receptor expressed by the T cell, and the polypeptide comprises VHH, a dimerization domain, and a TRAV domain from the N-terminus to the C-terminus.

[0044] In some embodiments, the T cell linker comprises VHH that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerizing domain, a TRAV domain, and VHH from the N-terminus to the C-terminus.

[0045] In some embodiments, the T cell linker comprises VHH that binds to a receptor expressed by the T cell, and the polypeptide comprises VHH, a dimerization domain, and a TRBV domain from the N-terminus to the C-terminus.

[0046] In some embodiments, the T cell linker comprises VHH that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerizing domain, a TRBV domain, and VHH from the N-terminus to the C-terminus.

[0047] In some embodiments, the T cell linkage comprises a first VHH domain and a second VHH domain configured to bind to the same receptor expressed by the T cell.

[0048] In some embodiments, the T cell linkage comprises a first VHH domain that binds to a first receptor expressed by the T cell and a second VHH domain that binds to a second receptor expressed by the T cell.

[0049] In some embodiments, the T cell linker comprises scFv that binds to a first T cell receptor and VHH that binds to a second T cell receptor, wherein scFv and VHH are connected by a peptide linker.

[0050] In some embodiments, the first polypeptide comprises a first binding domain of a T cell linkage that binds to an extracellular domain of a first receptor expressed by a T cell, and the second polypeptide comprises a second binding domain of a T cell linkage that binds to an extracellular domain of a second receptor expressed by a T cell.

[0051] In some embodiments, the first binding domain of the T cell linkage is a first VHH that is fused to the first polypeptide and binds to CD3, and the second binding domain of the T cell linkage is a second VHH that is fused to the second polypeptide and binds to CD2.

[0052] In some embodiments, the first binding domain of the T cell linkage is a first VHH that binds to CD2, and the second binding domain of the T cell linkage is a second VHH that binds to CD3.

[0053] In some embodiments, the T cell linker is fused with the first or second polypeptide through a peptide linker.

[0054] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multiple specific molecule is provided herein, wherein the multiple specific molecule comprises (i) an MHC-peptide complex linker comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell linker comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex linker and the T cell linker form a continuous single polypeptide chain comprising one or more binding domains of the T cell linker, a TRBV domain, a TRBC domain, a TRAV domain, and a TRAC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the multiple specific molecule further comprises a linker connecting the TRBC domain and the TRAV domain. In some embodiments, the linker is a non-cleavage linker. In some embodiments, the linker comprises a sequence of 20 to 38 amino acids. In some embodiments, the linker is a structure-directed flexible linker. In some embodiments, the linker comprises Gly, Ser, Ala, and / or Glu residues.

[0055] In some embodiments, the multiple-specific molecule comprises the sequence of SEQ ID NO. 862 excluding the signal peptide of SEQ ID NO. 668. In some embodiments, the multiple-specific molecule comprises one or more sequences of SEQ ID NO. 604, SEQ ID NO. 682, SEQ ID NO. 603, (SEQ ID NO. 683)(GGGGS), SEQ ID NO. 620, SEQ ID NO. 863, SEQ ID NO. 864, SEQ ID NO. 619, SEQ ID NO. 865, and SEQ ID NO. 866. In some embodiments, the multiple-specific molecule comprises the sequence of SEQ ID NO. 862 excluding the signal peptide of SEQ ID NO. 668.

[0056] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex linker comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell linker comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex linker and the T cell linker form a continuous single polypeptide chain comprising one or more binding domains of the T cell linker, a TRAV domain, a TRAC domain, a TRBV domain, and a TRBC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the multispecific molecule further comprises a linker connecting the TRAC domain and the TRBV domain. In some embodiments, the linker comprises 20 to 38 amino acids including Gly, Ser, Ala, and / or Glu amino acids. In some embodiments, one or more binding domains of the T cell linker comprise a single chain variable fragment [scFv]. In some embodiments, the scFv comprises a light chain variable domain (VL) followed by a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus. In some embodiments, the scFv comprises a light chain variable domain (VL) followed by a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus.

[0057] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex linker comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell linker comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex linker and the T cell linker form a continuous single polypeptide chain comprising a TRAV domain, a TRAC domain, one or more binding domains of the T cell linker, a TRBV domain, and a TRBC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the multispecific molecule further comprises a linker connecting the TRAC domain and one or more binding domains of the T cell linker. In some embodiments, one or more binding domains of the T cell linker comprise a short-chain variable fragment [scFv]. In some embodiments, scFv comprises a light chain variable domain (VL) followed by a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus. In some embodiments, scFv comprises a light chain variable domain (VL) followed by a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus.

[0058] In some embodiments, the multispecific molecule comprises one or more sequences selected from the group consisting of SEQ ID NO. 619, SEQ ID NO. 868, SEQ ID NO. 869, SEQ ID NO. 604, SEQ ID NO. 870, SEQ ID NO. 603, SEQ ID NO. 620, SEQ ID NO. 871, and SEQ ID NO. 866. In some embodiments, the multispecific molecule comprises the sequence of SEQ ID NO. 867 excluding the signal peptide of SEQ ID NO. 667.

[0059] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRBV domain, the TRBC domain, one or more binding domains of the T cell conjugate, the TRAV domain, and the TRAC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the multispecific molecule further comprises a linker connecting the TRBC domain and one or more binding domains of the T cell linker, wherein the linker comprises 20 to 37 amino acids and comprises Gly, Ser, Ala and / or Glu amino acids.

[0060] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule is provided herein, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the scFv, TRBV domain and TRAV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the recombinant nucleic acid further comprises a TCR alpha constant (TRAC) domain. In some embodiments, the recombinant nucleic acid further comprises a TCR beta constant (TRBC) domain. In some embodiments, the T cell linkage forms a continuous single polypeptide chain comprising scFv, TRBV domain, TRBC domain, TRAV domain, and TRAC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the recombinant nucleic acid, furthermore, the T cell linkage comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. In some embodiments, the recombinant nucleic acid, furthermore, the T cell linkage comprises a VH domain and a VL domain in the direction from the N-terminus to the C-terminus.

[0061] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule is provided herein, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the scFv, TRAV domain and TRBV domain in the direction from the N-terminus to the C-terminus.

[0062] In some embodiments, the recombinant nucleic acid further comprises a TCR alpha constant (TRAC) domain. In some embodiments, the recombinant nucleic acid further comprises a TCR beta constant (TRBC) domain. In some embodiments, the T cell linkage forms a continuous single polypeptide chain comprising scFv, a TRBV domain, a TRBC domain, a TRAV domain, and a TRAC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VH domain and a VL domain in the direction from the N-terminus to the C-terminus.

[0063] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule is provided herein, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRAV domain, scFv and TRBV domains in the direction from the N-terminus to the C-terminus. In some embodiments, the recombinant nucleic acid further comprises a TCR alpha constant (TRAC) domain. In some embodiments, the recombinant nucleic acid further comprises a TCR beta constant (TRBC) domain.

[0064] In some embodiments, the T cell linkage forms a continuous single polypeptide chain comprising scFv, a TRAV domain, a TRAC domain, a TRBV domain, and a TRBC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VH domain and a VL domain in the direction from the N-terminus to the C-terminus.

[0065] In one embodiment, a recombinant nucleic acid comprising a sequence encoding a multispecific molecule is provided herein, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRBV domain, scFv and TRAV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the recombinant nucleic acid further comprises a TCR alpha constant (TRAC) domain. In some embodiments, the recombinant nucleic acid further comprises a TCR beta constant (TRBC) domain. In some embodiments, the T cell linkage forms a continuous single polypeptide chain comprising scFv, a TRAV domain, a TRAC domain, a TRBV domain, and a TRBC domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises a VH domain and a VL domain in the direction from the N-terminus to the C-terminus.

[0066] In some embodiments, the recombinant nucleic acid further encodes a peptide linker, wherein the linker comprises an amino acid sequence having 90% sequence identity with the sequence of SEQ ID NO. 864, or 872, or 873. In some embodiments, the TRAC domain comprises a wild-type rat TRAC sequence or a wild-type human TRAC sequence. In some embodiments, the TRAC domain does not contain a mutation. In some embodiments, the TRAC domain comprises a mutation compared to the rat TRAC sequence or the wild-type human TRAC sequence. In some embodiments, the mutation is a stability-enhancing mutation. In some embodiments, the mutation is selected from the group consisting of S139F, T150I, and A190T, wherein the residue positions are numbered based on the Kavat numbering scheme.

[0067] In some embodiments, the TRBC domain comprises a wild-type rat TRBC sequence or a wild-type human TRBC sequence. In some embodiments, the TRBC domain does not contain mutations compared to the wild-type sequence. In some embodiments, the TRBC domain comprises mutations compared to the rat TRBC sequence or a wild-type human TRBC sequence. In some embodiments, the mutation is a stability-enhancing mutation. In some embodiments, the mutation is selected from the group consisting of E134K, H139R, D155P, and S170D, wherein the residue positions are numbered based on the Kavat numbering scheme.

[0068] In one embodiment, a composition comprising a multispecific molecule encoded by the sequence of a recombinant nucleic acid of any one of the aforementioned embodiments is provided herein.

[0069] In some embodiments, multispecific molecules are isolated or purified.

[0070] A pharmaceutical composition comprising any one of the aforementioned embodiments is provided herein.

[0071] In one embodiment, a method for treating a subject requiring cancer treatment is provided in the present invention, comprising the step of administering a therapeutically effective amount of the aforementioned drug.

[0072] In one embodiment, a method for producing a multispecific molecule is provided herein, wherein the multispecific molecule is (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the multispecific molecule is an engineered T cell receptor (TCR) construct comprising a post-translational modification, and the method comprises the step of producing a multispecific molecule comprising a mammalian post-translational modification signature by expressing the multispecific molecule from a recombinant nucleic acid comprising a sequence encoding the multispecific molecule in a mammalian cell. In one embodiment, the method further comprises the step of isolating or purifying the multispecific molecule comprising the mammalian post-translational modification signature. In some embodiments, the cell secretes a first polypeptide and a second polypeptide encoded by the sequence of the recombinant nucleic acid of the aforementioned composition.

[0073] A mammalian cell comprising a recombinant nucleic acid of any one of the aforementioned embodiments is provided herein.

[0074] Reference by reference

[0075] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as having been referenced by reference. To the extent that the publications and patents or patent applications referenced by reference conflict with the disclosures contained herein, this specification is intended to replace and / or supersede any such conflicting materials. Brief explanation of the drawing

[0076] The main features of the present invention are specifically described in the appended claims. The features and advantages of the present invention will be better understood by referring to the following specific details for carrying out the invention, which describe exemplary embodiments utilizing the principles of the present invention, and the following attached drawings (also referred to as "drawings" herein). Fig. 1a is a schematic diagram illustrating an overview of the work undertaken as a word diagram. Fig. 1b Figure 1 shows a schematic diagram of the plasmid, expression, and tetramer binding assay. SP, signal peptide. Flow cytometry scan is for illustrative purposes only and is not data. Fig. 1c represents the expression, enrichment, and tetramer analysis of the TCR library. Fig. 1d This represents a schematic analysis of tetramers and monomers. Fig. 2 This is a schematic diagram showing a general overview of various experimental bispecific sTCR scaffold designs. The central schematic diagram shows the p-MHC complex presented by the tumor cell at the top, and the multispecific linker (soluble TCR) on the left containing TRAV and TRBV domains (MHC-peptide complex linker) for binding with p-MHC, which is schematically indicated by a double arrow. The sTCR TRAC and TRBC domains form a scaffold, which is interconnected by disulfide bonds at positions 1 and 2 as shown in the schematic diagram. The T cell linker contains an anti-CD3 scFv capable of binding to CD3, a surface molecule on the T cell, and is attached to the variable domain via a short peptide linker. Top left, a bispecific sTCR in which a CD3-specific scFv is attached to a TRBV as a T cell linkage, as described above. Bottom left, similar to top left, but CD3-specific scFv is attached to TRAV as a T cell connector. Top right, a bispecific sTCR with a CD3-specific scFv attached to a TRBC as a T cell linkage, as described above. Bottom right, a bispecific sTCR in which a CD3-specific scFv is attached to a TRAC as a T cell linkage, as described above. Fig. 3It features schematic diagrams of 10 different sTCR scaffold structures that were constructed and tested. Scaffold 2, in which TRAV-TRBV forms a p-MHC linkage, is the basic structure for evaluating stability with the purification tag 10X HIS and the affinity receptor molecule Biotin Acceptor Protein (BAP) tag. Positions 1 and 2 represent interchain disulfide linkages. Scaffold 5 illustrates two sets of TCRs linked to an IgG domain. Each sTCR comprises two chains—an alpha chain containing TRAV-TRAC; and a beta chain containing TRBV and TRBC. Scaffold 6 contains TRAV and TRAC stabilized by two scaffold polypeptides having a single interchain disulfide linkage. The two scaffolding polypeptides are immunoglobulin Fv domains arranged within the Fv clasp. Scaffold 14 contains an sTCR fused to an immunoglobulin (Ig) Fc structure. The Fc portion is engineered to enhance stability in the binding form and includes a knob-and-hole configuration. Scaffold 19 contains a tagged sTCR molecule containing a TRAV-TRAC-TRBV-TRBC tetramer structure fused to an anti-CD3 scFv via a short linker peptide. Scaffold 20 has a basic structural similarity to Scaffold 6 and also contains a scFv attached to TRBV via a short linker peptide. Scaffold 21 contains a basic structure similar to Scaffold 14 and further contains a scFv attached to TRBV via a short linker peptide. Scaffold 36 contains the basic sTCR configuration of Scaffold 2 and is connected in series to two scFvs attached to TRBV via a peptide linker. Scaffold 37 contains the basic tetrameric sTCR configuration, where TRAC and TRBC are tagged with BAP, sorbase A, and 10X HIS tags.Scaffold 43 contains a basic tetrameric sTCR structure linked to an Ig Fc domain. The arrow indicates the protease target site used to cleave the sTCR at the Ig domain. Fig. 4 The top panel shows a schematic representation of the experimental protocol including the transfection and purification of the product. Representative gel filtration results for product purity verification are shown in the upper right. The bottom panel shows the workflow and timeline of tumor cell death by effector T cells in an in vitro assay. Fig. 5a and 5b Figure 5 shows exemplary representative data for the generation and purification of scaffold 2, and shows gel electrophoresis of purified sTCR in cell culture medium (Figure 5a) and a single protein peak at the intermediate time point of elution (Figure 5b), respectively. Fig. 6 Figure [] shows a schematic layout of the workflow for target cell cytotoxicity analysis to test the functional efficacy of soluble TCR constructs. T cells were obtained from peripheral blood samples from healthy donors. A375 cells transfected with polynucleotides encoding peptides and MHC and expressing polypeptides were used as target cells. Purified sTCR products were added to the cultures at indicated concentrations to analyze T cell activation and cytotoxicity, which were measured by the loss of GFP-positive target cells compared to untreated controls. Fig. 7a This represents the structural design of a scaffold 19 (Ros9a scaffold 19) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 7b Figure 6 shows cytotoxicity data using the method described in the workflow layout. Cytotoxicity was measured by the area containing the total number of GFP-positive cells (=total green objects), which is a measure of viable cells. Therefore, higher cytotoxicity is correlated with lower green cells. Fig. 8a This represents the structural design of a scaffold 20b (Ros9a scaffold 20b) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 8b represents cytotoxicity data. Fig. 9a This represents the structural design of a scaffold 21 (Ros9a scaffold 21) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 9b represents cytotoxicity data. Fig. 10a This represents the structural design of a scaffold 19 (Ros9d scaffold 19) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 10b represents cytotoxicity data. Fig. 11a This represents the structural design of a scaffold 21 (Ros9d scaffold 21) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 11b represents cytotoxicity data. Fig. 12a This represents the structural design of a scaffold 19 (Ros10 scaffold 19) construct having TCR alpha and beta variable domains capable of binding to a mutant RAS epitope bound to an MHC encoded by HLA-C:08:02. Fig. 12b represents cytotoxicity data. Fig. 13Figure [] shows a schematic layout of the workflow for target cell cytotoxicity analysis to test the functional efficacy of soluble TCR constructs. T cells were obtained from peripheral blood samples from healthy donors. Target cells were tumor cells of the HPAC cell line endogenously expressing the mutated Ras epitope and HLA-C:08:02. Purified sTCR products were added to the cultures at the indicated concentrations to analyze T cell activation and cytotoxicity, and were measured using target cells positive for the apoptosis markers Annexin V and Caspace 3 / 7 compared to untreated controls. Fig. 14a and 14b Figure 14 shows imaging (Figure 14a) and quantitative data (Figure 14b) obtained from a comparative imaging analysis between Ros9a scaffold 19 and Ros 10 scaffold 19 treatment groups, showing annexin-positive cells (orange) and caspace 3 / 7-positive cells (green) at different peptide concentrations. Fig. 14c represents imaging showing only Annexin V-positive cells. Figures 15a–15b It represents the cytotoxicity analysis of donor T cells that have rested for a shorter period, indicating an increase in target cell cytotoxicity. Fig. 16 Schematic diagrams of various multispecific linker scaffolds having additional T cell linkers including anti-CD3 scFv and CD8a ectodomains are shown. Fig. 17 Schematic diagrams of various multispecific linkage scaffolds having anti-CD3 or anti-CD2 VHH domains are illustrated. The amino acid sequences of scaffolds 53, 55, and 57 are disclosed elsewhere in this specification, for example, in Table 10. Figs. 18a–18b This illustrates schematic diagrams of various multispecific linker scaffolds designed for testing. They are fused to the basic tetrameric sTCR structure via short peptide linkers. Scaffolds 45–48 contain basic tetrameric structures in which TRAV and TRBV are linked to the immunoglobulin constant region. Both chains contain heteromeric Ig. Scaffolds 46 and 48 contain a disulfide bond located at position 2, which is omitted in scaffolds 46 and 48. Scaffolds 49–51 are designed to contain anti-CD3 scFv attached to TRAV or TRBV. In scaffold 50, TRAV and TRBV are initially linked but contain a cleavage sequence (F2A). Scaffold 49 contains two polypeptides, the first polypeptide having the orientation NH2-aCD3scFv_VH-TRAV-(linker+tag)-COOH, where aCD3scFv represents anti-CD3scFv, and the second polypeptide having the orientation NH2-TRBV-aCD3scFv_VL-(linker+tag)-COOH. Scaffold 50 is produced as a single polypeptide NH2-aCD3scFv_VH-TRAV-(F2A)-TRBV--aCD3scFv_VL-(linker+tag)-COOH having a cleavable sequence (F2A) between them. Scaffold 51 is a single polypeptide NH2-aCD3scFv_VH-TRAV-(linker)-TRBV--aCD3scFv_VL-(linker+tag)-COOH having a linker connecting two parallel chains. Scaffold 52 is a single polypeptide NH2-TRAV--aCD3scFv_VH--(linker)-aCD3scFv_VL-TRBV-(linker+tag)-COOH having a linker connecting two parallel chains. Fig. 19Schematic diagrams of various multispecific linker scaffolds designed for the experiment are shown. Scaffolds 103, 106, and 137, and 108 and 138 contain basic tetrameric sTCR structures. Among these, scaffold 138 has an additional domain that binds to TRBC1 in addition to anti-CD3VHH. Scaffolds 139 and 142 are newer experimental designs that do not contain basic tetrameric sTCR structures. In scaffold 139, TRAV and TRBV are each linked to anti-CD3VHH via a short peptide linker. Scaffold 142 contains an anti-CD3VHH domain attached to one of TRAV and TRBV and an anti-TRBC1 scFv attached to the other of TRAV and TRBV via a short peptide linker. Fig. 20 This shows schematic diagrams of various multispecific connector scaffolds designed for testing. Fig. 21 Figure 1 illustrates a schematic diagram of a multispecific linker scaffold having one or more VHH T cell linkages. In these scaffold designs, the linker between the VHH and the TCR is an extended linker. Fig. 22 Figure 22 shows a schematic layout of the workflow for target cell cytotoxicity analysis to test the functional efficacy of soluble TCR constructs built based on Scaffold 22. T cells were obtained from peripheral blood samples from healthy donors. A375 cells transfected with polynucleotides encoding peptides and MHC and expressing polypeptides were used as target cells. Purified sTCR products were added to cultures at indicated concentrations to analyze T cell activation and cytotoxicity, which were measured by the loss of GFP-positive target cells compared to untreated controls. Fig. 23 Figure 2 shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9a scaffold 22. The anti-CD3 arm is based on the TR66-VHVL structure. Fig. 24This shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9a scaffold 22. The anti-CD3 arm is based on the TR66-VLVH structure. Fig. 25 This shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9a scaffold 22. The anti-CD3 arm is based on the 210112-VHVL construct. Fig. 26 Figure 2 shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9a scaffold 22. The anti-CD3 arm is based on the 210112-VLVH construct. Fig. 27 Figure 2 shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9d scaffold 22. The anti-CD3 arm is based on the TR66-VHVL structure. Fig. 28 Figure 2 shows the structure and analysis results for testing the cytotoxic potential of the construct Ros9d scaffold 22. The anti-CD3 arm is based on the 210112-VHVL structure. Fig. 29 Figure 13 shows a schematic diagram of the workflow for analyzing the cytotoxic potential of sTCR constructs against tumor cell lines with endogenous KRAS G12D, which is an analysis similar to that shown in Figure 13. Fig. 30 This shows imaging of targeted HPAC tumor cells treated with the Ros9a scaffold 22 sTCR construct in the presence of T cells, showing Annexin-positive cells (orange) and Caspace 3 / 7-positive targeted apoptotic cells (green) at different peptide concentrations. Fig. 31 Figure 30 shows the quantitative evaluation results obtained by interpreting the cytotoxicity analysis. Fig. 32 This represents the results of the imaging analysis when target cells were stained with Annexin V alone in the analysis. Fig. 33 It shows an analysis similar to that shown in Figures 15a and 15b. The donor T cells rested for a shorter period than in the protocols of Figures 30–32, which enhances target cell cytotoxicity. Fig. 34Figure 33 illustrates a graph showing the quantitative evaluation of the analysis results. Fig. 35a This shows a graphical representation of the structure of a single-strand soluble bispecific TCR (BiTE) construct designed to enable mRNA formatting and delivery and to simplify development pipelines. In this design, TRBC is linked to the TRAV fragment to form a single polypeptide chain containing both the TCR alpha and beta chains, and another linker links the anti-CD3 scFv. The general framework of the structure is based on the primary scaffold structure described herein. In some cases, as shown in the figure, the linker between TRBC and TRAV is the GS linker, (G4S)7. Top left : Scaffold Name: Ros9a_SC107βα_antiCD3. Graphic representation of a construct having one or more point mutations in the constant (TRAC and TRBC) regions that enhance polypeptide stability. Additional mutations are designed to remove glycosylation sites. Top right : Scaffold name Ros9a_SC107wβα_anti-CD3. As shown in the top-left model, this is a graphical representation of the structure free of multiple mutations in the constant domain for the purpose of enhancing stability. An alphafold 3D model of the protein structure illustrating the spatial orientation of the polypeptide domains is shown at the bottom. Nt, N-terminus; Ct, C-terminus. The design leaves the anti-CD3 scFv N-terminus in a free state. The TCR has an orientation from the N-terminus to the C-terminus of its alpha and beta chains such that the beta chain is positioned toward the N-terminus of the alpha chain. Fig. 35b represents a schematic diagram of the short-chain availability TCR design, and Top left Scaffold name with stabilizing mutation in the invariant domain: Ros9a_SC111αβ_antiCD3; Top rightThe scaffold name that does not contain stability-enhancing invariant domain mutations is Ros9a_SC111wαβ_antiCD3. The bottom alphafold model image, in which a linker [e.g., (G4S)4] connects the N-terminus of the anti-CD3 scFv to the C-terminus of TRBC to form a short chain containing an internal anti-CD3 scFv, approaches the same position as the construct shown in Fig. 35a but is not restricted by the linker. In this case, the alpha chain is the N-terminus of the beta chain. Fig. 36 (Top) shows the design of a soluble BiTE (e.g., mRNA encoding a polypeptide) having a domain layout oriented from the N-terminus to the C-terminus, an N-terminal anti-CD3 scFv containing variable light chain [VL] and variable heavy chain [VH] domains followed by a linker connecting the scFv to a TCR beta chain containing a TRBV fused to TRBC, which is then connected to a TCR alpha chain containing a TRAV fused to TRAC by a second, longer linker. An exemplary construct, Ros9a_SC107wβα_anti-CD3. The exemplary construct Ros9a_SC107wβα_anti-CD3 may have the sequence of SEQ ID NO. 862. SEQ ID NO. 862 lacks the stability-enhancing mutations S>F, T>I, and A>T in the TCR alpha contact (TRAC) region as shown in Table 12. Fig. 36 (Bottom) shows a schematic diagram of the polypeptide structure. The dotted lines represent the linkers that generate the short chains. Fig. 37(Top) represents a soluble BiTE having a domain layout oriented from the N-terminus to the C-terminus, e.g., mRNA encoding a specific domain, wherein an N-terminal TCR alpha chain containing TRAV fused to TRAC is followed by a linker linking the TCR alpha with an anti-CD3 scFv containing variable light chain [VL] and variable heavy chain [VH] domains, followed by a linker linking the scFv with a TCR beta chain containing TRBV fused to TRBC. Exemplary construct Ros9a_SC111wαβ_antiCD3, Fig. 37 (Bottom) shows the schematic structure of the polypeptide. Fig. 38 (Left) shows the design of a single polypeptide chain sTCR having a peptide linker connecting the TCR alpha and beta chains, and an exemplary alphafold 3D modeling structure. Different linkers, H1 (linker 1), H2 (linker 2), or GS linker (35GS), were tested, and polypeptides with alpha-beta as well as beta-alpha orientations in the direction from the N-terminus to the C-terminus were tested. Cell surface expression and peptide binding analysis were performed for each construct. The results are shown in the graph on the right. Fig. 39a Figure [] represents a schematic protocol for analyzing the cytotoxic potential of BiTE sTCRs. A single polypeptide chain containing TCR alpha and beta domains and anti-CD3 scFv were co-cultured with T cells (effector) and target cancer cell lines (e.g., A375 cells), and apoptosis of the target cells was measured. PBMCs from three healthy donors were isolated and rested in inactivated medium for one day prior to analysis. In an alternative analysis setup, shorter resting periods (3–5 hours) were also tested. In an alternative set, T cells were isolated from PBMCs by negative selection ('uncontacted' purified T cells) and the analysis was performed. (E: T, effector cell to target cell ratio). Fig. 39bThis represents data illustrating a comparison of two sTCR BiTE constructs, one having Fc fusion in the TCR invariant domain (top figure), and the other not having Fc fusion (bottom figure). E, CD3 + (Effective) Cells were proliferated and rested overnight, and T, (Target cancer cell line) A375 was transduced to express KRAS G12D+HLA C*08:02. BiTE is a Ros9a sTCR-based construct added to the culture at concentrations ranging from 100 nM to 0.4 nM. The data indicate substantially lower cytotoxic potential of the Fc fusion construct. Fig. 40Data comparing the efficacy of the indicated single-chain sTCR construct versus the double-chain sTCR construct in promoting T cell-mediated target cell cytotoxicity. The top left shows T cell cytotoxicity data using the sTCR construct Ros9aS74_antiCD3, which contains two distinct TCR chains (alpha chain and beta chain), and is presented as a control to evaluate the cytotoxic potential of the single-chain construct designed herein. For example, the top right shows cytotoxicity data using the single-chain sTCR construct Ros9a_SC107_antiCD3, and the inset shows a graphic view of the Ros9a_SC107_antiCD3 polypeptide structure. The data indicate that Ros9a_SC107_antiCD3 has equivalent efficacy to Ros9aS74_antiCD3. Using a similar analysis, the cytotoxic potential of the short-chain sTCR construct Ros9a_SC111_antiCD3 (bottom left) was evaluated, which had a slightly lower potential to induce cytotoxicity than Ros9aS74_antiCD3. However, higher doses showed a significant improvement in cytotoxicity induction, and the bottom right shows the data for the sTCR Ros9a_SC113_antiCD3 construct. The effect was dose-dependent, which is indicated by the index in the right column. Target cells express green fluorescent protein, and its decrease was a rapid and easy measure of T cell-mediated cytotoxicity. Fig. 41 Figure 40 shows imaging data of target cell depletion in the presence of the sTCR BiTE construct in the analysis. Specific details for implementing the invention

[0077] All terms are intended to be understood as they are understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this disclosure pertains.

[0078] Section titles used herein are for organizational purposes only and should not be interpreted as limiting the subjects described.

[0079] While various features of the present disclosure may be described in the context of a single embodiment, features may also be provided separately or in any suitable combination. Conversely, for clarity, the present disclosure may be described herein in the context of a separate embodiment, but the present disclosure may also be implemented as a single embodiment.

[0080] The words 'comprising' (and any form of inclusion, e.g., 'comprise' and 'comprises'), 'having' (any form of having, e.g., 'have' and 'have'), 'including' (and any form of inclusion, e.g., 'includes' and 'include'), or 'containing' (any form of inclusion, e.g., 'containing' and 'containing') as used in this specification and claim(s) may be inclusive or open and do not exclude additional unmentioned elements or method steps. Any embodiment discussed in this specification may be implemented in connection with any method or composition of this disclosure, and vice versa. Additionally, the composition of this disclosure may be used to achieve the method of this disclosure.

[0081] When referring to measurable values ​​such as parameters, quantities, temporary durations, etc., the terms “about” or “approximately” as used herein are intended to encompass the specified values ​​and variations of + / -20% or less, + / -10% or less, + / -5% or less, or + / -1% or less from the specified values, insofar as such variation is appropriate to be performed in this disclosure. It should be understood that the values ​​themselves referred to by the modifiers “about” or “approximately” are also specifically disclosed.

[0082] "Agents" may include any type of molecule and include, but are not limited to, antibodies, peptides, proteins, polynucleotides (e.g., oligonucleotides, RNA, or DNA), small molecules, derivatives thereof, and analogs thereof.

[0083] "Biological sample" may be any tissue, cell, body fluid, or other material derived from an organism. As used herein, the term "sample" may include biological samples such as any tissue, cell, body fluid, or other material derived from an organism. The term "biological sample" may encompass various types of samples obtained from an organism and may be used for diagnostic or monitoring analysis. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as biopsy samples or tissue cultures, or cells and their progeny derived therefrom. The term encompasses samples that have been manipulated in any way after their acquisition, such as by treatment with reagents, solubilization, or concentration for specific components. The term encompasses clinical samples and also includes cells in cell cultures, cell supernatants, cell lysates, serum, plasma, biological body fluids, and tissue samples.

[0084] "Specifically binds" may refer to a state in which a compound (e.g., a peptide) recognizes and binds to a molecule (e.g., a peptide or polypeptide) but does not substantially recognize or bind to other molecules within a sample, e.g., a biological sample; that is, a state in which the compound exhibits selective binding to the molecule. The "binder" described herein includes, but is not limited to, a protein, polypeptide, or fragment thereof that exhibits specific binding to a homologous molecule. The binder may refer to an antigen-binding domain, such as a first binding domain of a bispecific or trispecific linker or a second antigen-binding domain of a bispecific or trispecific linker. In some cases, the binder may be any biomolecule or fragment thereof, such as a peptide or conjugated peptide or a ligand that exhibits specific binding to a portion of an exemplary linker by specifically binding to a receptor on a cell.

[0085] In some cases, 'immune response' may include T cell-mediated and / or B cell-mediated immune responses influenced by the regulation of T cell co-stimulation. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity. Additionally, the term immune response includes immune responses indirectly influenced by T cell activation, e.g., antibody production (humoral response) and activation of cytokine-responsive cells.

[0086] 'Functional derivatives' of a natural sequence polypeptide may be compounds having qualitative biological properties common to the natural sequence polypeptide. 'Functional derivatives' include, but are not limited to, fragments of the natural sequence and derivatives of the natural sequence polypeptide and its fragments, provided that they possess biological activity common to the corresponding natural sequence polypeptide. The term 'derivative' may encompass both amino acid sequence variants of the polypeptide and covalent modifications thereof.

[0087] In some cases, the term "binding material" may refer to a binding domain within a recombinant polypeptide produced by design as described herein. The binding domain sequence may be derived from a naturally occurring protein and manipulated into a recombinant polypeptide by recombinant DNA techniques. The binding domain may be selected based on its binding specificity to its target or cognate element. The binding domain may be derived from an antibody that binds to a target antigen or cognate molecule, or from a protein that is a functional fragment thereof. Desired characteristics of the binding domain may be high specificity to its target, high binding affinity, or both. The binding domain may be referred to as a linker in that it binds to the target molecule to which it binds. Thus, in some cases, the target for the binding material may refer to the protein, polypeptide, or biomolecule to which the binding domain binds. The target may be located in a cell different from the cell where the binding domain may be located. In some embodiments, the cell containing the target (e.g., the protein or biomolecule to which the binding material binds) may be referred to as the target cell. In some cases, the binding substance may not be located on the cell, for example, or may be acellular, and in such cases may be referred to as a soluble binding substance. The binding substance may be an antibody, or any fragment thereof, scFV, sdAb, VHH.

[0088] Often, as used herein, the term “antibody” may refer to an antibody, scFv, VHH, single domain antibody (sdAb), or a protein or polypeptide comprising an inactive antigen-binding domain, wherein the antigen-binding ability is designed to be blocked or inactive, for example, by binding to a cleavable antigen-binding polypeptide, until an active step is performed to convert the pro-antibody into its active form. In some embodiments, the active step comprises cleaving a substance that blocks the antigen-binding domain with a protease.

[0089] In many cases, the term 'affinity' can refer to the characteristic of a molecule's ability to bind to another molecule or a ligand with chemical specificity. Generally, molecule a, which has a higher affinity for binding to molecule b than to molecule c, will bind more strongly to b and c. Chemical specificity is the ability of a protein's binding site to bind to a specific ligand. The fewer ligands a protein can bind to, the greater its specificity. Specificity represents the binding strength between a specific protein and a ligand. This relationship can be explained based on the dissociation constant (KD) by the first scFv, which is specific to cell surface components, and characterizes the balance between the bound and unbound states of the protein-ligand system.

[0090] In many cases, the term 'antigen-presenting cell(s)' or its abbreviation 'APC(s)' may refer to cells or cells capable of endocellular adsorption, processing, and presentation of antigens. The term includes specialized antigen-presenting cells, e.g., B lymphocytes, monocytes, dendritic cells (DCs), and Langerhans cells, as well as other antigen-presenting cells, such as keratinocytes, endothelial cells, glial cells, fibroblasts, and oligodendrocytes. The term 'antigen presentation' may mean that an antigen appears on the cell surface as a peptide fragment bound to an MHC molecule. For example, many different types of cells, including monocytes or macrophages, B cells, follicular dendritic cells, and dendritic cells, can function as APCs. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that produce proteins recognized in relation to class I MHC molecules are generally proteins produced within the cell, and these antigens are processed and bind to class I MHC molecules.

[0091] "Epitope" may refer to a part of an antigen or other macromolecule capable of forming binding interactions with the variable region binding pocket of an antibody or TCR. The term includes any protein determinant capable of specifically binding to antibodies, antibody peptides, and / or antibody-like molecules as defined herein (including, but not limited to, T cell receptors). Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally possess specific charge characteristics as well as specific three-dimensional structural features.

[0092] In many cases, the term “antigen” may be any organic or inorganic molecule capable of stimulating an immune response. As used herein, the term “antigen” extends to, but is not limited to, any molecule capable of stimulating an immune response, such as peptides, polypeptides, proteins, nucleic acid molecules, carbohydrate molecules, organic or inorganic molecules.

[0093] In many cases, the 'antibody' or 'antibody moiety' may include, but is not limited to, any polypeptide chain-containing molecular structure that recognizes an epitope. The antibodies that may be used in the present invention may be polyclonal antibodies, but monoclonal antibodies are preferred because they can be reproduced through cell culture or recombination and can be modified to reduce their antigenicity. The term is intended to include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY, and to include whole antibodies including single-chain whole antibodies and their antigen-binding (Fab) fragments. The antigen-binding antibody fragments include Fab, Fab', and F(ab')2, Fd(V H and composed of CH1), single-chain variable fragment [scFv], single-chain antibody, disulfide-linked variable fragment [dsFv] and V L or V HFragments containing domains are included, but are not limited to. Antibodies may be of any animal origin. Antigen-binding antibody fragments, including short-chain antibodies, may include variable region(s) alone, or in combination with all or part of the hinge region, CH1, CH2, and CH3 domains. Any combination of variable region(s) and the hinge region, CH1, CH2, and CH3 domains is also included. Antibodies may be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to HLA-associated polypeptides or HLA-peptide complexes. A person skilled in the art will recognize that various immunoaffinity techniques are suitable for enriching soluble proteins, such as soluble HLA-peptide complexes or membrane-bound HLA-associated polypeptides, which are, for example, cleaved from the membrane by proteases. These include, for example, a technique in which (1) one or more antibodies capable of specifically binding to soluble proteins are immobilized on a fixation or transfer substrate (e.g., plastic wells or resin, latex, or paramagnetic beads), and (2) a solution containing soluble proteins derived from a biological sample is passed over the antibody-coated substrate to allow the soluble proteins to bind to the antibodies. The substrate containing the antibodies and the bound soluble proteins is separated from the solution, and optionally, the antibodies and soluble proteins are dissociated, for example, by changing the pH and / or ionic strength and / or ionic composition of the solution containing the antibodies. Alternatively, an immunoprecipitation technique may be used to cause the antibodies and soluble proteins to combine to form macromolecular aggregates. The macromolecular aggregates may be separated from the solution by size exclusion techniques or centrifugation.

[0094] The adaptive immune system responds to the molecular structures of invading organisms, namely antigens. Unlike the innate immune system, the adaptive immune system is highly specific to pathogens. Adaptive immunity can also provide long-term protection; for example, a person who has recovered from measles is now protected from measles for the rest of their life. There are two types of adaptive immune responses: humoral immunity and cell-mediated immunity. In humoral immunity, antibodies secreted into the body fluids by B cells bind to pathogen-derived antigens and eliminate the pathogens through various mechanisms, such as complement-mediated lysis. In cell-mediated immunity, T cells capable of destroying other cells are activated. For example, if disease-associated proteins are present within a cell, they are fragmented into peptides by proteases inside the cell. Specific cellular proteins then attach to the antigens or peptides formed in this way and are transported to the cell surface, where they are presented to molecular defense mechanisms in the body's T cells. Cytotoxic T cells recognize these antigens and kill the cells containing them.

[0095] In many cases, the terms 'major histocompatibility complex (MHC),' 'MHC molecule,' or 'MHC protein' may refer to proteins intended to bind to antigenic peptides produced by the proteolytic cleavage of protein antigens within phagocytes or antigen-presenting cells, and to present them to T lymphocytes for activation. These antigenic peptides may represent T cell epitopes. Human MHC is also called the HLA complex. Therefore, the terms 'human leukocyte antigen (HLA) system,' 'HLA molecule,' or 'HLA protein' may refer to the gene complex encoding MHC proteins in humans. In rodent species, the term MHC may be referred to as the 'H-2' complex. A person skilled in the art will recognize that the terms 'Major Histocompatibility Complex [MHC]', 'MHC molecule', 'MHC protein', and 'Human Leukocyte Antigen [HLA] System', 'HLA molecule', and 'HLA protein' are used interchangeably herein.

[0096] HLA proteins are typically classified into two types, referred to as HLA Class I and HLA Class II. While the protein structures of the two HLA classes are very similar, their functions may differ. Class I HLA proteins are present on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins are usually presented to unsensitized or cytotoxic T lymphocytes (CTLs) after being loaded with antigens originating from endogenous proteins or pathogens present inside the cell. HLA Class II proteins are present on antigen-presenting cells (APCs), which include, but are not limited to, dendritic cells, B cells, and monocytes or macrophages. They primarily present exogenous antigen sources, such as peptides processed outside the cell, to helper T cells. Most peptides bound by HLA Class I proteins originate from cytoplasmic proteins produced by the organism's own healthy host cells and do not normally stimulate an immune response.

[0097] In the HLA class II system, phagocytes, such as monocytes, macrophages, and immature dendritic cells, absorb material into phagosomes via phagocytosis—although B cells exhibit a more general endocytosis into endosomes—and these fuse with lysosomes, where acidic enzymes cleave the absorbed proteins into many different peptides. Autophagy is a source of HLA class II peptides. Encoded in the host genome, and through physicochemical dynamics in molecular interactions with the host's HLA class II variants, specific peptides exhibit immunodominance and are loaded onto HLA class II molecules. These migrate to the cell surface and are released externally. The most studied subclass II HLA genes are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0098] The presentation of peptides by HLA class II molecules to CD4+ helper T cells is necessary for the immune response to foreign antigens. Once activated, CD4+ T cells promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. HLA class II molecules are heterodimers of α and β chains that interact to form peptide binding grooves that are more open than class I peptide binding grooves. Peptides bound to HLA class II molecules are believed to possess a 9-amino acid binding core and lateral residues protruding from the groove on the N-terminal or C-terminal sides. These peptides are typically 12 to 16 amino acids long and often contain 3 to 4 anchor residues at binding register positions P1, P4, P6 / 7, and P9 (Reference [Rossjohn et al., 2015]).

[0099] HLA alleles are expressed in a codominant manner, meaning that alleles (variants) inherited from both parents are expressed equally. For example, since each person possesses two alleles for each of the three class I genes (HLA-A, HLA-B, and HLA-C), they can express six different types of class II HLA. At class II HLA loci, each person inherits a pair of HLA-DP genes (DPA1 and DPB1 encoding α and β chains), HLA-DQ (DQA1 and DQB1 for α and β chains), one gene HLA-DRα (DRA1), and one or more genes HLA-DRβ (DRB1 and DRB3, -4, or -5). For example, HLA-DRB1 has nearly 400 known alleles. This means that a heterozygous individual can inherit three or more functional class II HLA alleles from each parent, i.e., six or eight. Therefore, HLA genes are highly polymorphic, and many different alleles exist in different individuals within a population. There are various possible variations in the genes encoding HLA proteins, which allow each person's immune system to respond to a wide range of external invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is assigned a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, and HLA-E*01:01 (non-classical). In some embodiments, the class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[0100] Target-specific HLA alleles or the target's HLA genotype can be determined by any method known in the art. In an exemplary embodiment, the method includes the step of determining a polymorphic genotype, which may include: aligning reads extracted from a sequence analysis data set to a gene reference set containing allelic variants of a polymorphic gene; determining a first posterior probability or posterior probability derivation score for each allelic variant within the alignment; identifying an allelic variant having the maximum first posterior probability or posterior probability derivation score as the first allelic variant; identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants; determining a second posterior probability or posterior probability derivation score for one or more other allelic variants using weighting factors; identifying a second allelic variant by selecting an allelic variant having the maximum second posterior probability or posterior probability derivation score (the first and second allelic variants define the genotype for the polymorphic gene); and providing an output of the first and second allelic variants.

[0101] The term 'amino acid' as used herein may be intended to include both natural and synthetic amino acids, and both D and L amino acids. Synthetic amino acids also include chemically modified amino acids, such as salts, amino acid derivatives, and amides, which are included but not limited thereto. Amino acids present in the polypeptide of the present invention may be modified to form modified amino acids by methylation, amideation, acetylation, or substitution with other chemical groups, which may alter their cyclic half-life without adversely affecting their biological activity.

[0102] In some cases, ‘connected in an operable state’, ‘linked’, ‘fused’, or ‘connected’ may be used interchangeably to describe that two structural units or subunits are structurally connected to each other. The connection may be direct, that is, there may be no other components between the two, or indirect, provided that one or more linkers described elsewhere in this specification connect the two units of this description.

[0103] In many instances throughout this document, the terms 'peptide', 'polypeptide', and 'protein' may be used interchangeably herein to describe a series of at least two amino acids covalently bonded by a peptide bond or a modified peptide bond, e.g., an isocovalent body. There is no limit to the maximum number of amino acids that may comprise a peptide or protein. The terms 'oligomer' and 'oligopeptide' are also intended to mean the peptides described herein. Additionally, the term polypeptide extends to fragments, analogs, and derivatives of a peptide, said fragment, analog, or derivative retains the same biological functional activity as the peptide from which the fragment, derivative, or analog is derived.

[0104] The polypeptide used herein may be a 'protein' including, but not limited to, glycoproteins, lipoproteins, cellular proteins, or membrane proteins. The polypeptide may comprise one or more subunits of a protein. The polypeptide may be encoded by a recombinant nucleic acid. In some embodiments, the polypeptide described herein comprises one or more structurally distinct domains. In some embodiments, each domain of the polypeptide described herein may have a distinct function. Generally, a domain is a structural part of a protein or polypeptide that has a defined function. A moiety is a part of a polypeptide, protein, or nucleic acid having a specific structure or performing a specific function. For example, a signaling moiety is a specific unit within the larger structure of a polypeptide, protein, or recombinant nucleic acid that (or, in the case of a nucleic acid, the protein part encoded by it) is involved in a signaling process, e.g., phosphorylation. In some examples, two or more domains may be required to achieve a single function. In some cases, two or more domains required to achieve a single function may exist on two or more different polypeptides, and the function is performed only when a specific three-dimensional structure is achieved, for example, through the oligomerization and appropriate orientation of two or more different polypeptides. For example, the MHC-peptide complex linkage described herein may include a TRAV domain and a TRBV domain, each existing on a different polypeptide, and the dimerization of the two polypeptides facilitates achieving the stereochemical configuration required for binding to the MHC-peptide complex.

[0105] Throughout this document, p-MHC may refer to a structural unit of an antigenic peptide and its homologous MHC. MHC molecules are encoded by HLA genes and may be MHC class I alleles or MHC class II alleles. Generally, peptides bound to MHC class I molecules are shorter (9–11 amino acids) than peptides bound to MHC class II molecules. Peptides presented to MHC class I molecules are generally presented to and can activate CD8+ T cells. Peptides presented to MHC class II molecules are generally presented to and can activate CD4+ T cells. MHC class I peptides may refer to peptides bound to and presented to MHC class I molecules, and MHC class I bound peptides may be referred to interchangeably with CD8 peptides or semantic variants thereof. MHC class II peptides can likewise refer to peptides presented by binding to MHC class II molecules, and MHC class II binding peptides can be interchangeably referred to as CD4 peptides or semantic variants thereof.

[0106] As used herein, the term "recombinant nucleic acid molecule" may refer to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule may be any nucleic acid molecule containing a combined nucleic acid molecule that is derived from different origins and is not naturally attached together. Recombinant nucleic acids may be synthesized in the laboratory. Recombinant nucleic acids may be prepared using recombinant DNA techniques that utilize enzymatic modification of DNA, such as enzymatic restriction cutting, ligation, and DNA cloning. As used herein, the recombinant nucleic acid may be DNA or RNA. Recombinant DNA may be transcribed in vitro to produce messenger RNA (mRNA), and the recombinant mRNA may be isolated and purified to be used for transfecting cells. Recombinant nucleic acids may encode proteins or polypeptides. Recombinant nucleic acids may be introduced into living cells under suitable conditions and may be expressed within living cells. As used herein, "expression" of nucleic acids generally refers to the transcription and / or translation of nucleic acids. The product of nucleic acid expression is generally a protein, but may also be mRNA. The detection of mRNA encoded by recombinant nucleic acid in cells into which recombinant nucleic acid has been introduced is considered positive evidence that the nucleic acid is 'expressed' in the cell.

[0107] The process of inserting or introducing nucleic acids into a cell can be achieved through transformation, transfection, or transduction. Transformation is the process of bacterial cells absorbing foreign nucleic acids. This process is suitable for the proliferation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into bacterial cells capable of absorbing extracellular DNA from the environment. While some bacterial species naturally possess the ability to transform under specific environmental conditions, this ability is artificially induced in a laboratory setting. Transfection is the forced introduction of small molecules, such as DNA, RNA, or antibodies, into eukaryotic cells. What complicates the situation is that 'transfection' also refers to the introduction of bacteriophages into bacterial cells. 'Transduction' is primarily used to describe the introduction of recombinant viral vector particles into target cells, whereas 'infection' refers to natural infection of humans or animals by wild-type viruses.

[0108] As used herein, the term "vector" refers to any genetic construct capable of transferring nucleic acids between cells, such as plasmids, phages, transposable factors, cosmids, chromosomes, viruses, virions, etc. A vector may be capable of one or more of replication, expression, recombination, insertion, or integration, but is not required to possess each of these capabilities. Plasmids are species of the genus encompassed by the term "vector." A vector generally refers to a nucleic acid sequence containing replication origins and other elements necessary for replication and / or maintenance in a host cell. A vector capable of directing the expression of operably linked genes and / or nucleic acid sequences is referred to herein as an "expression vector." Generally, useful expression vectors are often in the form of "plasmids," which refers to a circular double-stranded DNA molecule that is not bound to a chromosome when in vector form and typically contains DNA encoding elements for stable or transient expression. Other expression vectors that may be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, and these vectors may be incorporated into the host genome or replicate autonomously within the cell. The vector may be a DNA or RNA vector. Other forms of expression vectors performing equivalent functions known to a person skilled in the art, such as self-replicating extrachromosomal vectors or vectors capable of being incorporated into the host genome, may also be used. Exemplary vectors are capable of autonomous replication and / or expression of the nucleic acids to which they are linked.

[0109] The terms 'spacer' or 'linker' used in relation to fusion proteins may refer to a peptide that binds to a protein containing the fusion protein. In some embodiments, the constituent amino acids of the spacer may be selected to influence some properties of the molecule, such as the folding, net charge, or hydrophobicity of the molecule. Linkers suitable for use in one embodiment of the present disclosure are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linker is used to separate the two antigen peptides by a sufficient distance, which, in some embodiments, ensures that each antigen peptide folds properly. An exemplary peptide linker sequence takes on a flexibly extended stereotype and does not tend to form an aligned secondary structure. Typical amino acids of the flexible protein region include Gly, Asn, and Ser. Almost any permutation of amino acid sequences containing Gly, Asn, and Ser is expected to satisfy the above criteria for the linker sequence. Other amino acids that are nearly neutral, such as Thr and Ala, may also be used in the linker sequence. In some cases, the linker may be designed as a combination of Gly(G) and Ser(S). For example, G4S would refer to a linker that may contain the sequence of GGGGS. For example, in some cases, the linker sequence is GGGGS, or, for example, in some cases, the linker sequence is a chain of GGGGS. The linkers used herein are not limited to those described above. Other amino acids that are nearly neutral, such as Thr and Ala, may also be used in the linker sequence. The linkers used herein may include linkers available in the public domain, for example, published sequences or commercially available linkers. The linkers used herein may include self-designed linkers.

[0110] In some embodiments, the peptide linker has more than one functional property, such as the property described herein. For example, the peptide linker links two or more functional domains, such as a binding domain. Additionally, the peptide linker may be a specific signal inducer when the linker comes into contact with an extracellular part of a cell, such as a receptor or a ligand-binding protein.

[0111] The term 'immunopurification (IP)' (or immunoaffinity purification or immunoprecipitation) may refer to a process well known in the art and is widely used to isolate a desired antigen from a sample. Generally, the process involves contacting a sample containing the desired antigen with an affinity matrix containing antibodies against the antigen covalently bonded to a solid phase. The antigen in the sample binds to the affinity matrix through immunochemical bonding. The affinity matrix is ​​then washed to remove unbound species. The antigen is removed from the affinity matrix by altering the chemical composition of the solution in contact with the affinity matrix. Immunopurification can be performed on a column containing the affinity matrix, in which case the solution is the eluent. Alternatively, immunopurification can be a batch process, in which case the affinity matrix is ​​maintained as a suspension in a solution. A critical step in this process is the removal of the antigen from the matrix. This is generally achieved by increasing the ionic strength of the solution in contact with the affinity matrix, for example, by the addition of inorganic salts. Changes in pH can also be effective in dissociating the immunochemical binding between the antigen and the affinity matrix.

[0112] As used herein, the terms 'determining,' 'evaluating,' 'analyzing,' 'measuring,' 'detecting,' and their grammatical equivalents refer to both quantitative and qualitative determinations; therefore, the term 'determining' may be used interchangeably with 'analyzing,' 'measuring,' etc., in this document. When a quantitative determination is intended, the phrase 'determining the amount of the analyte, etc.' is used. When a qualitative and / or quantitative determination is intended, the phrases 'determining the level of the analyte' or 'detecting the analyte' may be used.

[0113] A ‘fragment’ may be a part of a protein or nucleic acid substantially identical to the reference protein or nucleic acid. In some embodiments, the fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.

[0114] The terms 'isolated,' 'purified,' or 'biologically pure,' and their grammatical equivalents refer to a substance that is freed to varying degrees from the components typically associated with it when found in its natural state. 'Isolated' refers to the degree of separation from the original source or surroundings. 'Purified' refers to a higher level of separation than isolation. A 'purified' or 'biologically pure' protein is sufficiently free from other materials so that any impurities do not substantially affect the biological properties of the protein or result in adverse consequences. That is, the nucleic acid or peptide of the present disclosure is purified if it is substantially freed from cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or from chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are generally determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term 'purified' may indicate that the nucleic acid or protein essentially produces a single band on an electrophoretic gel. In the case of proteins that may undergo modifications, for example, phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified individually.

[0115] "Cancer" may refer to any disease caused by or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is a non-limiting example of a neoplasm or cancer. The terms "cancer," "tumor," or "hyperproliferative disorder" refer to the presence of cells possessing typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and specific morphological features. Cancer cells are often in the form of tumors, but these cells may exist alone in animals or be non-tumorigenic cancer cells, such as leukemia cells.

[0116] As used herein, the term 'MHC-peptide complex linkage' may refer to one or more domains that bind to an MHC-peptide complex. The MHC-peptide complex linkage described herein may include a TRAV domain and a TRBV domain, each existing on a different polypeptide, and the dimerization of the two polypeptides facilitates achieving the stereochemical configuration necessary for binding to the MHC-peptide complex.

[0117] As used herein, the term 'T cell linker' may refer to a domain that binds to T cell molecules, such as proteins expressed by T cells. The T cell linker described herein may include a short-chain variable fragment [scFv] capable of binding to a T cell target antigen / molecule, such as a protein expressed by T cells. The T cell linker described herein may include a VHH capable of binding to a T cell molecule, such as a protein expressed by T cells. In one case, where the T cell molecule or the protein expressed by the T cell is a receptor, the T cell linker may be a ligand capable of binding to the receptor.

[0118] As used herein, the term "pharmaceuticalally acceptable" may refer to those approved or eligible to be approved by federal or state regulatory agencies for use in animals, including humans, or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias. "Pharmaceuticalally acceptable excipients, carriers, or diluents" refers to excipients, carriers, or diluents that can be administered to a subject with the agent without impairing its pharmaceutical activity and are non-toxic when administered at a dose sufficient to deliver a therapeutic amount of the agent. As mentioned herein, "pharmaceuticalally acceptable salts" of pooled disease-specific antigens may be acid or base salts generally considered in the art to be suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications. These salts include inorganic and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Certain pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromide, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanyl acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethane disulonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, parmoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodide, phenylacetic acid, alkaline acids, such as acetic acid, HOOC-(CH2)n-COOH where n is 0 to 4. Likewise, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. A person skilled in the art, based on the present disclosure and knowledge of the art, would consider the literature [Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.Additional pharmaceutically acceptable salts for pooled disease-specific antigens provided herein, including those listed in 1418 (1985)] will be recognized.

[0119] Nucleic acid molecules useful for the method of the present disclosure may include any nucleic acid molecule encoding the polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having substantial identity with respect to the endogenous sequence may generally hybridize with at least one strand of a double-stranded nucleic acid molecule. 'Hybridize' refers to a nucleic acid molecule pairing with a complementary polynucleotide sequence or a portion thereof under various strictness conditions to form a double-stranded molecule. For example, strictness salt concentrations may typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low severity hybridization can be obtained in the absence of organic solvents, e.g., formamide, whereas high severity hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Severity temperature conditions may typically include temperatures of at least about 30 °C, at least about 37 °C, or at least about 42 °C. Various additional parameters, such as hybridization time, concentration of the cleaning agent, e.g., inclusion or exclusion of sodium dodecyl sulfate (SDS) and carrier DNA, are well known to those skilled in the art. Various levels of severity are achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization may take place at 30 °C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.In another exemplary embodiment, hybridization may occur at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization may occur at 42 °C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations to these conditions will be obvious to a person skilled in the art. In most applications, the severity of the washing step after hybridization may also vary. Washing severity conditions can be defined by salt concentration and temperature. As described above, washing severity can be increased by decreasing the salt concentration or increasing the temperature. For example, strict salt concentrations for the washing step may be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Strict temperature conditions for the washing step may include temperatures of at least about 25 °C, at least about 42 °C, or at least about 68 °C. In an exemplary embodiment, the washing step may take place at 25 °C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the washing step may take place at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another exemplary embodiment, the washing step may take place at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations to these conditions will be obvious to a person skilled in the art. Hybridization techniques are well known to ordinary technicians, for example in the literature [Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc.Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); 및 Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York]에 기재되어 있다.

[0120] "Substantially identical" may be used in relation to a comparison between the sequences of two or more polypeptides or nucleic acid molecules, for example, the sequence of one polypeptide may be described as exhibiting at least 50% identity with respect to a reference amino acid sequence. The reference sequence may be a sequence disclosed herein or may be referred to as one disclosed in other publicly available materials. Such sequences may be at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical to the sequence used for comparison at the amino acid level or in nucleic acid. Sequence identity may generally be measured using sequencing software (e.g., the sequencing software package of the Genetics Computing Group at the Center for Biotechnology at the University of Wisconsin-Madison, 53705, 1710 University Avenue, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include variations within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program may be used, and probability scores between e-3 and em° indicate closely related sequences. 'Reference' is the standard for comparison.

[0121] In many cases, the term 'scaffold' may refer to a molecular platform structure that can be further adjusted to suit a specific purpose. In the context described herein, the scaffold may refer to a multispecific T cell linker, which may comprise at least one arm that binds to a cell presenting an epitope of interest on its surface and at least a second arm that binds to a T cell capable of binding to the cell presenting the epitope and performing one or more of cell destruction, activation of an epitope-specific immune response, or cell inactivation.

[0122] The term ‘subject’ or ‘patient’ refers to an animal that is the subject of treatment, observation, or experiment. By example only, the individual includes, but is not limited to, human or non-human mammals, such as non-human primates, rodents, cattle, horses, dogs, sheep, or cats.

[0123] The alpha(a) and beta(b) chains of an abTCR are generally considered to each have two 'domains', namely variable and constant domains. The variable domain may consist of the concatenation of a variable region and a binding region, and is generally located at the terminal portion of the TCR chain. Thus, the TCR alpha variable domain may refer to the concatenation of the TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region or the C-terminal truncated TRAC sequence. Likewise, the TCR beta variable domain may refer to the concatenation of the TRBV and TRBD / TRBJ regions, and the TCR beta constant domain may refer to the extracellular TRBC region or the C-terminal truncated TRBC sequence.

[0124] The terms 'treat,' 'treated,' 'treating,' 'treatment,' 'to treat,' 'relieve,' or 'to alleviate' refer to therapeutic measures that cure, / or slow down, reduce / reduce symptoms, or halt the progression of a diagnosed pathological condition or disorder. Therefore, those requiring treatment may include individuals who already possess a disorder. In some cases, treatment may refer to reducing or alleviating associated disorders and / or symptoms (e.g., neoplasms or tumors, infectious agents, or autoimmune diseases). 'Treatment' may refer to administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer, infection by an infectious agent, or autoimmune disease). 'Treatment' includes the concept of 'relief,' which refers to reducing the frequency or severity of any symptoms or other adverse effects associated with the disease and / or side effects associated with the therapy. The term 'treatment' may also encompass the concept of 'management,' which refers to reducing the severity of a disease or disability in a patient, for example, extending the lifespan or increasing the ability to survive of a patient with the disease, or delaying its recurrence, for example, extending the remission period in a patient who has suffered from the disease. It should be noted that, although not excluded, treatment of a disability or condition does not require the complete elimination of the disability, condition, or associated symptoms.

[0125] The term 'prevent' or 'prevention' refers to preventive or protective measures that delay the onset of a targeted pathological condition or disorder. Therefore, those who require prevention include those who are susceptible to the disorder and those for whom the disorder must be prevented.

[0126] The term “therapeutic effect” refers to alleviating, to some extent, one or more symptoms of a disorder (e.g., neoplasm, tumor, infection by an infectious agent, or autoimmune disease) or associated pathology. As used herein, “therapeutic effective dose” may refer to an amount of agent effective, when administered as a single or multiple doses to a cell or target, in extending the survival of a patient suffering from such disorder beyond what would be expected in the absence of such treatment, or reducing, preventing, or delaying one or more signs or symptoms of the disorder. “Therapeutic effective dose” is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian with ordinary skill in the art can readily determine and prescribe the “therapeutic effective dose” (e.g., ED50) of the necessary pharmaceutical composition.

[0127] References in the specification to ‘some embodiments,’ ‘one embodiment,’ ‘one embodiment,’ or ‘another embodiment’ mean that the features, structures, or characteristics described in connection with the embodiments are included in at least some embodiments of the present disclosure, but are not necessarily so in all embodiments.

[0128] Although cancer cells or tumor cells may be repeatedly referred to as target cells herein, the concept described herein may be applicable to any type of target cell, such as infected cells or specific disease cell types that need to be removed by immune cells, provided that the binding domain to the cell surface components of the cancer cells is appropriately replaced with a binding domain to the cell surface components specific to the target cells.

[0129] T cell receptors (TCRs) on T cells can bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of TCRs triggers a signal cascade within the T cell, inducing proliferation and differentiation into mature effector T cells. To target a vast variety of antigens, T cell receptors must possess tremendous diversity.

[0130] This diversity is obtained through the genetic rearrangement of different discontinuous segments of genes encoding different structural regions of the TCR. The TCR consists of one α chain and one β chain, or one γ chain and one δ chain. The TCR α / β chain consists of a highly polymorphic variable region at the N-terminus involved in antigen recognition and an invariant region. At the genetic level, these chains are divided into several regions: a variable (V) region, a diversity (D) region (only the β chain and δ chain), a binding (J) region, and a invariant (C) region. The human β chain gene contains more than 60 variable (V) segments, 2 diversity (D) segments, more than 10 binding (J) segments, and 2 invariant region segments (C). The human α chain gene contains more than 50 V segments and more than 60 J segments, but does not contain a D segment and contains one C segment. The β-chain gene contains more than 30 variable (V), 2 diversity (D), more than 10 binding (J) segments, and 2 constant region segments (C). The α-chain gene contains nearly 100 V segments and 60 J segments, but contains no D segments and one C segment. During T cell differentiation, specific T cell receptor genes are generated by rearranging one V, one D (only the β-chain and δ-chain), one J, and one C region gene. TCR diversity is further amplified by incorrect V-(D)-J rearrangements, where random nucleotides are introduced and / or deleted at the recombination site. Because rearrangements of TCR loci occur within the genome during T cell maturation, each mature T cell expresses only one specific α / β TCR or γ / δ TCR.

[0131] MHC and antigen binding are mediated by complementary determinants 1, 2, and 3 (CDR1, CDR2, CDR3) of the TCR. CDR3 of the β chain, which is most important for antigen recognition and binding, is encoded by the VDJ junction of the rearranged TCR β chain gene.

[0132] The TCR is part of a complex signaling mechanism, which includes a heterodimer complex of TCR α and β chains, the co-receptor CD4 or CD8, and the CD3 signaling module. While the CD3 chain transmits activation signals into the cell, the TCR α / β heterodimer is entirely responsible for antigen recognition. Therefore, the delivery of the TCR α / β chain provides an opportunity to redirect T cells toward any antigen of interest.

[0133] Some natural TCRs may have weak affinity and low stability for peptide:MHC complexes, which limits their potential to reach effective therapeutic effects as soluble drugs. Although the buried surface area generated at the TCR:pHLA protein-protein interface is large (average approximately 2000 Angstroms) 2), and naturally measured binding affinities were observed to be relatively weak compared to other Ig-like proteins (KD approximately 0.1–1000 μM) (Reference [Bridgeman JS, Sewell AK, Miles JJ, Price DA & Cole DK (2012) Structural and biophysical determinants of alphabeta T-cell antigen recognition. Immunology 135, 9-18]). TCRs selective for tumor-associated pHLA tend to bind to weaker ends in this range, which may indicate thymic deletions of TCRs that bind with strong affinity to autologous tumor peptides, adding to the difficulties regarding TCR selection and utility as therapeutic agents in this disease field. To overcome these limitations and fully utilize TCRs, several genetic and protein engineering solutions have been applied to TCRs to improve both their stability and affinity. Antigen-specific CD8+ T cell-mediated immune responses depend on the proper recognition of αβ T cell receptors (TCRs) of peptide-major histocompatibility class I (MHC I) molecular complexes. The binding sites of the TCR contain three complementarity determining regions (CDRs) for each chain, where CDR3 is the most diverse and important CDR in antigen recognition. Methods to improve TCR affinity include introducing amino acid sequence variations into the TCR complementarity determining regions (CDRs).

[0134] TCR-mimicking antibodies against peptide HLA are large-scale (>10 11Phage / yeast displays require extensive panning from VH / VL libraries and / or animal (i.e., camels, mice) immunization and screening. Unlike TCRs, antibodies do not start with a 'pHLA-oriented' stance; affinity maturation must occur from the beginning. Personalized medicine is time-consuming and expensive. 'Off-the-shelf' approaches are not an option.

[0135] In one embodiment, a soluble TCR may be considered, designed, manufactured, and tested herein. A soluble TCR is a TCR that includes alpha and beta chains corresponding to a natural TCR but is not membrane-immobilized. A soluble TCR may be fabricated as a nucleic acid construct, introduced into a cell of interest, then translated and released extracellularly. A soluble TCR may bind to one, two, or three different targets, wherein the targets are antigens, antigens complexed with MHC, or various combinations thereof as designed, wherein the targets are located in one or more very close cells. For example, a soluble TCR designed to bind to two different targets may bind cytotoxic T cells to cancer cells by having a first target that binds to cytotoxic T cells and a second target that binds to cancer antigens on cancer cells. For example, in one embodiment, if a soluble TCR is designed to bind to a target on a desired cell, the binding may activate the target cell by generating signal transduction within the target cell.

[0136] Manipulating T cells is another option receiving tremendous interest and being researched. T cells have been engineered to express TCRs designed for higher specificity to target epitopes. However, for the purposes of this study, safely and effectively manipulating T cells is not a simple task. We are seeking a customizable, off-the-shelf platform that is easier to generate and manufacture, and effective as a therapeutic agent. Developing technologies to stabilize and enhance the activity of soluble TCRs extracellularly can create new therapeutic modalities and expand patient access to treatment.

[0137] Soluble TCRs engineered in this manner can offer significant advantages. High-affinity soluble TCR bispecific or multispecific molecules enable off-the-shelf precision immunotherapy. Mammalian cell displays can identify candidate TCRs that exhibit high expression and unique folding within mammalian cells and can be safely used for therapeutic purposes. An exemplary overview of the workflow for sTCR generation is Figs. 1a–1d It is provided for. It also provides an opportunity to create new formulations. The method described herein is designed to create an enhanced TCR platform that bypasses the manipulation of individual TCRs and enables the plug-and-play use of any functional TCR, which combines two basic ideas: enhancement of TCR / MHC binding by a divalent CD8 co-receptor and increased binding affinity to T cells by divalent or polyvalent binding. This method utilizes rational structural induction and scanning-induced mutagenesis at the CDR3ab (CDR3 alpha and CDR3 beta) loop positions to enhance TCR affinity, which aims to enhance affinity without impairing specificity by significantly reducing the search space to investigate, preserving CDR3 positions that provide strong peptide:HLA (pHLA) recognition, and mutagenesis at weak / neutral positions.

[0138] Developing technologies to stabilize and enhance the activity of soluble TCRs outside the cell can create new therapeutic modalities and expand patient access to treatment.

[0139] Multispecific T cell binding molecules

[0140] An attractive and promising alternative to TCR-based therapies is to re-induce natural T cells into target tumor cells using carefully and rationally designed soluble TCRs. The present disclosure provides methods and compositions that provide soluble TCRs (sTCRs) for specific antigens and bind to T cells of appropriate antigen specificity to generate robust activation and antigen-specific cytotoxicity. In one embodiment, the methods and compositions provided herein enable a time- and cost-effective method for generating personalized antigen-specific sTCRs. T cell binding molecules can be engineered to enhance affinity, and this process induces affinity maturation. While TCR domains can be matured through phage and yeast displays, these techniques share the disadvantages of non-human glycosylation patterns and the need for subsequent reconstitution into a final bispecific format. More importantly, to successfully select specific TCRs with high affinity and stability, very large display libraries (usually >10 11It requires a variant. The combination of antigen recognition and T cell binding domains enables polyclonal activation of T cells independently of the T cell's TCR specificity or the affinity of the T cell's p-MHC:TCR. Even though there is a high probability that T cells located very close to a target cell within a biological system will possess at least minimal binding potential, even if transient, to the target cell's antigen peptide, the sTCR of the present disclosure can facilitate sustained binding and activation of T cells located close to the target cell (particularly when the desired target provides a p:MHC complex with low affinity for natural TCR binding by the T cell), thereby converting the T cell into an active effector T cell.

[0141] One of the attractive concepts disclosed herein is a 'plug-and-play' approach, which creates platform designs for customized linker specificity, which, for example, provide, but are not limited to, personalized immunotherapy using sTCRs. The methods and compositions described herein provide such platforms by creating sTCR scaffold(s) that provide structural characteristics effective for inserting a TRAV:TRBV domain of interest, or, for example, introducing sequences for CDR1, CDR2, and CDR3 of alpha and beta variable domains effectively designed for human therapeutic use targeting any antigen of interest.

[0142] The present disclosure provides a method utilizing the activation of T cells by a soluble recombinant TCR (sTCR), the sTCR being described herein. The sTCR disclosed herein is designed to comprise (I) an MHC-peptide complex linkage (used interchangeably with the term MHC linkage herein) and (II) a T cell linkage that structurally binds an antigen-bearing target cell (e.g., a peptide-MHC-bearing cell, e.g., a target cell) and an effector T cell to both cells and cross-links them to induce them to be in very close proximity, thereby increasing interaction, as well as activating T cells toward a target-specific response. The effector T cell may be a T cell having an endogenous TCR specific to the peptide (e.g., a homologous TCR for the peptide-MHC presented by the antigen-bearing target cell). The effector T cell may be a cytotoxic T cell. The effector T cell may be a CD8+ T cell. Antigen-containing target cells may be cancer cells. Target-specific reactions may be cytotoxic reactions.

[0143] In one embodiment, a recombinant sTCR is provided comprising a recombinant multispecific protein molecule, namely, at least two binding substances or linkages, (i) an MHC-peptide complex linkage capable of binding to a complex comprising a specific antigen peptide that forms a complex with MHC for antigen presentation, and (ii) a T cell linkage that binds to an extracellular domain of a receptor (or cell surface ligand) expressed by a T cell. The designer recombinant sTCR described herein may have various target specificities based on the sequences on the binding domains, in particular the TRAV and TRBV sequences of the MHC-peptide complex linkage and the sequence of the T cell linkage domain. Each of the TRAV and TRBV domains comprises a supervariable complementarity determining region [CDR] 1, 2, 3 (CDR1, CDR2, CDR3). The present disclosure provides a method using reasonable structural induction and scanning-induced mutagenesis of only the CDR3αβ ring position, which can significantly reduce the search space to be investigated, as well as preserve the CDR3 position that provides strong p-HLA recognition and induce mutagenesis at weak / neutral positions to enhance affinity without impairing specificity. The specificity of the T cell linker is conferred by a variable sequence which may be an antibody or a fragment thereof, a scFV or VHH domain, which is characterized by the presence of CDR1, 2 and 3 supervariable domains.

[0144] The present disclosure provides a method comprising another advantage. Conventional methods use the production of soluble TCRs (sTCRs) in non-mammalian cells, for example, the generation of sTCRs in bacterial cells. The present method is performed in mammalian cells containing recombinant nucleic acid and vector designs optimized for expression within mammalian cells, thereby conferring appropriate expression and activation in a mammalian environment by maintaining glycosylation and other post-translational signatures. Post-translational modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis, and affect the cellular function of the protein, including expression and activity. Additionally, glycosylated and acetylated peptides have been shown to be more immunogenic in similar tests. In some embodiments, multispecific linker molecules are generated in mammalian cells in vitro at production scale. Recombinant nucleic acid is introduced into mammalian cells cultured in vitro, and multispecific linker protein molecules at production and / or production scale are isolated, harvested, and purified. In some embodiments, the multispecific linker molecule contains a mammalian post-translational modification signature upon isolation. In some embodiments, the mammalian cell is a human cell, for example, a human cell line cultured in vitro. In some embodiments, the multispecific molecule contains a post-translational modification. In some embodiments, the multispecific molecule contains a mammalian glycosylation signature. In some embodiments, the multispecific molecule contains a mammalian phosphorylation signature. In some embodiments, the multispecific molecule contains a mammalian acetylation signature.

[0145] Accordingly, a composition comprising a recombinant nucleic acid having a sequence encoding a multispecific molecule is provided herein, wherein the multispecific molecule is an engineered T cell receptor (TCR) construct comprising (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising an extracellular domain of the T cell receptor or a binding domain that binds to a T cell ligand, wherein the multispecific molecule is an engineered T cell receptor (TCR) construct comprising a T cell conjugate comprising a post-translational modification of a human cell.

[0146] In some embodiments, the recombinant multiple-specific molecule is soluble. The soluble protein molecule may be a molecule that is not introduced into the cell membrane or localized within the cell in its mature form. For example, the multiple-specific molecule lacks a transmembrane domain. In some embodiments, the multiple-specific molecule may be secreted from the cell.

[0147] In some embodiments, the multispecific molecule comprises two polypeptides, for example, a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and the second polypeptide comprise one or more multimerizing domains or stabilizing domains that essentially hold the two polypeptides together within a structural assembly. In some embodiments, the first polypeptide and the second polypeptide comprise one or more disulfide bonds between the two polypeptides.

[0148] In one embodiment, the present disclosure provides one or more synthetic or recombinant biomolecules, such as proteins or polypeptides, capable of binding to and activating T cells to trigger an immune response against target cells, such as tumor cells. In some embodiments, the synthetic or recombinant biomolecule binds (a) to a cell surface molecule (i.e., antigen) in relation to an MHC complex on a target cell on one side, and (b) to a cell surface molecule (i.e., antigen or receptor) on a T cell on the other side, thereby effectively bringing at least two cells (target and effector cells, respectively) into close proximity so that other cell receptors and membrane components on both cells can interact, thereby enabling the effector T cell to trigger the death of the target cell. Such synthetic or recombinant biomolecules may be referred to as bispecific conjugates, or bispecific T cell conjugates, or BiTEs. In one or more embodiments, the bispecific conjugate comprises two antigen-binding domains ('binding substances'). At least one of the two binding substances is designed to bind to a protein expressed on the surface of an effector T cell, and at least one of the two binding substances is designed to bind to an antigen peptide-MHC complex on a target cell. In some embodiments, the antigen binding domain is an antibody or a fragment thereof. In some embodiments, the binding substance may be a ligand that binds to a receptor on a cell surface, such as a T cell or a receptor on a target cell.

[0149] In one embodiment, the present disclosure provides a therapeutic composition comprising one or more synthetic or recombinant biomolecules, such as proteins or polypeptides, capable of binding to and activating T cells to trigger apoptosis and immune responses against target cells, such as cancer cells, wherein the synthetic or recombinant biomolecules comprise two or more binding substances. Accordingly, in some embodiments, a therapeutic agent is provided herein, wherein the therapeutic agent comprises a first binding domain (or first binding substance) which may be a first TCR antigen-binding domain or a functional fragment thereof that specifically interacts with an antigen or surface molecule in relation to a peptide-MHC complex on a target cell, and a second binding domain (or second binding substance) which may be an antibody or functional fragment, ligand, or receptor that specifically interacts with a T cell.

[0150] In one embodiment, the recombinant biomolecule comprises three binding substances, each of which exhibits a specific binding to a surface molecule, and thus the recombinant biomolecule may exhibit binding to three elements on two or more cells. In one embodiment, the recombinant biomolecule having three binding substances may bind to one or more antigens on T cells or target cells. As described herein, the recombinant biomolecule having three binding substances is referred to as a trispecific T cell engager (TriTE). In some embodiments, the TriTE may bind to or bind to two or more different cells, e.g., at least one T cell and at least one target cell, e.g., a cancer cell. In some embodiments, the BiTE or TriTE may bind to more than one antigen or surface molecule on the T cell or cancer cell to activate the T cell or inhibit the function of the cancer cell. In some embodiments, the bispecific, trispecific, or multispecific conjugate may include a second trigger, that is, a second signal that initiates an immune or inflammatory response that not only induces the death of target cells by T cells but also activates other immune cells for sustained response and the generation of immune memory. In some embodiments, the bispecific, trispecific, or multispecific conjugate is a chimeric molecule.

[0151] By using the methods and compositions described herein, T cells can be induced to activate the immune response cycle regardless of their effects in the tumor microenvironment. T cells can be induced to activate subsequent immune response processes that kill target cells and generate a successful and sustained adaptive immune response and immune memory against the target.

[0152] The present disclosure provides a method and a composition for in vivo use. In some embodiments, a method for in vivo administration of a recombinant nucleic acid construct encoding a soluble TCR described herein is provided herein.

[0153] A composition containing a therapeutic agent is described in the section below.

[0154] I. MHC-peptide complex linkage

[0155] Significant progress has been made in identifying tumor-specific antigens capable of triggering an immune response. Tumor-specific antigens (TSAs) can be neoantigens. Neoantigens may be uniquely present only in cancer cells and absent in normal tissues, such as in cancer tissue versus non-cancer tissue within a single human subject, making them attractive targets for immunotherapy. While most somatic mutations are random passenger mutations unique to individual patients, KRAS , PIK3CA or ERBB2 Tumor-genic driver genes such as, and TP53 and PTEN A subset of mutations within tumor suppressors, including [specific antigens], forms a class of shared antigens common among patient cohorts. Some of these shared antigens, which are immunogenic and restricted to common HLAs, can often be considered driver mutations. Examples of shared antigens currently described include KRAS G12D / G12V, commonly found in 60–70% of pancreatic adenocarcinomas and 20–30% of colorectal cancers, and PIK3CA H1047L, detected in approximately 5% of metastatic breast cancers. High-affinity soluble TCRs that specifically target these shared common antigens enable off-the-shelf precision immunotherapy for cancer patients.

[0156] A composition comprising a recombinant nucleic acid having a sequence encoding a multispecific molecule, e.g., a multispecific linker molecule, is provided herein, wherein the multispecific molecule comprises (i) an MHC-peptide complex linker comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain, wherein the peptide within the MHC-peptide complex is an oncological peptide, e.g., a RAS peptide sequence; and (ii) a T cell linker comprising a binding domain that binds to an extracellular domain of a receptor expressed on a T cell. A recombinant nucleic acid having a sequence encoding a multispecific molecule that can be used for therapeutic purposes is provided herein for in vivo use in a human subject requiring treatment, wherein the recombinant nucleic acid having a sequence encoding a multispecific molecule is administered to a human subject, the recombinant nucleic acid is absorbed by a cell in vivo, and the multispecific linker molecule encoded by the recombinant nucleic acid is expressed in vivo. In some embodiments, a multispecific linker molecule is administered to a subject, wherein the multispecific molecule includes a post-translational modification of a human cell.

[0157] In some embodiments, the multispecific molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a TRAV domain and the second polypeptide comprises a TRBV domain.

[0158] In some embodiments, the peptide of the MHC-peptide complex is a peptide derived from a cancer antigen.

[0159] In some embodiments, the peptides of the MHC-peptide complex contain mutations.

[0160] In some embodiments, the MHC in the MHC-peptide complex binds to the wild-type peptide of the MHC-peptide complex with a lower affinity compared to the peptide containing the mutation.

[0161] In some embodiments, the peptide of the MHC-peptide complex does not contain the amino acid sequence YLEPGPVTA.

[0162] In some embodiments, the MHC of the MHC-peptide complex includes a class I MHC.

[0163] In some embodiments, the MHC of the MHC-peptide complex is a class I MHC polypeptide.

[0164] The MHC of the MHC-peptide complex is encoded by HLA. Generally, about six HLA alleles are expressed in all humans. Typically, an allele is described as one of two or more versions of a DNA sequence (a single nucleotide or a segment of a nucleotide) at a specific genomic location. An individual inherits two alleles, one from each parent, for any given genomic location where these variations exist. If the two alleles are identical, the individual is homozygous for that allele. If the alleles are different, the individual is heterozygous. The distribution and frequency of HLA antigens vary significantly among different ethnic groups. Genes encoding HLA heterodimers are highly polymorphic, and over 12,000 Class I and over 4,000 Class II allelic variants have been identified across human populations. It has been hypothesized that this diversity in HLA polymorphism evolved under unique selective pressures in different geographical regions. Some HLA alleles exist at higher frequencies in specific human populations than others. Therefore, certain HLA alleles may appear at a higher frequency in a given population than other HLA alleles. For the purposes of discussion, low-frequency HLA alleles may be alleles occurring in less than 1% of a given human population (e.g., a human population cohort described by race or geographic area). The White population or the North American population may be mentioned for illustrative purposes only.

[0165] In some embodiments, the MHC of the MHC-peptide complex has a peptide binding affinity greater than at least 50 nM. In some embodiments, the MHC of the MHC-peptide complex binds to peptide antigens that are present in low abundance in the disease system. For example, cancer antigens may belong to proteins that are expressed in low abundance in cells, e.g., cancer cells, but may be antigens that induce a potent immunogenic response, e.g., T cell activation. For example, particularly low-abundance proteins are proteins that have fewer copies in the cell compared to the typical copy number of other proteins of intermediate or high expression. In one embodiment, an sTCR system may be developed, wherein the MHC-peptide complex linkage recognizes low-abundance proteins in the p-MHC complex.

[0166] In the completely unmodified form of the TCR, the alpha variable domain (Vα) and the beta variable chain domain (Vβ) are separate polypeptide chains, that is, located within the α chain and the β chain, respectively. When the TCR α chain and β chain interact, particularly when the Vα and Vβ domains interact, one of the epitope binding sites of the TCR is formed.

[0167] In some embodiments, the binding domain may be further modified to increase its binding specificity or binding affinity or both. A person skilled in the art may use existing techniques to improve the binding properties of the binding region, and such modifications are considered within the scope of this disclosure.

[0168] In some exemplary embodiments, TRAV:TRBV discovery may lead to the analysis of target-specific cancer-specific neoantigen epitopes. (a) A neoantigen for a single target may be obtained by analyzing gene expression at the whole-genome or exome scale; (b) the epitopes differentially expressed in the target’s cancer cells versus non-cancer cells may be HLA-matched to the MHC of high affinity encoded by the target’s HLA using a suitable MHC-peptide prediction algorithm, e.g., RECON 3.0, NeonMHC2, NetMHCpan, IEDB; (c) T cells may then be screened for high-affinity binding to each target-specific cancer-specific neoantigen peptide; and (d) the TCR variable regions of the screened T cells may be subsequently sequenced. The TRAV and TRBV sequences thus obtained may be used to generate recombinant sTCRs having the characteristics described herein. The generalized method can be effectively modified by using one or more specific improvements described herein. Incorporating a selected TRAV:TRBV pair into a suitably well-designed scaffold structure can generate a new and effective sTCR for in vivo therapy.

[0169] In some embodiments, a suitable MHC-peptide prediction algorithm is a machine learning HLA peptide presentation prediction model. In some embodiments, the machine learning HLA peptide presentation prediction model is trained using training data containing sequence information of training peptide sequences identified by mass spectrometry to be presented by HLA proteins expressed in training cells. In some embodiments, the method comprises the step of ranking at least two peptides identified as being presented by at least one of one or more proteins encoded by a class II HLA allele of a target cell based on presentation predictions. For example, prior application PCTUS201968084, filed by the applicant on December 20, 2019 and published as WO2020132586 on June 25, 2020, provides guidance to the subject matter and is incorporated herein in its entirety.

[0170] In some embodiments, the method includes the step of selecting one or more peptides from two or more ranked peptides.

[0171] In some embodiments, the method includes the step of selecting one or more peptides from a plurality of peptides identified as being presented by at least one of one or more proteins encoded by a class II HLA allele of a target cell.

[0172] In some embodiments, the method includes the step of selecting one or more peptides from two or more ranked peptides based on the presented prediction.

[0173] In some embodiments, a machine learning HLA peptide presentation prediction model has a positive predictive value (PPV) of at least 0.07 when amino acid information of a plurality of test peptide sequences is processed to generate a plurality of test presentation predictions, and each test presentation prediction indicates the possibility that one or more proteins encoded by a class II HLA allele of a target cell may present a predetermined test peptide sequence among the plurality of test peptide sequences, wherein the plurality of test peptide sequences comprises at least 500 test peptide sequences including (i) at least one hit peptide sequence confirmed by mass spectrometry to be presented by an HLA protein expressed in a cell and (ii) at least 499 decoy peptide sequences contained within a protein encoded by the genome of an organism, wherein the organism and the target are of the same species, and the plurality of test peptide sequences comprise a ratio of at least one hit peptide sequence to at least 499 decoy peptide sequences, and the plurality of test The top percentage of peptide sequences is predicted to be presented by HLA proteins expressed in cells by a machine learning HLA peptide presentation prediction model.

[0174] In some embodiments, a machine learning HLA peptide presentation prediction model has a positive predictive value [PPV] of at least 0.1 when amino acid information of a plurality of test peptide sequences is processed to generate a plurality of test binding predictions, and each test binding prediction indicates the probability that one or more proteins encoded by a class II HLA allele of a target cell will bind to a predetermined test peptide sequence among the plurality of test peptide sequences, wherein the plurality of test peptide sequences comprises at least 20 test peptide sequences including (i) at least one hit peptide sequence identified by mass spectrometry as being presented by an HLA protein expressed in a cell, and (ii) at least 19 decoy peptide sequences contained within a protein including at least one peptide sequence identified by mass spectrometry as being presented by an HLA protein expressed in a cell, e.g., a single HLA protein expressed in a cell (e.g., a single-allele cell), and wherein the plurality of test peptide sequences comprises at least one hit peptide sequence versus at least 19 decoy peptides The sequences include a ratio of 1:19, and the top percentage of multiple test peptide sequences are predicted to bind to HLA proteins expressed in cells by a machine learning HLA peptide presentation prediction model.

[0175] In some embodiments, there is no amino acid sequence overlap between at least one hit peptide sequence and a decoy peptide sequence.

[0176] In some embodiments, the machine learning HLA peptide presentation prediction model has a positive predictive value [PPV] of at least 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, It is 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

[0177] In some embodiments, the top percentage is the top 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%, or 40%.

[0178] In some embodiments, one of the bispecific or trispecific linkages binds to the MHC on the target cell, which forms a complex with the cancer-specific antigen peptide on the cancer cell. In some embodiments, the MHC protein is encoded by HLA, wherein the HLA is Class I HLA (human leukocyte antigen [HLA]-A, -B, -C). In some embodiments, the MHC protein of the complex is a protein encoded by HLA, wherein the HLA is Class II HLA (HLA-DP, -DQ, or -DR). In some embodiments, the MHC protein within the MHC complex is encoded by HLA-A, -B, -C, -DP, -DQ, or -DR.In some embodiments, the MHC protein encoded by HLA is HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA-DQB1*02:01 / HLA-DQA1*05:01, HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02, HLA-DQB1*06:04 / HLA-DQA1*01:02, HLA-DRB1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1*08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*12:02, HLA-DRB1*13:01, HLA-DRB1*13:02, HLA-DRB1*13:03, HLA-DRB1*14:01, HLA-DRB1*15:01, HLA-DRB1*15:02, HLA-DRB1*15:03, HLA-DRB1*16:01, HLA-DRB3*01:01, HLA-DRB3*02:02, It includes HLA class II proteins selected from the group consisting of HLA-DRB3*03:01, HLA-DRB4*01:01, and HLA-DRB5*01:01.

[0179] In some embodiments, HLA-DR is paired with DRA*01:01.

[0180] In some embodiments, the MHC protein encoded by HLA is DPA*01:03 / DPB*04:01, DRB1*01:01, DRB1*01:02, DRB1*03:01, DRB1*04:01, DRB1*04:02, DRB1*04:04, DRB1*04:05, DRB1*07:01, DRB1*08:01, DRB1*08:02, DRB1*08:03, DRB1*09:01, DRB1*11:01, DRB1*11:02, DRB1*11:04, DRB1*12:01, DRB1*13:01, DRB1*13:02, DRB1*13:03, DRB1*14:01, DRB1*15:01, It is an HLA class II protein selected from the group consisting of DRB1*15:02, DRB1*15:03, DRB1*16:02, DRB3*01:01, DRB3*02:01, DRB3*02:02, DRB3*03:01, DRB4*01:01, DRB4*01:03, and DRB5*01:01.

[0181] In some embodiments, the HLA-DR protein contains DRA*01:01 within the dimer.

[0182] In some embodiments, the MHC protein encoded by HLA comprises an HLA-DP protein selected from the group consisting of DPB1*01:01, DPB1*02:01, DPB1*02:02, DPB1*03:01, DPB1*04:01, DPB1*04:02, DPB1*05:01, DPB1*06:01, DPB1*11:01, DPB1*13:01, and DPB1*17:01.

[0183] In some embodiments, the HLA-DP protein is paired with DPA1*01:03.

[0184] In some embodiments, the MHC protein encoded by HLA comprises an HLA-DQ protein complex selected from the group consisting of A1*01:01+B1*05:01, A1*01:02+B1*06:02, A1*01:02+B1*06:04, A1*01:03+B1*06:03, A1*02:01+B1*02:02, A1*02:01+B1*03:03, A1*03:01+B1*03:02, A1*03:03+B1*03:01, A1*05:01+B1*02:01, and A1*05:05+B1*03:01.

[0185] Sequences of Class I and Class II HLA alleles can be identified in the IPD-IMGT / HLA database. In some embodiments, HLA alleles are selected to correspond to genotypes of interest. In some embodiments, HLA alleles are mutated HLA alleles, which may be non-naturally occurring alleles or naturally occurring alleles in affected patients. The method disclosed herein has the additional advantage of identifying HLA binding peptides for HLA alleles associated with various disorders as well as for alleles present at low frequencies. For example, in some embodiments, HLA alleles are present at a frequency of less than 1% within a population, such as within a Caucasian population.

[0186] In some embodiments, compositions comprising a recombinant polynucleic acid comprising a sequence encoding one or more polypeptides, for example, a first polypeptide and a second polypeptide, are provided herein. In some cases, throughout the description, the terms 'first polypeptide' and 'first polypeptide chain' may be used interchangeably, and the terms 'second polypeptide' and 'second polypeptide chain' may be used interchangeably.

[0187] In some embodiments, the polypeptide described herein comprises a first binding domain and a second binding domain operably connected to the first domain, for example, the first domain is fused to the second domain. For example, the first binding domain and the second binding domain are operably connected when the C-terminus of the first domain is fused to the N-terminus of the second domain, or alternatively, when the N-terminus of the first domain is fused to the C-terminus of the second domain. For example, the first binding domain and the second binding domain are operably connected when the C-terminus of the first domain is connected to the N-terminus of the second domain via a linker, for example, a peptide linker or a chemical linker, or alternatively, when the N-terminus of the first domain is connected to the C-terminus of the second domain via a linker, for example, a peptide linker. In some embodiments, the first polypeptide chain is configured to oligomerize with the second polypeptide chain. The second polypeptide may include one or more domains connected in an operable state as described above, wherein the first binding domain specifically interacts with the antigen peptide in relation to the MHC complex on the target cell, and the second binding domain specifically interacts with the first extracellular protein of the T cell.

[0188] In one embodiment, the linker comprises two domains located on two different polypeptide chains, and the assembly of the functional linker is achieved when the two polypeptide chains are structurally aligned in an appropriate arrangement after dimerization. Dimerization is achieved by one or more dimerization domains present on each polypeptide. In some embodiments, dimerization is enhanced by manipulating the polypeptides, such as by introducing one or a mutation into the first or second polypeptide, or both.

[0189] In some embodiments, the MHC linkage comprises a binding substance that binds to an MHC-peptide complex, wherein the peptide is presented by the MHC. The MHC linkage comprises at least variable domains of a T cell receptor (TCR), for example, a T cell alpha variable domain (TRAV) and a T cell beta variable domain (TRBV). In one embodiment, the MHC linkage comprises alpha and beta T cell receptor variable domains (TRAV and TRBV), wherein the TRAV domain and the TRBV domain arise from separate polypeptides, and a functional MHC linkage is produced when the polypeptides are dimerized. For example, TRAV constitutes a domain on the first polypeptide, and TRBV constitutes a domain on the second polypeptide. In some embodiments, the TRAV domain comprises a human TRAV domain. In some embodiments, the TRBV domain comprises a human TRBV domain.

[0190] In some embodiments, the TRAV or TRBV domain each contains one or more mutations for the human wild-type TRAV or TRBV domain.

[0191] Accordingly, a multispecific conjugate comprising a first polypeptide and a second polypeptide is provided herein, wherein the first polypeptide comprises an MHC conjugate domain, e.g., a TRAV domain, and the second polypeptide comprises an MHC conjugate domain, e.g., a TRBV domain and a T cell conjugate. In one embodiment, the first polypeptide comprises a TRAV domain and a multimerization domain, and the second polypeptide comprises a TRBV domain and a multimerization domain, wherein the TRAV and TRBV domains are aligned or juxtaposed upon dimerization of the first polypeptide and the second polypeptide to form an MHC conjugate.

[0192] The multimerization domain of the first polypeptide and the multimerization domain of the second polypeptide dimerize with each other after translation and may include one or more disulfide bridges.

[0193] In some embodiments, the multimerization domain may comprise a TCR alpha contact domain (TRAC) on the first polypeptide and a TCR beta constant domain (TRBC) on the second polypeptide. Various other exemplary dimerization and oligomerization domains are disclosed herein.

[0194] In some embodiments, the TRAC domain and the TRBC domain include human TRAC and human TRBC domain invariant domains.

[0195] In some embodiments, the TRAC domain or TRBC domain includes one or more mutations for the wild-type human TRAC and TRBC domains.

[0196] In one embodiment, a recombinant polypeptide construct comprising two or more polypeptides is provided herein, comprising: a first polypeptide comprising a binding domain and a dimerization domain; a second polypeptide comprising a binding domain and a dimerization domain; for example, a first polypeptide comprising a TRAV domain and a TRAC domain; and a second polypeptide comprising a TRBV domain and a TRBC domain, wherein the TRBV domain or the TRBC domain may further comprise a T cell linker domain.

[0197] In an alternative embodiment, a recombinant polypeptide construct comprising two or more polypeptides is provided herein, comprising: a first polypeptide comprising a binding domain and a dimerization domain; a second polypeptide comprising a binding domain and a dimerization domain; for example, a first polypeptide comprising a TRAV domain and a TRAC domain; and a second polypeptide comprising a TRBV domain and a TRBC domain, wherein the TRAV domain or the TRAC domain may further comprise a T cell linker domain.

[0198] A bispecific recombinant construct comprising a first polypeptide and a second polypeptide is provided herein, comprising: a first linker comprising an MHC binding material domain comprising a first binding domain on the first polypeptide and a second binding domain on the second polypeptide; and a second linker comprising a T cell linker present on the second polypeptide.

[0199] In some embodiments, for example, the cancer-specific antigen peptide for target cancer cells is a mutated / cancer antigen derived from a mutated protein, e.g., KRAS, GATA3, MAGE1, PRAME, NYESO-1, ABL1, EGFR, ERBB3, FGR3, BCR, ALK, p53, CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinogen embryonic antigen, CTLA4, crypto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin av3, LAG-3, Lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, It may be one or more of tenasin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, or VEGFR-3.In some embodiments, one of the aforementioned targets may be considered for generating a target cell-specific binding material or soluble TCR having a linker comprising a TRAV / TRBV domain, which specifically binds to an antigen peptide of one of the aforementioned exemplary gene products presented by an appropriate MHC protein, and the binding material comprises a second linker that binds to a cell surface component of a cell, e.g., a T cell.

[0200] a. MHC:peptide (p:MHC) complex

[0201] In some embodiments, the MHC-peptide complex linkage is designed based on the identification of the cancer-specific antigen to be targeted. In some embodiments, for example, the cancer-specific antigen peptide is a KRAS antigen. In some embodiments, the cancer-specific KRAS antigen includes mutations to the KRAS antigen present in non-cancerous healthy cells and normal cells and tissues. In some embodiments, (i) the activating oncogene mutation is a KRAS mutation and / or, (ii) the KRAS mutation is a G12 mutation, optionally the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations and / or, (iii) the KRAS mutation is a G13 mutation, optionally the G13 KRAS mutation is a G13D KRAS mutation and / or, or (iv) the activating oncogene mutation is an H-RAS or N-RAS mutation. KRAS antigens or epitopes, and corresponding MHC molecules encoded by HLA alleles that can bind to epitopes and be presented to T cells, can be obtained from prior research in the industry. For example, the mutant RAS peptide sequence is DTAGHEEY, TAGHEEYSAM, DILDTAGHE, DILDTAGH, ILDTAGHEE, ILDTAGHE, DILDTAGHEEY, DTAGHEEYS, LLDILDTAGH, DILDTAGRE, DILDTAGR, ILDTAGREE, ILDTAGRE, CLLDILDTAGR, TAGREEYSAM, REEYSAMRD, DTAGKEEYSAM, CLLDILDTAGK, DTAGKEEY, LLDILDTAGK, ILDTAGKE, ILDTAGKEE, DTAGLEEY, ILDTAGLE, DILDTAGL, ILDTAGLEE, GLEEYSAMRDQY, LLDILDTAGLE, LDILDTAGL, DILDTAGLE, DILDTAGLEEY, AGVGKSAL, GAAGVGKSAL, AAGVGKSAL, CGVGKSAL, ACGVGKSAL, DGVGKSAL, ADGVGKSAL,DGVGKSALTI, GARGVGKSA, KLVVVGARGV, VVVGARGV, SGVGKSAL, VVVGASGVGK, GASGVGKSAL, VGVGKSAL, VVVGAGCVGK, KLVVVGAGC, GDVGKSAL, DVGKSALTI, VVVGAGDVGK, TAGKEYSAM, DTAGHEEYSAM, TAGHEEYSA, DTAGREEYSAM, TAGKEEYSA, AAGVGKSA, AGCVGKSAL, AGDVGKSAL, AGKEEYSAMR, AGVGKSALTI, ARGVGKSAL, ASGVGKSA, ASGVGKSAL, AVGVGKSA, CVGKSALTI, DILDTAGK, DILDTAGREEY, DTAGKEEYSAMR, DTAGKEEYS, DTAGKEEYSAMR, DTAGLEEYS, DTAGLEEYSA, DTAGLEEYSAMR, DTAGREEYS, DTAGREEYSAMR, GAAGVGKSA, GACGVGKSA, GACGVGKSAL, GADGVGKS, GAGDVGKSA, GAGDVGKSAL, GASGVGKSA, GCVGKSAL, GCVGKSALTI, GHEEYSAM, GKEEYSAM, GLEEYSAMR, GREEYSAM, GREEYSAMR, HEEYSAMRD, KEEYSAMRD, KLVVVGASG, LDILDTAGR, LEEYSAMRD, LVVVGARGV, LVVVGASGV, REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK, VGASGVGKSA, The group consisting of VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD, YKLVVVGAR, and DILDTAGKE; or (b) may be selected from at least one polynucleotide encoding at least one polypeptide.

[0202] In some embodiments, the mutant RAS peptide sequence may be selected from the group consisting of KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and / or VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK.

[0203] In some embodiments, the mutant RAS peptide sequence may be selected from the group consisting of LVVVGACGV, KLVVVGACGV, LVVVGADGV, KLVVVGADGV, LVVVGAVGV, KLVVVGAVGV, VVGACGVGK, VVVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, VVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, and VVVGAVGVGK.

[0204] In some embodiments, the mutant RAS peptide sequence may be selected from the group consisting of VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, and VVVGAVGVGK.

[0205] In some embodiments, the mutant RAS peptide sequence may be selected from the group consisting of GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, or GAVGVGKSA.

[0206] In some embodiments, the mutant RAS peptide sequence comprises at least 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, or 40 amino acids.

[0207] In some embodiments, the mutant RAS peptide sequence is predicted to bind to or bind to a protein encoded by the HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele and / or HLA-C08:02 allele.

[0208] In some embodiments, the mutant RAS peptide sequence is the HLA-A02:01 allele and the HLA-A03:01 allele, the HLA-A11:01 allele, the HLA-A03:02 allele, the HLA-A30:01 allele, the HLA-A31:01 allele, the HLA-A33:01 allele, the HLA-A33:03 allele, the HLA-A68:01 allele, or the HLA-A74:01 allele; the HLA-A02:01 allele and the HLA-C08:02 allele; HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and HLA-C08:02 allele; or to the protein encoded by the HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and alleles HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele It is predicted that they will combine or combine.

[0209] In some embodiments, at least one polypeptide comprises at least one mutant RAS peptide sequence that binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM.

[0210] In some embodiments, the binding potential of a covalent antigen, such as a KRAS mutation, is predicted by MHC-peptide binding prediction analysis software, which ranks peptide:MHC pairs according to predicted binding affinities, with 1st place having the highest affinity.

[0211] Peptide sequences containing the RAS Q61H mutation, corresponding HLA alleles, and binding potential rankings. The binding potential ranking is a measure of MHC:peptide binding efficiency converted into arbitrary integer values, where '1' represents the strongest binding strength and the binding strength gradually decreases as the value increases. In some embodiments, the peptide sequences provided in Table 1 are bound to or predicted to bind to proteins encoded by HLA alleles, and those alleles are provided in the corresponding columns next to the peptide sequences in Table 1. peptide Allele Combined potential ranking ILDTAGHEEY HLA-A36:01 1 ILDTAGHEEY HLA-A01:01 2 DTAGHEEYSAM HLA-A26:01 3 DTAGHEEYSAM HLA-A25:01 4 GHEEYSAM HLA-B15:09 4 DTAGHEEY HLA-A26:01 5 ILDTAGHEE HLA-C08:02 5 AGHEEYSAM HLA-C01:02 6 AGHEEYSAM HLA-B46:01 6 DTAGHEEY HLA-A25:01 6 DTAGHEEY HLA-A01:01 6 DTAGHEEY HLA-B18:01 7 DTAGHEEY HLA-A36:01 7 ILDTAGHEE HLA-C05:01 7 ILDTAGHEE HLA-A02:07 7 ILDTAGHEEY HLA-A29:02 7 ILDTAGHEEY HLA-C08:02 7 HEEYSAMRD HLA-B49:01 8 TAGHEEYSA HLA-B35:03 8 DTAGHEEYS HLA-A68:02 9 DTAGHEEYSAMR HLA-A68:01 9 GHEEYSAM HLA-B39:01 9 ILDTAGHEE HLA-A01:01 9 LDTAGHEEY HLA-B53:01 9 HEEYSAMRD HLA-B41:01 10 ILDTAGHEE HLA-A36:01 10 DTAGHEEY HLA-B58:01 11 LLDILDTAGH HLA-A01:01 12 TAGHEEYSAM HLA-B35:03 12 LDTAGHEEY HLA-B35:01 13 DILDTAGHE HLA-A26:01 14 DTAGHEEY HLA-C12:03 14 ILDTAGHEEY HLA-C05:01 14 AGHEEYSAM HLA-A30:02 15 DILDTAGHEEY HLA-A25:01 15 DTAGHEEY HLA-C02:02 15 ILDTAGHEE HLA-C04:01 15 DILDTAGH HLA-A26:01 16 ILDTAGHEE HLA-A02:01 16 LDTAGHEEY HLA-A29:02 16 ILDTAGHE HLA-A01:01 17 LDTAGHEEY HLA-B18:01 17 AGHEEYSAM HLA-C14:03 18 DILDTAGHEEY HLA-A29:02 18 DTAGHEEYS HLA-A26:01 18 ILDTAGHEEY HLA-B15:01 18 DTAGHEEYSA HLA-A68:02 19 ILDTAGHE HLA-C05:01 19 ILDTAGHEEY HLA-A02:07 19 ILDTAGHEEY HLA-A30:02 19 LDTAGHEEY HLA-A36:01 19 AGHEEYSAM HLA-C14:02 20 AGHEEYSAM HLA-B15:03 20 LLDILDTAGH HLA-A02:07 20

[0212] In some embodiments, the peptide containing the RAS Q61R mutation comprises the sequence TCLLDILDTAGREEYSAMRDQYM. In some embodiments, the peptide containing the RAS Q61R mutation comprises the sequence provided in Table 2.

[0213] Peptide sequence containing the RAS Q61R mutation, the corresponding HLA allele, and the ranking of binding potentials obtained from the Neon MHC program 펩타이드 대립유전자 결합 잠재력 순위 ILDTAGREEY HLA-A36:01 1 ILDTAGREEY HLA-A01:01 2 DTAGREEYSAM HLA-A26:01 3 DILDTAGR HLA-A33:03 4 DILDTAGR HLA-A68:01 5 DTAGREEY HLA-A26:01 6 DTAGREEYSAM HLA-A25:01 6 CLLDILDTAGR HLA-A74:01 7 DTAGREEY HLA-A01:01 7 REEYSAMRD HLA-B41:01 7 GREEYSAMR HLA-B27:05 8 ILDTAGREE HLA-C08:02 8 ILDTAGREEY HLA-A29:02 8 REEYSAMRD HLA-B49:01 8 AGREEYSAM HLA-B46:01 9 DTAGREEY HLA-B18:01 9 DTAGREEY HLA-A25:01 9 DTAGREEY HLA-A36:01 9 DILDTAGR HLA-A74:01 10 DILDTAGRE HLA-A26:01 10 ILDTAGREE HLA-C05:01 10 DILDTAGR HLA-A26:01 11 GREEYSAM HLA-B39:01 11 AGREEYSAM HLA-B15:03 12 GREEYSAM HLA-C07:02 12 ILDTAGREE HLA-A01:01 12 TAGREEYSA HLA-B35:03 12 ILDTAGREEY HLA-A30:02 13 DTAGREEYS HLA-A68:02 14 ILDTAGRE HLA-A01:01 14 CLLDILDTAGR HLA-A31:01 15 DTAGREEYSAMR HLA-A68:01 15 LLDILDTAGR HLA-A01:01 15 DTAGREEY HLA-B58:01 16 ILDTAGREEY HLA-C08:02 16 DILDTAGR HLA-A31:01 17 ILDTAGREE HLA-C04:01 17 ILDTAGREEY HLA-A32:01 17 LLDILDTAGR HLA-A74:01 17 TAGREEYSAM HLA-B35:03 17 DILDTAGREEY HLA-A32:01 18 ILDTAGRE HLA-C05:01 18 ILDTAGREE HLA-A02:07 18 REEYSAMRD HLA-B40:01 18 AGREEYSAM HLA-B15:01 19 AGREEYSAMR HLA-A31:01 19 ILDTAGRE HLA-A36:01 19 LDILDTAGR HLA-A68:01 19 LDTAGREEY HLA-A29:02 19 LDTAGREEY HLA-B35:01 19 REEYSAMRD HLA-B45:01 19 REEYSAMRDQY HLA-A36:01 19 DTAGREEY HLA-C02:02 20

[0214] In some embodiments, the peptide containing the RAS Q61K mutation comprises the sequence TCLLDILDTAGKEEYSAMRDQYM. In some embodiments, the peptide containing the RAS Q61K mutation comprises the sequence provided in Table 3. In some embodiments, the peptide sequence provided in Table 3 binds to or is predicted to bind to a protein encoded by an HLA allele, and that allele is provided in the corresponding column next to the peptide sequence in Table 3.

[0215] Peptide sequence containing the RAS Q61K mutation, corresponding HLA allele, and binding potential ranking 펩타이드 대립유전자 결합 잠재력 순위 ILDTAGKEEY HLA-A36:01 1 ILDTAGKEEY HLA-A01:01 2 DTAGKEEYSAM HLA-A26:01 3 CLLDILDTAGK HLA-A03:01 4 DTAGKEEY HLA-A01:01 5 DTAGKEEY HLA-A26:01 5 DTAGKEEYSAM HLA-A25:01 5 AGKEEYSAM HLA-B46:01 6 DILDTAGKE HLA-A26:01 7 KEEYSAMRD HLA-B41:01 7 DTAGKEEY HLA-B18:01 8 GKEEYSAM HLA-B15:03 8 ILDTAGKEE HLA-C08:02 8 ILDTAGKEEY HLA-A29:02 8 DTAGKEEYS HLA-A68:02 9 LDTAGKEEY HLA-B53:01 9 TAGKEEYSA HLA-B35:03 9 DILDTAGK HLA-A68:01 10 DTAGKEEY HLA-A36:01 10 KEEYSAMRD HLA-B49:01 10 LDTAGKEEY HLA-C07:01 10 DTAGKEEYSAMR HLA-A68:01 11 ILDTAGKEE HLA-C05:01 11 ILDTAGKEEY HLA-C08:02 11 LLDILDTAGK HLA-A01:01 12 AGKEEYSAM HLA-A30:02 13 DTAGKEEY HLA-A25:01 13 DTAGKEEYS HLA-A26:01 13 ILDTAGKE HLA-C05:01 13 LDTAGKEEY HLA-B35:01 13 AGKEEYSAMR HLA-A31:01 14 DILDTAGK HLA-A33:03 14 ILDTAGKE HLA-A01:01 14 ILDTAGKEE HLA-A01:01 14 ILDTAGKEE HLA-A02:07 14 TAGKEEYSAM HLA-B35:03 14 AGKEEYSAM HLA-B15:01 15 ILDTAGKEEY HLA-A30:02 15 LDTAGKEEY HLA-B46:01 15 DTAGKEEY HLA-B58:01 16 ILDTAGKEEY HLA-C05:01 17 AGKEEYSAM HLA-A30:01 18 AGKEEYSAM HLA-B15:03 18 DTAGKEEY HLA-C02:02 18 LDTAGKEEY HLA-A29:02 18

[0216] In some embodiments, the peptide containing the RAS Q61L mutation comprises the sequence TCLLDILDTAGLEEYSAMRDQYM. In some embodiments, the peptide containing the RAS Q61L mutation comprises the sequence provided in Table 4. In some embodiments, the peptide sequence provided in Table 4 binds to or is predicted to bind to a protein encoded by an HLA allele, and that allele is provided in the corresponding column next to the peptide sequence in Table 4.

[0217] Peptide sequence containing the RAS Q61L mutation, corresponding HLA allele, and binding potential ranking 펩타이드 대립유전자 결합 잠재력 순위 ILDTAGLEEY HLA-A36:01 1 ILDTAGLEEY HLA-A01:01 2 LLDILDTAGL HLA-A02:07 3 GLEEYSAMRDQY HLA-A36:01 4 DTAGLEEY HLA-A25:01 5 DTAGLEEY HLA-A26:01 5 DTAGLEEYSAM HLA-A26:01 5 DTAGLEEY HLA-A01:01 6 ILDTAGLEE HLA-C08:02 6 ILDTAGLEE HLA-A01:01 6 CLLDILDTAGL HLA-A02:04 7 ILDTAGLEE HLA-A36:01 7 LLDILDTAGL HLA-A01:01 7 DILDTAGL HLA-B14:02 8 DILDTAGLEEY HLA-A25:01 8 DTAGLEEYS HLA-A68:02 8 DTAGLEEYSAM HLA-A25:01 8 GLEEYSAMR HLA-A74:01 8 ILDTAGLE HLA-A01:01 8 DILDTAGLEEY HLA-A26:01 9 DTAGLEEY HLA-A36:01 9 ILDTAGLEEY HLA-A29:02 9 DILDTAGL HLA-B08:01 10 DTAGLEEY HLA-B18:01 10 ILDTAGLEE HLA-A02:07 10 LDTAGLEEY HLA-B35:01 10 CLLDILDTAGL HLA-A02:01 11 DTAGLEEY HLA-C02:02 11 ILDTAGLEE HLA-C05:01 11 ILDTAGLEEY HLA-C08:02 11 ILDTAGLEEY HLA-A02:07 11 LLDILDTAGL HLA-C08:02 11 DILDTAGL HLA-A26:01 12 LDTAGLEEY HLA-B53:01 12 DTAGLEEY HLA-C03:02 13 DTAGLEEY HLA-B58:01 13 ILDTAGLEEY HLA-A30:02 13 LLDILDTAGL HLA-C05:01 13 LLDILDTAGL HLA-C04:01 13 DTAGLEEYSAMR HLA-A68:01 14 ILDTAGLE HLA-A36:01 15 LLDILDTAGL HLA-A02:01 15 AGLEEYSAM HLA-B15:03 16 DTAGLEEYSA HLA-A68:02 16 GLEEYSAMRDQY HLA-A01:01 16 ILDTAGLE HLA-C04:01 16 ILDTAGLEEY HLA-B15:01 16 LDILDTAGL HLA-B37:01 16 AGLEEYSAM HLA-A30:02 17 AGLEEYSAM HLA-B48:01 17 AGLEEYSAMR HLA-A31:01 17 ILDTAGLEE HLA-C04:01 17 LDTAGLEEY HLA-C03:02 17 AGLEEYSAM HLA-C14:02 18 GLEEYSAMR HLA-A31:01 18 LEEYSAMRD HLA-B41:01 18 LLDILDTAGLE HLA-A01:01 18 AGLEEYSAM HLA-C14:03 19 LDILDTAGL HLA-B40:02 19 LDTAGLEEY HLA-A29:02 19 DILDTAGLE HLA-A26:01 20 DTAGLEEY HLA-B15:01 20 ILDTAGLEEY HLA-A02:01 20 LDTAGLEEY HLA-A36:01 20 LDTAGLEEY HLA-B46:01 20 DTAGLEEY HLA-A68:02 21 DTAGLEEY HLA-C12:03 21 ILDTAGLE HLA-C05:01 21 LDTAGLEEY HLA-B18:01 21 LEEYSAMRD HLA-B49:01 21 TAGLEEYSA HLA-B54:01 21 DILDTAGLEEY HLA-A29:02 22 GLEEYSAM HLA-C05:01 22

[0218] In some embodiments, the variable domain sequence may be derived from the TCR sequence isolated and characterized in the initial study. The TCR includes a variable complementarity determining region [CDR] as well as a framework region (FR) and an invariant region. The amino acid sequence of the third complementarity determining region [CDR3] loop of the α and β chain variable domains primarily determines the sequence diversity of αβ T cells resulting from recombination between variable (Vβ), diversity (Dβ), and junction (Jβ) gene segments within the β chain locus, and recombination between similar Vα and Jα gene segments within the α chain locus, respectively. The presence of these multiple gene segments at the TCR α and β chain loci allows many distinct CDR3 sequences to be encoded. Independent addition and deletion of nucleotides at the Vβ-Dβ, Dβ-Jβ, and Vα-Jα junctions during the TCR gene rearrangement process further increase CDR3 sequence diversity. In this respect, immunogenicity is reflected in the diversity of the TCR.

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[0262]

[0263] In some embodiments, the antigen is an ovarian cancer antigen or a T lymphoma antigen.

[0264] In some embodiments, TRAV:TRBV comprises a sequence for binding to a G12D mutated RAS antigen (GADGVGKSA) presented on an MHC protein encoded by HLA:C*08:02. TRAV is a sequence

[0265] Includes LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSD (Sequence No. 619). Each CDR sequence is as follows.

[0266] CDR1: NIATNDY (Sequence No. 874)

[0267] CDR2: GYKTK (Sequence No. 875)

[0268] CDR3: LVGDMDQAGTALI(Sequence No. 876): (CDR sequence according to IMGT)

[0269] TRBV is sequence

[0270] Includes AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVV (Sequence No. 620). Each CDR sequence is as follows.

[0271] CDR1: SGHDT (Sequence No. 877)

[0272] CDR2: YYEEEE(Sequence No. 878)

[0273] CDR3: ASSLGEGRVDGYT(Sequence No. 879); (CDR sequence according to IMGT)

[0274] In some embodiments, TRAV:TRBV may include a sequence for binding specificity to any other non-RAS epitope as needed. For example, TRAV:TRBV for a multispecific recombinant soluble polypeptide may include binding specificity to a MART1 epitope presented on an MHC protein encoded by HLA:A*02:01. In this case, the antigen binding affinity (KD) of pHLA is 16 nM. For example, the TCR comprises a TRAV domain having the sequence of QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAGGGGADGLTFGKGTHLIIQP (SEQ ID NO. 621) or a sequence that is at least 80% identical to SEQ ID NO. 621; and includes a TRBV domain having the sequence of GITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSQGLAGAGELFFGEGSRLTVL (SEQ ID NO. 622) or a sequence that is at least 80% identical to SEQ ID NO. 622, wherein TRAV:TRBV binds to a MART1 epitope.

[0275] In some embodiments, TRAV:TRBV comprises a sequence for binding to a claudin epitope. In one embodiment, the claudin is expressed in a cancer cell. In one embodiment, the claudin is expressed on the surface of a cancer cell. In one embodiment, the claudin is selected from the group consisting of claudin 18.2 and claudin 6. In one embodiment, the first binding domain binds to an extracellular domain of the claudin. In one embodiment, the first binding domain binds to a natural epitope on the surface of a living cell. In one embodiment, the binder comprises a variable domain (VH) [VH(CLDN)] of the heavy chain of an immunoglobulin having specificity for the claudin antigen, and a variable domain (VL) [VL(CLDN)] of the light chain of an immunoglobulin having specificity for the claudin antigen.

[0276] II. T 세포연결체

[0277] In some embodiments, bispecific T cell linkages are designed to enhance TCR-MHC synapses of T cell:cancer cell interactions and promote T cell activation sufficiently to facilitate the activation and death of target cancer cells. The fundamental observations requiring the work disclosed herein are derived from the general observation that TCR-MHC peptide interactions are low-affinity interactions. TCR binding (binding strength, dissociation rate) facilitated by CD4 / CD8 co-receptors to MHC. Synapse formation and death require the integration of one or more signals over time (repetitive binding). Furthermore, low density of MHC:peptides is sufficient to enable recognition / death. Adhesion molecule interactions prolong and facilitate T cell target interactions. Bispecific or trispecific linkages prolong and facilitate T cell target interactions through additional fixation. Generally, co-stimulatory receptor interactions fine-tune the quality of the T cell response. One function sought to be achieved herein is sufficient activation of T cells to generate an effective response that destroys target cells. Therefore, the linker of the bispecific or trispecific linker is a T cell activator, for example, a ligand.

[0278] In some embodiments, the bispecific linker comprises one or more binding domains, wherein the binding domains comprise scFv, sdAb, VHH, or a combination thereof.

[0279] In contrast, in scFv constructs, the antibody's V L and V H The domain is contained in a single polypeptide chain. The two domains are V L and V H The domain is separated by a flexible linker long enough to self-assemble into a functional epitope binding site.

[0280] In some embodiments, one or more binding domains of a T cell linkage bind to an extracellular domain of an endogenous receptor expressed by a T cell. In some embodiments, one or more binding domains of a T cell linkage bind to an extracellular domain of a receptor selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICAM-1, and CD8. In some embodiments, one or more binding domains of a T cell linkage bind to CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, a first binding domain of a T cell linkage that binds to an extracellular domain of a first receptor expressed by a T cell comprises a first VHH, and a second binding domain of a T cell linkage that binds to an extracellular domain of a second receptor expressed by a T cell comprises a second VHH. In some embodiments, (i) the C-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the C-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. In some embodiments, (i) the N-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the N-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. In some embodiments, the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by the T cell comprises scFv, and the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by the T cell comprises VHH. In some embodiments, a first binding domain that binds to an extracellular domain of a first receptor expressed by a T cell and a second binding domain that binds to an extracellular domain of a second receptor expressed by a T cell are connected by a peptide linker.

[0281] In some embodiments, a first binding domain on a T cell linker may be connected to a second binding domain through a flexible peptide linker. In some embodiments, the flexible linker may have a length of 2 to 50 amino acids.

[0282] scFv comprises a light chain variable domain and a heavy chain variable domain. In some embodiments, the light chain variable domain and the heavy chain variable domain may comprise a short peptide linker comprising at least 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 or more amino acids between the light chain variable domain and the heavy chain variable domain.

[0283] In some embodiments, scFv is humanized.

[0284] Humanized scFv contains a 'complementary determining region' [CDR] that exists on the framework of an immunoglobulin of a different species compared to the framework of the maternal immunoglobulin from which the CDR is derived. For example, a 'humanized antibody' can be produced by transplanting a rat CDR into the framework region of a human antibody.

[0285] In some embodiments, a short linker connecting (a) and (b) to an operable state may have additional functions. In some embodiments, the peptide may bind to a specific cell surface receptor, such as a CD3 receptor, and activate a receptor-mediated cell signaling pathway in T cells. In some embodiments, the linker is designed to bind to and activate at least one inflammatory pathway within a T cell or to enhance T cell-mediated apoptosis of the target cell. In some embodiments, the linker peptide may have the function of blocking or inhibiting the downregulation of target cell-mediated T cell function. In some embodiments, a nucleic acid construct for expressing a bispecific scFv linker comprises an N-terminal signaling peptide sequence for the secretion of the bispecific scFv linker.

[0286] In one embodiment, the exemplary CD3 binding domain is a scFv. In one embodiment, the anti-CD3 binding material is a scFv, and specific domains and subdomains, their respective sequences and / or variants are listed in Table 6A. In one embodiment, the anti-CD3 scFv comprises a sequence that is at least 80% identical to SEQ ID NO. 601. In some embodiments, the exemplary CD3 binding domain is a scFv comprising a sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 601. In one embodiment, the anti-CD3 scFv comprises a sequence that is at least 80% identical to SEQ ID NO. 602. In some embodiments, the exemplary CD3 binding domain is a scFv containing a sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 602. In one embodiment, the anti-CD3 scFv contains a sequence that is at least 80% identical to SEQ ID NO. 612, 613, or 614. In some embodiments, the exemplary CD3 binding domain is a scFv containing a sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 612. In some embodiments, the exemplary CD3 binding domain is a scFv containing a sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 613. In some embodiments, the exemplary CD3 binding domain is a scFv containing a sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 614.In some embodiments, an exemplary CD3 binding domain is an scFv comprising the sequence of SEQ ID NO. 601, 602, 612, 613, or 614. In some embodiments, an exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 80% identical to SEQ ID NO. 603. In some embodiments, an exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 85% identical to SEQ ID NO. 603. In some embodiments, an exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO. 603. In some embodiments, an exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 603. In some embodiments, an exemplary CD3 binding domain comprises a heavy chain variable domain having the sequence of SEQ ID NO. 603. In some embodiments, the exemplary CD3 binding domain comprises a light chain variable domain that is at least 80% identical to SEQ ID NO. 604. In some embodiments, the exemplary CD3 binding domain comprises a light chain variable domain that is at least 90% identical to SEQ ID NO. 604. In some embodiments, the exemplary CD3 binding domain comprises a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 604.

[0287] [Table 6A]

[0288] CD3 결합 도메인 및 서열

[0289]

[0290]

[0291] In one embodiment, the exemplary CD3 binding domain is VHH. In one embodiment, the anti-CD3 binding material is VHH, and specific domains and subdomains, their respective sequences and / or variants are listed in Table 6B.

[0292] In some embodiments, the exemplary CD3 binding domain is VHH. In some embodiments, the CD3 binding VHH comprises a heavy chain variable domain that is at least 80% identical to SEQ ID NO. 615. In some embodiments, the exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO. 615. In some embodiments, the exemplary CD3 binding domain comprises a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO. 615.

[0293] In some embodiments, the exemplary CD3 binding material is VHH, and the CD3 binding domain comprises HCDR3 of SEQ ID NO. 618. In some embodiments, the exemplary CD3 VHH comprises HCDR1 having the sequence of SEQ ID NO. 616 and HCDR2 having the sequence of SEQ ID NO. 617.

[0294] [Table 6B]

[0295] CD3 결합 도메인 및 서열

[0296]

[0297] [Table 6C]

[0298] CD3 결합 도메인 및 서열

[0299]

[0300]

[0301] In one embodiment, a scFv, e.g., the scFv of SEQ ID NO. 669 or SEQ ID NO. 670, is designed from the anti-CD3 antibody TR66. In one embodiment, the anti-CD3 antibody sequence TR66 comprises the sequence of SEQ ID NO. 673. Several synthetic constructs were constructed and examined to determine whether unpaired cysteine ​​residues occurring within the primary sequence of the Ig domain would interfere with the correct formation of intra-chain and / or inter-chain disulfide bonds, which are essential for the proper folding and stability of the resulting antibody fragment. Such unpaired cysteine ​​should be avoided as it can impair the efficacy, homogeneity, productivity, and stability of the final protein product. In addition to the 'standard' set of cysteine ​​involved in disulfide pair formation, free cysteine ​​residues may be present within the variable domain. For example, in the VH domain of the OKT3 antibody, a conserved cysteine ​​located three residues ahead of the CDR-H3 start site is present at position H92 and forms a structural disulfide bond with position H22. However, another cysteine ​​(Cys) at the H100A position (CDR-H3) of this molecule enables a misfolding phenomenon in which H100A forms a disulfide bond with H22 instead of H92, which can produce a misfolded, insoluble, and non-functional product. To overcome this potential mispairing of cysteine ​​residues, a positional substitution for free cysteine ​​was performed (reference [Kipriyanov, Protein Engineering 10:445-453, 1997]). Through this single substitution, a significant increase in the productivity and stability of scFv derived from OKT3 was achieved while maintaining overall binding activity. The VH domain of the current unmodified anti-CD3 antibody TR66 (SEQ No. 679) contains this free cysteine ​​at position 114 of the sequence shown in SEQ No. 669. Comparing the VH domain of the anti-CD3 antibody TR66 with the VH domain of the anti-CD3 antibody OKT3 reveals 96.6% sequence homology.

[0302] Substitution of free cysteine ​​by a serine residue within CDR-H3 of the VH domain of the anti-CD3 antibody TR66 against bi-scFv proteins targeting CD3 was performed. This is reflected in SEQ ID NO. 669, or SEQ ID NO. 670, or SEQ ID NO. 672, or SEQ ID NO. 717. In some embodiments, the anti-CD3 antibody is HCDR3YYDDHY according to the Kavat nomenclature. S The underlined position of LDY (sequence number 717) contains serine (C114S) instead of cysteine.

[0303] In some embodiments, cysteine ​​is not substituted with serine. In some embodiments, the unsubstituted cysteine ​​is the HCDR3 sequence YYDDHY C It is evident in the sequence containing LDY (sequence number 675).

[0304] In some embodiments, the anti-CD3 binding material is QVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYCLDY WGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTC RASSSVSYMN WYQQKSGTSPKRWIY DTSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT It is a scFv containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of FGAGTKLELK (Sequence No. 725).

[0305] In some embodiments, the anti-CD3 binding material is QVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTC RASSSVSYMN WYQQKSGTSPKRWIY DTSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT It is a scFv comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of FGAGTKLELK (SEQ No. 669). This orientation is a VH-VL arrangement from the N-terminus to the C-terminus. In some embodiments, the scFv comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ No. 669. In some embodiments, the scFv comprises the amino acid sequence of SEQ No. 669.

[0306] In some embodiments, the anti-CD3 binding material is QIVLTQSPAIMSASPGEKVTMTC RASSSVSYMN WYQQKSGTSPKRWIY DTSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT FGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDY It is a scFv comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of WGQGTTLTVSS (SEQ No. 670). This orientation is a VL-VH arrangement from the N-terminus to the C-terminus. In some embodiments, the scFv comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ No. 670. In some embodiments, the scFv comprises the amino acid sequence of SEQ No. 670.

[0307] In some embodiments, the CD3 binding domain is the sequence QVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDY WGQGTTLTVSS(sequence number 671); or

[0308] QVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYCLDY It includes a heavy chain variable domain that is at least 80% identical to WGQGTTLTVSS (SEQ ID NO. 726), at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical.

[0309] In some embodiments, the CD3 binding domain is the sequence QIVLTQSPAIMSASPGEKVTMTC RASSSVSYMN WYQQKSGTSPKRWIY DTSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT It includes a light chain variable domain that is at least 80% identical to FGAGTKLELK (SEQ ID NO. 672), at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical.

[0310] In some embodiments, the CD3 binding material scFv may have an array of VH-linker-VL having a sequence that is at least 80% identical to the sequence QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (Sequence No. 727).

[0311] In some embodiments, the CD3 binding material scFv may have an array of VL-linker-VH having a sequence that is at least 80% identical to the sequence QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS (Sequence No. 728).

[0312] In some embodiments, the CD3 binding domain comprises HCDR1, 2 and 3 and LCDR1, 2 and 3 as shown below, according to the Kavat and IMGT nomenclature as shown below.

[0313] [Table 6D]

[0314] CDR sequence and variant of TR66 antibody.

[0315]

[0316] In some embodiments, the anti-CD3 binding domain (scFv) comprises the sequence QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (Sequence No. 725).

[0317] In some embodiments, the anti-CD3 binding domain (scFv) comprises the sequence QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 728).

[0318] In some embodiments, the anti-CD3 binding domain (scFv) comprises the sequence KLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 679).

[0319] In some embodiments, compositions for a bispecific sTCR or a multispecific sTCR having a novel anti-CD3 binding agent are provided herein. In some embodiments, the anti-CD3 binding agent is a scFv. The anti-CD3 binding agent comprises a variable heavy chain domain (VH domain). The anti-CD3 binding agent comprises a variable light chain domain (VL domain).

[0320] [Table 6E]

[0321] CD3 binding domain and sequence

[0322]

[0323]

[0324] In one embodiment, the anti-CD3 binding domain comprises the heavy chain CDR3 sequence GIYSDSSDFIGNL (SEQ No. 707). In some embodiments, the heavy chain CDR1 sequence of SEQ No. 705. In some embodiments, the heavy chain CDR2 sequence of SEQ No. 706. In one embodiment, the anti-CD3 binding domain comprises the light chain CDR3 sequence QQYNTIINVDRT (SEQ No. 710). In one embodiment, the anti-CD3 binding domain comprises the light chain CDR1 sequence (SEQ No. 708). In some embodiments, the light chain CDR2 sequence of NNYYMC (SEQ No. 709).

[0325] In some embodiments, the anti-CD3 scFv comprises an anti-CD3 binding domain, wherein the binding domain comprises at least 60% of the heavy chain variable domain sequence of the sequence of SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 70% sequence identity with SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 75% sequence identity with SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 80% sequence identity with SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 85% sequence identity with SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 90% sequence identity with SEQ ID NO. 703. In some embodiments, the heavy chain sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO. 703.

[0326] In some embodiments, the anti-CD3 scFv comprises an anti-CD3 binding domain, wherein the binding domain comprises at least 60% of the light chain variable domain sequence of the sequence of SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 70% sequence identity with SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 75% sequence identity with SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 80% sequence identity with SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 85% sequence identity with SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 90% sequence identity with SEQ ID NO. 704. In some embodiments, the light chain sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO. 704.

[0327] In some embodiments, the T cell linkage comprises one or more binding domains for T cell surface molecules, for example, receptors. In some embodiments, the T cell linkage comprises a scFv for binding to a first receptor expressed on the T cell and a VHH for binding to a second receptor also expressed on the same T cell. An exemplary sTCR design may comprise a TCR linkage comprising, for example, a first binding domain that is an anti-CD3 scFv and a second binding domain that is an anti-CD2 VHH, or vice versa. In some embodiments, the first binding domain may be anti-CD3 VHH and the second binding domain may be anti-CD2 VHH. In some embodiments, the first binding domain of the T cell linkage may be anti-CD2 VHH and the second binding domain may be anti-CD3 VHH. In some embodiments, the exemplary CD2 binding domain is an anti-CD2 VHH comprising the sequence QVQLVESGGGLVQAGGSLRLSCAASGLTFSSYGMAWFRRALGREREFVGAIRPGTIPYYSESVKGRFTVSKDNAKNTVSLQMNSLKPEDTAVYYCAAVRDYVGMPYYSGSAYEYWGQGTQVTVSS (SEQ No. 623) or a heavy chain variable domain having a sequence that is at least 80% identical to SEQ No. 623. In some embodiments, the T cell linker comprises an anti-CD2 binding domain comprising a heavy chain variable region [VH] comprising the sequence of heavy chain complementarity determining region 3 [HC CDR3] of AAVRDYVGMPYYSGSAYEY (SEQ No. 749) or VRDYVGMPYYSGSAYEY (SEQ No. 750). In some embodiments, the VH of the anti-CD2 binding domain comprises the HC CDR2 sequence of IRPGTIP (SEQ No. 751) or AIRPGTIPYYSESVKG (SEQ No. 752). In some embodiments, the VH of the anti-CD2 binding domain comprises the HC CDR1 sequence of GLTFSSYG (SEQ No. 753) or SYGMA (SEQ No. 754).

[0328] In some embodiments, the anti-CD2 binding domain is a VHH domain. In some embodiments, the anti-CD2 binding domain comprises the HC CDR1 sequence of GLTFSSYG (SEQ No. 753), the HC CDR2 sequence of IRPGTIP (SEQ No. 751), and the HC CDR3 sequence of AAVRDYVGMPYYSGSAYEY (SEQ No. 749). In some embodiments, the anti-CD2 binding domain comprises the HC CDR1 sequence of SYGMA (SEQ No. 754), the HC CDR2 sequence of AIRPGTIPYYSESVKG (SEQ No. 752), and the HC CDR3 sequence of VRDYVGMPYYSGSAYEY (SEQ No. 750).

[0329] In some embodiments, the T cell linkage component of the multispecific linkage comprises a binding domain capable of specifically binding to a T cell receptor constant domain. For example, it is a TCR alpha constant (TRAC) domain or a TCR beta constant (TRBC) domain. An exemplary T cell linker comprises an anti-TRBC1 scFv domain as an arm of a multispecific molecule and may comprise a scFv known as JOVI-1 having the sequence EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (SEQ ID No. 624) or a fragment thereof. In some embodiments, the TRBC1 binding domain comprises a sequence that is at least 80% identical to SEQ ID No. 624. In some embodiments, the TRBC1 binding domain comprises a sequence that is at least 90% identical to SEQ ID NO. 624. In some embodiments, TRBC1 comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 985%, and 99% identical to SEQ ID NO. 624. In some embodiments, the TRBC1 binding domain comprises a heavy chain variable domain comprising the sequence EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (SEQ ID NO. 625) or a sequence that is at least 80% identical to SEQ ID NO. 625.In some embodiments, the anti-TRBC1 scFv domain comprises a heavy chain CDR that is HCDR1: GYVMH (SEQ No. 737), HCDR2: FINPYNDDIQSNERFRG (SEQ No. 738); HCDR3: GAGYNFDGAYRFEDF (SEQ No. 739) as described according to Kavat nomenclature, or HCDR1: GYTFTGYV (SEQ No. 740), HCDR2: INPYNDDI (SEQ No. 741); and HCDR3: ARGAGYNFDGAYRFEDF (SEQ No. 742) as described according to IMGT nomenclature.

[0330] In some embodiments, the TRBC1 binding domain comprises the sequence DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (SEQ No. 626) or a light chain variable domain comprising a sequence that is at least 80% identical to SEQ No. 626. In some embodiments, the anti-TRBC1 scFv domain comprises LCDR1: RSSQRLVHSNGNTYLH (SEQ No. 743), LCDR2: RVSNRFP (SEQ No. 744); LCDR3: SQSTHVPYT (SEQ No. 745) as described according to the Kavat nomenclature; or LCDR1: QRLVHSNGNTY (SEQ No. 746), LCDR2: RVS (SEQ No. 747) as described according to the IMGT nomenclature; and includes a light chain CDR which is LCDR3: SQSTHVPYT (sequence number 745).

[0331] In some embodiments, the TRAC1 binding scFv comprises the sequence DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (SEQ ID NO. 748) or a sequence that is at least 80% identical to SEQ ID NO. 748. The CDR sequences are VH CDR 1: GYTFTGYV (sequence number 740), VH CDR2: INPYNDDI (sequence number 741), VH CDR3: ARGAGYNFDGAYRFEDF (sequence number 742); VL CDR1: QRLVHSNGNTY (sequence number 746), VL CDR2: RVS (sequence number 747), VL CDR3: SQSTHVPYT (sequence number 745)(IMGT).

[0332] In some embodiments, the T cell linkage component of the multispecific linkage comprises a binding domain capable of specifically binding to CD8. In some embodiments, the T cell linkage component of the multispecific linkage comprises a binding domain capable of specifically binding to CD8a. In some embodiments, the CD8a binding domain is scFv. In some embodiments, the CD8a binding domain is sdAb. In some embodiments, the CD8a binding domain is VHH.

[0333] Scaffold Design Manipulation for Tumor Cell-Specific Multispecific Soluble TCR Linkers

[0334] A composition comprising a recombinant nucleic acid expressed in mammalian cells is provided herein, wherein the recombinant nucleic acid comprises a sequence encoding a multispecific molecule, and the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the composition is an engineered T cell receptor (TCR) construct. In one embodiment, a composition comprising a recombinant nucleic acid having a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain, wherein the peptide within the MHC-peptide complex comprises a RAS peptide sequence, and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell. The multispecific T cell conjugate does not have any transmembrane domains.

[0335] In some embodiments, the multispecific molecule is soluble.

[0336] In some embodiments, the multispecific molecule lacks a transmembrane domain.

[0337] In some embodiments, the multispecific molecule consists of two polypeptides, namely a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and the second polypeptide comprise one or more disulfide bonds between the two polypeptides. In some embodiments, the multispecific molecule comprises the first polypeptide and the second polypeptide, wherein the first polypeptide comprises a TRAV domain and the second polypeptide comprises a TRBV domain. In some embodiments, the peptide of the MHC-peptide complex is a peptide derived from a cancer antigen. In some embodiments, the peptide of the MHC-peptide complex is a mutant peptide, and the MHC of the MHC-peptide complex binds to the mutant peptide with a higher affinity compared to the corresponding wild-type peptide. In some embodiments, the MHC of the MHC-peptide complex comprises a class I MHC. In some embodiments, the MHC of the MHC-peptide complex is a class I MHC polypeptide. In some embodiments, the MHC of the MHC-peptide complex is encoded by HLA that appears in less than 1% of the human population. In some embodiments, the MHC of the MHC-peptide complex has a peptide binding affinity greater than at least 50 nM. In some embodiments, one or more binding domains of the T cell linkage bind to the extracellular domain of an endogenous receptor expressed by the T cell.

[0338] In one embodiment, one or more binding domains of a T cell linkage bind to an extracellular domain of a receptor selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICAM-1, and CD8. In some embodiments, one or more binding domains of a T cell linkage bind to CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, one or more binding domains of a T cell linkage comprise an antibody domain or an antigen-binding fragment thereof. In some embodiments, the binding domain of a T cell linkage comprises scFv or sdAb. In some embodiments, one or more binding domains of a T cell linkage comprise VHH.

[0339] In the scaffold design considered herein, one or more binding domains of a T cell linkage comprise a first binding domain that binds to an extracellular domain of a first receptor expressed by a T cell and a second binding domain that binds to an extracellular domain of a second receptor expressed by a T cell. In some embodiments, the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by a T cell comprises a first VHH, and the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by a T cell comprises a second VHH.

[0340] In some embodiments, (i) the C-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the C-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. Or, in some embodiments, (i) the N-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the N-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage.

[0341] In some embodiments, the first binding domain of a T cell linker that binds to the extracellular domain of a first receptor expressed by a T cell comprises scFv, and the second binding domain of a T cell linker that binds to the extracellular domain of a second receptor expressed by a T cell comprises VHH. In some embodiments, the first binding domain that binds to the extracellular domain of a first receptor expressed by a T cell and the second binding domain that binds to the extracellular domain of a second receptor expressed by a T cell are connected by a peptide linker.

[0342] In some embodiments, the first binding domain binds to the extracellular domain of a first receptor expressed by a T cell, selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8, and the second binding domain binds to the extracellular domain of a second receptor expressed by a T cell, selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8. In some embodiments, the first receptor expressed by a T cell and the second receptor expressed by a T cell are different. In some embodiments, the first receptor expressed by a T cell is CD3, and the second receptor expressed by a T cell is CD2. In some embodiments, the first receptor expressed by a T cell is CD2, and the second receptor expressed by a T cell is CD3. In some embodiments, the first binding domain and the second binding domain of the T cell linkage are configured to bind to the same receptor expressed by a T cell. In some embodiments, the T cell linkage comprises an anti-CD3 binding domain. In some embodiments, the T cell linkage comprises two or more anti-CD3 binding domains. In some embodiments, the T cell linkage comprises an anti-TRBC1 binding domain. In some embodiments, the T cell linkage comprises an anti-CD2 binding domain. In some embodiments, the T cell linkage comprises (i) a first anti-CD3 binding domain, (ii) a second anti-CD3 binding domain, (iii) an anti-TRBC1 binding domain, (iv) an anti-CD2 binding domain, or (v) any combination of (i) to (iv).

[0343] The examples described in the previous section can be used in various ways to design and construct multispecific linkages, including modifications and variations that can be easily conceptualized by a person of ordinary skill using simple molecular biology techniques. For example, the VH and VL domain sequences within scFv can be taken for redesign into sdAb, VHH, etc. Various technical considerations were taken into account and utilized in parts of the resulting structure, including, for example, exchanging the N-terminal domain of the linker sequence to the C-terminal regarding the linker.

[0344] For example, the literature [Arndt et al. (Biochemistry 37:12918-12926, 1998)] describes so-called domain exchange as a possible explanation for the emergence of non-covalent oligomers of scFv fragments. Under this model, the protein state is placed in a possible thermodynamic equilibrium between the monomeric form and the dimeric / oligomeric form due to intramolecular and intermolecular exchanges at the continuously occurring VL / VH interface contacts. These oligomers may already be present in the cell culture supernatant and must be removed during the purification process. However, these molecular species can also be formed during the storage of the purified monomeric species. The preferred energy state of a protein is significantly influenced by the overall design (primary sequence, linker length, VL / VH orientation, etc.). The literature [Worn and Pltickthun (JMB 305:989-1010, 1999)] noted that using a linker with more than 20 residues can yield forms with a higher content of monomeric species. The literature [Desplancq et al., (Protein Eng. 7:1027-1033, 1994)] indicated that variable domain orientation can also influence the formation of dimeric and polymeric forms. In the same publication, Desplancq showed that linkers of 25 or 30 amino acids (amino acids, aa) exhibited the optimal ratio of monomers to dimers of their specific antibodies. The distance between the C-terminus of VL and the N-terminus of VH is approximately 39–43 Å, and the distance between the C-terminus of VH and the N-terminus of VL is 32–34 Å (Reference [Pltickthun et al., From PCR to fermentation. (J. McCafferty, HRHoogenboom, & DJ Chriswell, Eds.). In: (IRL Press., pp. 203-252, 1996)]. To obtain similar molecular properties, the linker for the oriented VL-VH must be longer than the VH-VL linker.The literature [Plockthun et al., (From PCR to fermentation. (J. McCafferty, HR Hoogenboom, & DJ Chriswell, Eds.). In: (IRL Press., pp. 203-252, 1996)] recommends using 15 or 20 amino acid-long linkers in VH / VL orientations and 20 or 25 amino acid-long linkers in VL / VH orientations. Another possibility to force monomer formation and stabilize VH / VL domain interactions is to manipulate interfacial disulfide bonds as contact surfaces between the two domains. The introduction of disulfide bridges at positions H44–L100 (Kavart numbering) has been most frequently used in scFvs with satisfactory results (Brinkmann et al., PNAS. 90:7538-7542, 1993; Worn and Plockthun, Biochemistry 38: [8739-8750, 1999; Weatherill et al., PEDS. 25:321-329, 2012]). This strategy has been successfully used to stabilize IgG-like bispecific antibodies combining scFv fused to full-length IgG (Michaelson et al., mAbs 1: 128-141, 2009; Schanzer et al., Antimicrob. Agents. Chemother. 55:2369-2378, 2011]). [Weatherill et al., (PEDS 25:321-239, 2012)] stabilized human scFv [VH-(G4S)4-VL and VL-(G4S)4-VH] via disulfide binding between the VH44 and VL-100 positions. Additionally, this publication [weatherill et al., (PEDS 25:321-239, 2012)] showed different results at different loading volumes and concentrations. By performing SE-HPLC experiments, we address the problem of domain exchange that may occur in scFvs that do not contain interfacial disulfide bonds.In the case of unstabilized scFv, the analysis results varied depending on the sample loading conditions; however, regardless of the conditions used, molecules stabilized by disulfide bonds eluted like monomers. Zhao et al. (Int. J. Mol. Sci. 12:1-11, 2011) observed that the stability of the stabilized molecules was higher after introducing the same mutant into scFv and storing it at 37°C for 20 hours. For the bispecific formulation using scFv fused to full-length IgG, Schanzer et al. (Antimicrob. Agents Chemother. 55:2369-2378 2011) compared the effects of linker length and interfacial disulfide bonding. They fused parent scFv or scdFv[VH-(G4S)3-VL] to the C-terminal or N-terminal portions of the heavy or light chains. For different linker lengths (20, 25, and 30 amino acids), the parent scFv was fused to the C-terminal portion of the heavy or light chain. The results obtained with different linker lengths confirmed that the 30 aa peptide is a more desirable linker for the production of stable monomers. After storage at 40°C for 7 days, the aggregate levels were 50% for scFv15, 18% for scFv20, 8% for scFv25, and 6% for scFv30. However, the disulfide scFv15 stabilized by interfacial disulfide bonding was slightly superior to scFv30. The same approach was used by the literature [Michaelson et al., (mAbs, 1:128-141 2009)], and they improved their IgG-like bispecific parent antibody (40% aggregate production) containing a scFv with a 15 aa linker oriented in VH / VL by increasing the linker length of the scFv to 20 aa and introducing an interfacial disulfide bond between the VH44 and VL-100 positions. The resulting molecule produced more than 98% stable monomers after 3 months at 4°C.The authors decided to undertake improvements to the scFv molecule before moving on to the bispecific form.

[0345] For the anti-RAS specific bispecific proteins described in this disclosure, it is not known whether the formation of dimeric and macromolecular forms may occur and what the effect is on the anti-RAS and / or anti-CD3 scFv molecules. To evaluate the optimal whole molecule for the anti-RAS specific bi-scFv protein for each isolated scFv, the following modifications are evaluated: domain orientation, linker length, introduction of interfacial disulfide bonds, and combinations of the three modifications.

[0346] In another example, a person skilled in the art would readily conceive of adding a self-cleavable sequence, such as T2A, P2A, or F2A, between adjacent domains, within a linker, or between two amino acid chains to produce a mature protein. An exemplary self-cleavable sequence is T2A: TNFSLLKQAGDVEENPGP (Sequence No. 627). A person skilled in the art would also readily conceive of concatenating or inserting a tag sequence, such as a HIS tag, to any region of the composition. Considering the sequence and description, a person skilled in the art could easily achieve the functionalization of the linker sequence as known in the art.

[0347] In some embodiments, a recombinant nucleic acid sequence encoding a first polypeptide and a second polypeptide for a bispecific molecule is described herein, and the molecule comprises: (a) a nucleic acid sequence encoding a TRAV / TRBV domain fused to a nucleic acid sequence encoding TRAC / TRBC, which binds to a cancer-specific peptide within an MHC complex on a cancer cell; (b) a VHH domain that binds to a cell surface component of an effector cell, e.g., a T cell; and (c) a short linker connecting (a) and (b) in an operable state.

[0348] In some embodiments, a nucleic acid sequence encoding a polypeptide containing a VHH or scFv binding domain may be inserted into a suitable expression vector under one or more promoters, for example, a CMV at the 5' end and a polyadenylation signal at the 3' end of the sequence encoding the polypeptide.

[0349] In some embodiments, the composition may include an internal ribosomal entry site (IRES), and, for example, the IRES may be located before a nucleic acid sequence encoding one or more polypeptides.

[0350] In some embodiments, the nucleic acid sequence encoding one of the polypeptides may be placed under the control of a separate promoter from the rest of the sequences that are expressed.

[0351] In some embodiments, the trispecific linker is a fusion construct of three binding material domains, and is a p-MHC linker comprising, for example, a TRAV / TRBV domain specific to a cancer-specific peptide within an MHC complex on a cancer cell in this case, a first T cell linker arm specific to a cell surface component on a T cell (e.g., a binding domain, scFv, or VHH), and a second T cell linker arm specific to another cell surface component on the same T cell (a binding domain, e.g., scFv or VHH). In some embodiments, the trispecific linker is designed so that a cell surface component on a T cell to which the third binding domain can bind provides an additional activation signal to the T cell, thereby triggering apoptosis of the target cell. In some embodiments, the third binding domain binds to another T cell receptor on the T cell. In some embodiments, the third binding domain binds to a cytokine receptor that activates the receptor and triggers intracellular signaling of the T cell. In some embodiments, the third binding domain is CD3 or a fragment thereof.

[0352] In some embodiments, the multispecific molecule may include one or more linkers or spacers. The linker or spacer may consist of 2 to 50 amino acids. In some embodiments, the linker may include 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 or more amino acids. In some embodiments, the linker comprises 3 to 30 amino acids. In some embodiments, the linker is a peptide with a length of 4 to 20 or 5 to 10 amino acids. In some embodiments, the peptide linker has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptide linker, or two linker peptides having an anchor or clasp, together span a length of 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, 35 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less.In some embodiments, two linker peptides having a peptide linker, or an anchor or clasp, are together over a length of 25 amino acids or less, 24 amino acids or less, 23 amino acids or less, 22 amino acids or less, 21 amino acids or less, 20 amino acids or less, 19 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, or 5 amino acids or less.

[0353] In some embodiments, the linker may include one or more amino acids, each amino acid being connected to an adjacent amino acid through a natural peptide bond, for example, a bond between the amino group of one amino acid and the carboxyl group of an adjacent amino acid. In some embodiments, the antigen-binding domain, scFv, or binding domain is connected to one another by the linker. In some embodiments, if there is more than one scFv, the more than one scFv are connected to one another by the linker.

[0354] In some embodiments, the linker is flexible. In some embodiments, the linker includes a hinge region. The linker is generally a short peptide sequence. In some embodiments, the linker is a stretch of glycine and one or more serine residues. Other amino acids preferred for the peptide linker include, but are not limited to, threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and alanine (Ala), arginine (Arg), phenylalanine (Phe), and glutamic acid (Glu). Among these, Pro, Thr, and Gln are amino acids frequently used as natural linkers. Pro is a unique amino acid with a cyclic side chain, which results in a very restricted structure. Pro-rich sequences can be used as inter-domain linkers, including the linker between the lipoyl and E3 binding domain of pyruvate dehydrogenase (GA2PA3PAKQEA3PAPA2KAEAPA3PA2KA) (SEQ No. 628). Experimental linkers may be flexible linkers, rigid linkers, and cleavage linkers. Sequences such as (G4S)x (where x is a multiple copy of a moiety designated as 1, 2, 3, 4, etc.) contain flexible linker sequences. Other flexible sequences used herein include multiple repeats of glycine, e.g., (Gly)6 or (Gly)8. On the other hand, rigid linkers may be used, for example, the linker (EAAAK)x (where x is an integer, 1, 2, 3, 4, etc.) creates a rigid linker. In some embodiments, the linker or spacer may consist of a non-reactive amino acid moiety, for example, a series of glycine, serine, or alanine residues. An exemplary linker may include the amino acid sequence GSGS, or SGGG, or SGGGGSG (SEQN 629). An exemplary linker may include the amino acid sequence SSGGGGSGGGGSGGGGS (SEQN 630).

[0355] The length of the linker peptide can be important in the design of a multispecific linker. For example, limiting the linker peptide length to fewer than 10 amino acids restricts binding between two adjacent domains. In some embodiments, the linker may include a fixing or latch function and may include a crosslinking moiety. The crosslinking moiety may be a peptide or a chemical crosslinking moiety.

[0356] In some embodiments, the peptide linker may additionally function as a conditionally cleavable linker. Conditionally cleavable can be understood as the peptide being cleaved when an agent cleaving the peptide is available. Conditionally cleavable can be understood as the peptide being cleavable when the agent cleaving the peptide is activated, for example, when the enzyme is activated if the agent cleaving the peptide is an enzyme. Conditionally cleavable can be understood as the peptide being cleavable when the agent cleaving the peptide is in an environment that enables the activation of the agent, or when a substance that activates the agent is present.

[0357] In some embodiments, the peptide linker may additionally function as a targeting peptide. Any one or more peptide linkers may include special functions, such as forming a dimer, trimer, or multimer. In some embodiments, one or more linkers may include a leucine zipper sequence.

[0358] In some embodiments, the synthetic clasp of the linker is a non-peptide crosslinking agent.

[0359] In some embodiments, mutually complementary bonding of homologous peptides may be achieved through chemical bonding, such as crosslinking. Chemical crosslinking agents may be useful for activating crosslinking in vitro. There are commercially available homologous and heterofunctional protein crosslinking agents. Examples include the BS2G crosslinking agent (BS), which is an amine-reactive, water-soluble, homofunctional crosslinking agent (in which both bonding units at opposite ends of the spacer arm have the same reactive group). 2 G; bis[sulfosuccinimidyl]glutarate), or its membrane-permeable version DSG (di(N-succinimidyl)glutarate), is included, and BS2G crosslinker (bis[sulfosuccinimidyl]suberate; sulfo-DSS; BSSS) or DST crosslinker (disuccinimidyl tartrate) belong to other homofunctional crosslinkers for peptides, while BMPS (N-(β-maleimidopropyloxy)succinimide ester); MBS crosslinker (m-maleimidobenzoyl-N-hydroxysuccinimide ester); PDPH crosslinker (3-[2-pyridyldithio]propionyl hydrazide) provide examples of some heterofunctional crosslinkers.

[0360] In some embodiments, variable light chains arranged in series within a multispecific connector [V L ] Subunit and variable heavy chain[V H The ] region can be connected via two linkers having homologous peptide fixation or latch elements. The length of the linker is a specific V L -V H It can restrict or promote binding. For example, restricting the linker peptide length to fewer than 10 amino acids is adjacent V L and V H Restricts coupling between domains.

[0361] In some embodiments, the fixing or latching element is 5x10 -6 Less than M, or 10 -6 Less than M, 5x10 -7Less than M, or 4x10 -7 Less than M, or 3x10 -7 Less than M, or 2x10 -7 Less than M; or 10 -7 Less than M, or 9x10 -8 Less than M, or 8x10 -8 Less than M, or 7x10 -8 Less than M, or 6x10 -8 Less than M, or 5x10 -8 Less than M, or 4x10 -8 Less than M, or 3x10 -8 Less than M, or 2x10 -8 Less than M, or 10 -8 Less than M, or 10 -8 Less than M, or 10 -10 K less than M D Indicates an affinity having or a higher affinity.

[0362] In addition, including additional fixation or heteropolymerization domains in these higher-order multispecific links (e.g., links having multiple binding domains) formed by the assembly of heteromeric or heteropolymeric units aids in the production, folding, stability, and tissue availability of the multispecific links.

[0363] In some embodiments, the linker may be a flexible linker. For example, the linker may include an amino acid that provides flexibility to the linker. Thus, the flexible linker enables the free movement required within the polypeptide so that the binding domain can be stretched, contracted, or twisted, and allows for the accommodation of spatial constraints upon binding to the target molecule. The linker may link TRAV with TRAC. The linker may link the TRAV or TRAC binding material domain of a bispecific or trispecific linker with the anti-TR binding domain. The linker generally serves as a structural element connecting the sites of effective binding material. In some embodiments, the linker may be flexible. In some embodiments, the linker may be rigid. The length of the linker is adjusted according to design requirements and the length that is optimal or required to separate the binding material from the opposite end. In some embodiments, the linker may include a peptide having a unique function other than connecting the two domains.

[0364] In one embodiment, a T cell conjugate that binds to CD3 of a TCR complex on a T cell is provided herein. The T cell conjugate is an anti-CD3 scFv.

[0365] The TRAV:TRBV interaction / stabilization domain is a component of the sTCR scaffold designed herein. In some embodiments, the stabilization domain is a polypeptide that individually links TRAV and TRBV, which helps TRAV:TRBV to be ideally positioned and aligned to interact with an antigen, e.g., a p-MHC complex. In some embodiments, the stabilization domain comprises one or more polypeptides. In some embodiments, the TRAC and TRBC domains comprise the dimerization and stabilization domains of two polypeptides of a multispecific molecule. The TRAC and TRBC domains may comprise one or more engineered disulfide bridges connecting the two. For example, the Thr 48 residue of TRAC and the Ser 57 residue of TRBC1 or TRBC2 may be replaced with cysteine ​​residues, said cysteine ​​forming a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the polypeptide. In some embodiments, the ab heterodimer TCRs described herein (polypeptides comprising a TRAV domain and a TRAC domain and polypeptides comprising a TRBV domain and a TRBC domain) may comprise a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence may be connected by a natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2 in addition to or in the absence of the engineered disulfide bond as described above.

[0366] In addition, the molecular design of bispecific and trispecific molecules may include scaffold structures and cofactors that aid the ability of the linker to bind modularly and simultaneously to multiple targets. These designs include additional anchoring or latch elements for two or more binding domains and linkers, which can promote and improve stability and flexibility in binding multiple domains. The additional anchoring or latch elements occur as homologous pairs, so that one of the homologous pairs of anchoring or latch forms is attached to one of the linker's binding domains, and the other of the pair is attached to the other of the homologous pairs.

[0367] In some embodiments, the linker is a recombinant protein comprising a plurality of binding domains described throughout this specification, each having individual binding specificity, which are each linked together by a linker having a homologous peptide anchoring or clasp element that exhibits mutually complementary binding. For example, one binding domain of the recombinant protein is fused with the first of a pair of homologous peptides, and the remaining binding domain is fused with the second of the peptide pair, wherein the peptide pair exhibits mutually complementary binding and the homologous peptide pair exhibits mutually complementary binding, comprising a leucine zipper domain. For example, a leucine zipper that occurs naturally in protein-protein interactions, such as a zipper sequence within the binding region of the c-Fos and c-Jun proteins. In some embodiments, the pair of homologous peptides may comprise synthetic peptides designed to bind specifically to each other via synthetic clasps.

[0368] In some embodiments, the therapeutic agent is a recombinant protein comprising a plurality of binding fragments configured to promote accelerated binding to one another through leucine zipper peptide pairs contained in the recombinant protein. Leucine zipper sequences often contain heptad-leucine repeats and form an adhesion peptide pair when two peptides have a leucine zipper structure. Among naturally occurring leucine zippers, the c-Fos and c-Jun pairs are the most widely known. They are K D : 5.4x10 -8 They exhibit a strong bonding affinity with M. They form parallel coils. In some embodiments, the leucine zipper coil is a coil of c-Fos:c-Jun pairs.

[0369] An exemplary LZA leucine zipper domain may include the following amino acid sequence AQLEKELQALEKENAQLEWELQALEKELAQK (Sequence No. 631) or a sequence having at least 80% sequence identity with Sequence No. 631.

[0370] An exemplary LZB leucine zipper domain may include the following amino acid sequence AQLKKKLQALKKKNAQLKWKLQALKKKLAQK (Sequence No. 632) or a sequence having at least 80% sequence identity with Sequence No. 632.

[0371] In some embodiments, the fixing or latch element exhibits a specific heterodimerization ability and does not exhibit homodimerization.

[0372] In some embodiments, the therapeutic agent is a recombinant protein comprising a plurality of binding fragments configured to promote accelerated binding to one another through synthetic clasps. In some embodiments, synthetic fixation or clasp elements are designed to heteromerize and prevent homomerization.

[0373] In one embodiment, a multispecific linker or a multispecific linker molecule encoded by the recombinant nucleic acid described herein is provided herein. The multispecific linker molecule may be considered as a polypeptide or protein comprising two or more binding domains or binding substances, for example, two binding substances, wherein each binding substance binds to a specific target, and each of the two binding substances may bind to a distinct target (in this case, the linker is a bispecific linker). In some embodiments, the multispecific linker comprises three binding substances, each binding to a specific target distinct from one another, and such a multispecific linker is a trispecific linker. In one embodiment, at least one linker of the multispecific linker disclosed herein is a T cell linker comprising a T cell-specific binding substance, for example, a binding substance that binds to a cell surface molecule expressed on a T cell. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the cell surface molecule is a co-receptor. In some embodiments, the cell surface molecule is a ligand. In some embodiments, at least one link of the multispecific link is a link comprising a binding domain for a disease-specific epitope within a complex with MHC expressed on a target cell. The target cell may be a cancer cell.

[0374] In some embodiments, improvements to the binding domain are provided herein, which are intended to increase the efficacy and effectiveness of bispecific T cell engagers (BiTEs) and / or trispecific T cell engagers (TRITEs), as binding materials that anchor effector cells, e.g., T cells, to target cells, e.g., infected cells or tumor cells, and juxtapose the two; secondly, to specifically activate T cells to attack and destroy target cells by lysing them; and thirdly, in addition to the two preceding functions, to activate an immune response cascade to antigen epitopes derived from target cells. To this end, a potent primary response is required, including target cell death by cytotoxicity and the release of cytokines and chemokines by effector T cells. Several improvements are designed to produce highly effective BiTEs and TRiTEs.

[0375] In one embodiment, the BiTE or TRiTE comprises at least two polypeptides, for example, a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and the second polypeptide are oligomerized to promote the orientation of separate binding domains in a manner that achieves the stereochemical structure required to bind to a specific target molecule, for example, an MHC-peptide complex.

[0376] In some embodiments, oligomerization increases the possibility of contact with effectors and target cells. In one embodiment, a cell protein or fragment thereof that naturally oligomerizes (e.g., dimerization or trimerization, etc.) may be included in a BiTE or TRiTE polypeptide construct to facilitate the oligomerization of BiTE or TRiTE units. Accordingly, in one embodiment, the BiTE or TRiTE of such construct comprises a first monomer comprising a first polypeptide comprising one or more binding domains (binding substances), wherein at least one binding substance is a first binding domain capable of binding to an antigen peptide within an MHC complex on a target cell only when paired with a second monomer comprising one or more binding domains comprising a second polypeptide comprising a second binding domain that binds to an antigen peptide within an MHC complex and is correctly oriented. In some embodiments, the BiTE or TRiTE construct may comprise an oligomerizable protein or fragment thereof (oligomerization domain). In some embodiments, the oligomerization domain may be fused to one or more binding domains. In one embodiment, the T cell linker is a domain capable of binding to a T cell surface component, for example, a receptor on the T cell. In one embodiment, the T cell linker domain is a ligand for the T cell surface receptor. In one embodiment, the T cell linker domain is an antibody or a fragment thereof that binds to the T cell surface receptor. In some embodiments, the T cell linker domain is a scFv that binds to the T cell surface receptor. In some embodiments, the T cell linker domain is a VHH domain that binds to the T cell surface receptor. In some embodiments, the T cell linker domain comprises one or more VHH domains, each of which binds to the T cell surface receptor.In some embodiments, the T cell linker domain comprises one or more VHH domains, each of which binds to a different T cell surface receptor expressed on the same T cell. In some embodiments, one or more VHH domains may be on a single polypeptide chain and may be joined via a linker. The linker may be a peptide linker, for example, a flexible peptide linker comprising 2 to 100 amino acids.

[0377] In some embodiments, the multispecific molecule includes a post-translational modification. In some embodiments, the multispecific molecule includes a mammalian glycosylation signature. In some embodiments, the multispecific molecule is soluble and lacks a transmembrane domain. In some embodiments, the multispecific molecule consists of two polypeptides, namely a first polypeptide and a second polypeptide.

[0378] With the intention of designing a large library or repertoire of sTCRs capable of inserting or exchanging CDRs and related specificities required for antigen binding to facilitate the generation of appropriate molecules for therapeutic application, the applicant has designed a number of such TCRs having generic structures and backbones designed and optimized to work with placeholder antigen specificity (e.g., CDR) sequences, which may be referred to as scaffolds. Essentially, scaffolds may be the available TCR designs of the present disclosure.

[0379] In one embodiment, the recombinant nucleic acid encoding the sTCR scaffold may comprise a single polypeptide, a sequence encoding a TRAV domain, wherein the TRAV framework sequence is optimized and CDR1, CDR2, and CDR3 are interchangeable depending on the target antigen, wherein TRAV is linked in an operable state to a sequence encoding TRAC - which is subsequently linked to a sequence encoding a polypeptide containing TRBC, which is linked in an operable state to TRBV via a post-translational self-cleavage sequence, e.g., a sequence encoding P2A, wherein the TRBV framework sequence is optimized and CDR1, CDR2, and CDR3 are interchangeable depending on the target antigen. Post-translational cleavage induces two polypeptides, which are aligned with each other in such a way that TRAV forms a TRBV-specific binding domain TRAV:TRBV, and TRAC and TRBC cross-react to enhance and stabilize the TRAV:TRBV alignment. In some embodiments, the recombinant nucleic acid encoding an exemplary sTCR generated in the scaffold may comprise a sequence encoding a polypeptide, the polypeptide comprising, from the N-terminus to the C-terminus, the following: an N-terminal natural signal sequence; a first insertion sequence comprising a TRAV that binds to TRBV and specifically binds to a desired antigen; a sequence for a TRAC that binds to TRBC included in the polypeptide chain and forms a stabilization domain; the TRAC may be further linked at the C-terminus to any marker or tag peptide, e.g., a FLAG peptide, or any other functional domain; a P2A sequence; a second insertion sequence comprising the natural signal sequence and a TRBV sequence that binds to TRAV in the polypeptide chain and specifically binds to a desired antigen; a cleavage site; a sequence for a TRBC stabilization domain; which may be linked at the C-terminus to any marker or tag peptide, e.g., a hexa-HIS peptide or other functional domain.In some embodiments, a linker sequence comprising approximately 1 to 10 amino acids may exist between any two proximal domains. Upon post-translational cleavage, the mature protein may comprise a self-assembled TRAV:TRBV domain on a separate polypeptide, wherein TRAV is operably linked to TRAC and TRBV is operably linked to TRBC, where TRAC:TRBC forms a stabilizing domain. In some embodiments, the TRAC and TRBC domains comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more point mutations that increase the stabilization of the domains. In some embodiments, the human TRAC comprises a disulfide locking site between T48C and T166C on the human TRAC single polypeptide domain. In some embodiments, the TRBC comprises a disulfide locking site S57C or S173C. In some embodiments, this scaffold structure may be approximately 312 amino acids in length.

[0380] In one embodiment, an exemplary recombinant nucleic acid encoding an sTCR polypeptide uses a basic scaffold design as exemplified above, but includes a leucine zipper stabilizing domain for increased TRAV:TRBV stability. Accordingly, the recombinant nucleic acid includes a sequence encoding a polypeptide comprising the following in the direction from the N-terminus to the C-terminus: an N-terminal natural signal sequence; a first insertion sequence containing TRAV that binds to TRBV and specifically binds to a desired antigen; a TRAC sequence optionally connected to a tag sequence (e.g., a FLAG tag); a sequence for a leucine zipper chain; a P2A sequence; a natural signal sequence; a second insertion sequence containing TRBV; a TRBC domain; a leucine zipper sequence that oligomerizes with another leucine zipper chain within the polypeptide, optionally connected to a tag sequence, a marker or tag peptide, e.g., a hexa-HIS tag, or other functional domain at the C-terminus. This type of basic scaffold design may include a 442-amino acid polypeptide.

[0381] In some embodiments, the scaffold comprises a human TRAC engineered to include one or more of stability-enhancing mutations, namely S139F, T150I, and A190T, and the TRBC comprises E134K, H139R, D155P, and S170D.

[0382] In some embodiments, the scaffold may include an Fc domain in addition to the TCR constant region for additional stabilization. In some embodiments, the Fc domain includes a knob-in-hole configuration. In some embodiments, the FcA domain includes the T366W 'knob' mutation. In some embodiments, the FcB domain includes the T336S 'hole' mutation. In some embodiments, the FcB domain includes the L368A 'hole' mutation. In some embodiments, the FcB domain includes the Y407V 'hole' mutation. In some embodiments, the scaffold includes the Fc region of human IgG1. In some embodiments, the FcB includes one or more amino acid substitutions selected from M252E, I253A, and H435A substitutions.

[0383] In some embodiments, the scaffold includes the basic configuration described in the previous paragraph and includes the FcA-FcB domain of the aforementioned Knop-and-Hole configuration, but lacks the TRAC and TRBC domains.

[0384] In some embodiments, the human TRAC may be linked to a 10X His tag via a short linker and then linked to a purine cleavage site. In some embodiments, the self-cleavable sequence may be an F2A sequence, and the human TRBC sequence may be linked to a BAP tag.

[0385] The stabilization domain may include a disulfide bond between human TRAC formed by cysteine ​​engineered at T166 (T166C) and human TRBC formed by cysteine ​​engineered at S173 (S173C). A natural disulfide bond exists between TRAC (C213) and TRBC (C247). One or more glycation sites may be removed from TRAC and TRBC.

[0386] In some embodiments, the scaffold comprises a human TRAC engineered to include one or more stability-enhancing mutations, namely S139F, T150I, and A190T, and the TRBC comprises S170D, C191A, or N205D. In some embodiments, both the TRAC and TRBC sequences may include one or more N>Q substitutions.

[0387] The numbering of amino acid residues in the TRAC and TRBC domains used in this document follows the Kavat numbering system (Reference [Kabat, et al. Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH]).

[0388] In one embodiment, a human TRAC may be fused to human IgG1 or a portion thereof comprising the Fc of IgG1 following the upper hinge of IgG1, the central hinge of IgG1, and the lower hinge of IgG1. Accordingly, in an embodiment, the recombinant nucleic acid encoding such an exemplary sTCR comprises a heavy chain (364 aa length) and a light chain (154 aa length) polypeptide, wherein the heavy chain comprises an N-terminal natural signal sequence in the direction from the N-terminus to the C-terminus; a first insertion sequence comprising a TRAV that binds to TRBV and specifically binds to a desired antigen; and a TRAC sequence fused to the upper hinge domain of human IgG1 at the C-terminus. A 10X-HIS tag may be attached to the C-terminus of human IgG1. In some embodiments, the light chain comprises a signal sequence and a second insertion sequence comprising a TRBV that is operably linked to human TRBC. In one embodiment, the human TRAC comprises one, two, or three N>Q substitutions. In some embodiments, one N>Q substitution is upstream of the disulfide lock position 1 (T48C or T166C), and two N>Q substitutions are downstream. The human IgG1 sequence has a disulfide site having a TRBC C-terminal cysteine. In some embodiments, the light chain includes a disulfide lock position formed by S57C or S173C on the human TRBC sequence. In some embodiments, a manipulated disulfide bond exists between the IgG1 hinge region and the human TRBC (C247).

[0389] In some embodiments, the dimerization or stabilization domain comprises SARAH domain A and SARAH domain B. In some embodiments, a recombinant nucleic acid encoding a engineered T cell receptor (TCR) construct comprises: (a) a first polypeptide comprising a TCR alpha variable (TRAV) domain comprising CDR1a, CDR2a, and CDR3a; one or more TRAV framework region (TRAV-FR) sequences; and (b) a second polypeptide comprising a TCR beta variable (TRBV) domain comprising CDR1b, CDR2b, and CDR3b. (a) one or more TRBV framework region (TRBV-FR) sequences (the first polypeptide and the second polypeptide recognize and bind to the first HLA-antigen complex), (c) a third polypeptide comprising a first alpha helical dimerization motif operably linked to the first polypeptide, the third polypeptide being selected from helical domains found in MST, RASSF, and WW45, proteins involved in the Hippo signaling pathway; and (d) a fourth polypeptide comprising a second alpha helical dimerization motif that dimerizes with the third polypeptide to form an Fv clasp. In some embodiments, the first alpha helical dimerization motif and the second alpha helical dimerization motif are heterogeneous. In some embodiments, the first alpha helical dimerization motif and the second alpha helical dimerization motif are homogeneous. In some embodiments, the first alpha helical dimerization motif and the second alpha helical dimerization motif are antiparallel in orientation. In some embodiments, the first alpha helical dimerization motif is operably connected to the C-terminus of the TRAV domain, and the second alpha helical dimerization motif is operably connected to the C-terminus of the TRBV domain. In some embodiments, the first alpha helical dimerization motif and the second alpha helical dimerization motif are fragments of human Mst1 kinase.In some embodiments, the first alpha helical dimerization motif or the second alpha helical dimerization motif is the SARAH domain of human Mst1 kinase or a fragment thereof, wherein the first alpha helical dimerization motif is the SARAH A domain and the second alpha helical dimerization motif is the SARAH B domain. In some embodiments, the first polypeptide further comprises a partial TRAJ motif at the C-terminus of the TRAV domain, which is connected to the SARAH A domain via a first GS linker, and the second polypeptide further comprises a partial TRBJ motif at the C-terminus of the TRBV domain, which is connected to the SARAH B domain via a second GS linker.

[0390] In some embodiments, the Fv clasp is used to enhance dimerization between TRAV and TRBV.

[0391] In some embodiments, TRAV is engineered to include a stability-enhancing mutation comprising X96L, where X is any amino acid at position 96 of the TCR alpha variable domain determined by the IMGT numbering scheme. In some embodiments, TRBV is engineered to include a stability-enhancing mutation comprising one or more of X9R and X10Y, where X is any amino acid at position 9 or 10 of the TCR beta variable domain determined by the IMGT numbering scheme.

[0392] In some embodiments, the N-glycosylated site may be removed from the TRAC and TRBC domains.

[0393] In some embodiments, one or more scaffold designs may include a single engineered disulfide bridge between TRAC (T166C) and TRBC (S173C), whereas the naturally occurring disulfide bond between TRAC (C213) and TRBC (C247) is removed by mutation of cysteine ​​to alanine or other amino acids.

[0394] In some embodiments, the exemplary scaffold may comprise stability-enhancing TRAC and TRBC mutants, namely TRAC(S139F, T150I, A190T); TRBC(E134K, H139R, D155P, S170D), wherein the N-glycosylation sites are removed from TRAC and TRBC. The scaffold may comprise Fc fused to each polypeptide and may comprise both engineered {TRAC(T166C) and TRBC(S173C)} and natural {TRAC(C213) and TRBC(C247)} disulfide bridges (Scaffold 14).

[0395] In some embodiments, the TRAV and TRBV framework regions are further optimized for stability and efficient expression in mammalian cells. In some embodiments, TRAV and TRBV are codon optimized.

[0396] One of the mutated RAS epitope-responsive TCRs used in the present study for scaffold design is Ros9a having the TRAV amino acid sequence LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSD (SEQN 619), wherein

[0397] CDR1: NIATNDY (Sequence No. 874)

[0398] CDR2: GYKTK (Sequence No. 875)

[0399] CDR3: LVGDMDQAGTALI (Sequence No. 876). (CDR sequence according to IMGT).

[0400] The corresponding TRBV is AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVV (sequence number 620),

[0401] CDR1: SGHDT(Sequence No. 877);

[0402] CDR2: YYEEEE(Sequence No. 878);

[0403] CDR3: ASSLGEGRVDGYT (Sequence No. 879). (CDR sequence according to IMGT).

[0404] TRAV:TRBV can bind to KRAS G12D / C*08:02 with high affinity.

[0405] Dimerization and oligomerization, stabilization domain.

[0406] In one embodiment, the first polypeptide further comprises a dimerization domain fused to a TRAV domain, and the second polypeptide comprises a dimerization domain fused to a TRBV domain. In some embodiments, the first polypeptide comprises a dimerization domain, a peptide linker, and a TRAV domain of the first polypeptide in the direction from the N-terminus to the C-terminus, and the second polypeptide comprises a dimerization domain, a peptide linker, and a TRBV domain of the second polypeptide in the direction from the N-terminus to the C-terminus. In some embodiments, the dimerization domain of the first polypeptide comprises a TCR alpha constant (TRAC) domain or a part thereof, and the dimerization domain of the second polypeptide comprises a TCR beta constant (TRBC) domain or a part thereof. In some embodiments, the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by one or more disulfide bridges.

[0407] In some embodiments, the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by a single disulfide bridge. In some embodiments, the first polypeptide comprises a T cell linkage. In some embodiments, the T cell linkage is connected to the dimerization domain of the first polypeptide. In some embodiments, the first polypeptide comprises a T cell linkage, a dimerization domain, and a TRAV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the first polypeptide comprises a TRAV domain and a T cell linkage in the direction from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises a T cell linkage. In some embodiments, the second polypeptide comprises a T cell linkage, a dimerization domain, and a TRBV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises a TRBV domain and a T cell linkage in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises scFv, a dimerization domain, and a TRAV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerization domain, a TRAV domain, and scFv in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises scFv, a dimerization domain, and a TRBV domain in the direction from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises scFv that binds to a receptor expressed by the T cell, and the polypeptide comprises a dimerization domain, a TRBV domain, and scFv in the direction from the N-terminus to the C-terminus.In some embodiments, the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises VHH, a dimerization domain, and a TRAV domain from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises a dimerization domain, a TRAV domain, and VHH from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises VHH, a dimerization domain, and a TRBV domain from the N-terminus to the C-terminus. In some embodiments, the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises a dimerization domain, a TRBV domain, and VHH from the N-terminus to the C-terminus.

[0408] In some embodiments, the T cell linkage comprises a first VHH domain and a second VHH domain configured to bind to the same receptor expressed by the T cell.

[0409] In some embodiments, the T cell linkage comprises a first VHH domain that binds to a first receptor expressed by the T cell and a second VHH domain that binds to a second receptor expressed by the T cell.

[0410] Accordingly, a composition comprising a recombinant polynucleic acid comprising a sequence encoding a first polypeptide chain, comprising a first binding domain, a first ligand or a functional fragment thereof that specifically interacts with an antigen peptide on a target cell; and a first immunoglobulin (Ig) domain, wherein the first binding domain, the first ligand or a functional fragment thereof and the first Ig domain are operably connected.

[0411] In some embodiments, the second binding domain specifically interacts with the first extracellular protein of the T cell. In some embodiments, the second ligand is configured to form a multimer with the first ligand. In some embodiments, the second ligand interacts with the second extracellular protein of the T cell, wherein the second extracellular protein is different from the first extracellular protein with which the second binding domain specifically interacts. In some embodiments, the first ligand interacts with the second extracellular protein of the T cell, wherein the second extracellular protein is different from the first extracellular protein with which the second binding domain specifically interacts. In some embodiments, the second polypeptide chain further comprises an additional binding domain that specifically interacts with an antigen peptide in relation to an MHC complex on a target cell. In some embodiments, the additional binding domain of the second polypeptide chain specifically interacts with the antigen peptide of the target cell with which the first binding domain specifically interacts. In some embodiments, the second polypeptide chain further comprises a second Ig domain. In some embodiments, the Ig domain is operably linked to a second binding domain and a second ligand. In some embodiments, an additional binding domain of the first polypeptide chain specifically interacts with a first extracellular protein to which the second binding domain specifically interacts. In some embodiments, the first Ig domain and / or the second Ig domain comprises an Fc domain. In some embodiments, the first Ig domain and / or the second Ig domain is an IgG Fc domain. In some embodiments, the IgG domain is selected from the group consisting of IgG1, IgG4, and IgGA. The immunoglobulin chain or Fc domain selected for the BiTE or TRiTE generally forms a dimer. The Ig Fc chain may form a homodimer. Or, the Ig Fc chain may be selected or configured to form a heterodimer.In one embodiment, the IgG polypeptide chain(s) are modified so that the modification helps increase the stability of the polypeptide. In some embodiments, the IgG polypeptide chain(s) are modified to increase the flexibility of the polypeptide. In some embodiments, the modification is a point mutation. In some embodiments, the modification includes the addition, deletion, or alteration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids within the polypeptide. In some embodiments, the modification includes structurally altering the polypeptide.

[0412] For example, the Ig Fc chain is modified to include a knop-and-hole bond between two Fc chains. In some embodiments, one or more amino acids may be suitably modified or altered to enable bonding between adjacent polypeptide chains. Such modification may include amino acid substitution with cysteine ​​to introduce a disulfide bond between two adjacent polypeptides or within the polypeptide. In some embodiments, the first Ig domain includes a knop, for example, the knop includes IgG1 Fc (F409R). In some embodiments, the second Ig domain includes a hole, for example, the hole includes IgG1 Fc (K405L).

[0413] In some embodiments, any of the exemplary scaffold designs may be used as a modular platform for a person skilled in the art who wishes to generate a soluble multispecific linker construct. The construct provided below is one of the basic structural motifs for a multispecific linker and comprises TCR-alpha and TCR-beta chains and one or more tags (HIS tag and BAP tag), to which one or more T cell linkers may be conjugated to obtain a bispecific or multispecific linker. In some embodiments, the scaffold is the sequence METDTLLLWVLLLWVPGSTGD LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSD YIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSC GSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMETDTLLLWVLLLWVPGSTGDD AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVVEDLKNVFPP EVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GSGGLNDIFEAQKIEWHE (SEQ No. 633) comprises a linked molecule that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical,

[0414] The TRAV sequence is LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSD (sequence number 619), and the TRAC sequence is YIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESS,

[0415] The TRBV sequence is AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVV (Sequence No. 620), and the TRBC sequence is EDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGSGGLNDIFEAQKIEWHE. TRAV and TRAC form the first polypeptide, and TRBV and TRBC form the second polypeptide.

[0416] In some embodiments, TRBV is the N-terminus of TRAV. In some embodiments, TRAV is the N-terminus relative to TRBV. In some embodiments, TRAV is associated with a tag, e.g., a HIS tag. In some embodiments, TRBV is associated with a tag, e.g., a HIS tag or a BAP tag. In some embodiments, TRAC is associated with a HIS tag or a BAP tag. In some embodiments, TRAC is associated with a HIS tag or a BAP tag. In some embodiments, TRAC is associated with a HIS tag or a BAP tag. In some embodiments, TRBC is associated with a HIS tag or a BAP tag.

[0417] In some embodiments, the alpha chain comprises one or more tags, a signal peptide, and a cleavage domain. In some embodiments, the beta chain comprises one or more tags, a signal peptide, and a cleavage domain.

[0418] In some embodiments, there is a cleavable sequence between the alpha chain and the beta chain. In some embodiments, the cleavable sequence is a P2A, F2A, or T2A sequence.

[0419] In some embodiments, the scaffold comprises a linker molecule comprising TRAV and TRBV domains connected to a TRAC domain and a TRBC domain, respectively, and further comprises an immunoglobulin chain conjugated to TCR alpha and beta chains. An exemplary representative drawing is Fig. 3 at Scaffold 5 It is represented as. In some embodiments, this scaffold is the sequence METLLGVSLVILWLQLARVNS QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLLIRDSQPSDSATYLCAGGGGADGLTFGKGTHLIIQP YIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESS VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGHHHHHHHHH( Scaffold 5, As an example having binding affinity for MART1 / A*02:01, it may include a sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to 199.16 TCR) (SEQ ID No. 634), and the corresponding beta chain is the sequence MGTRLLCWAALCLLGAELTE AGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSQGLAGAGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC It may contain sequences that are at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to (Sequence No. 635). TRAV sequences are shown in bold, and TRBV sequences are shown underlined. The 10X histidine tag, HHHHHHHHH (Sequence No. 636), is conjugated at the ends.

[0420] In some embodiments, the scaffold comprises a linker molecule comprising TRAV and TRBV domains and further comprises a molecular clasp or linker between the two chains. In some embodiments, the molecular clasp is an Fv clasp ( 도 3, 스캐폴드 6 ). In some embodiments, the scaffold comprises a linker molecule comprising two stabilizer polypeptide segments connected via TRAV, TRBV, and Fv clasps. In some embodiments, this scaffold comprises the sequence MWWRLWWLLLLLLLLWPMVWAA QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAGGGGADGLTFGKGTHLIICP GSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAKGSGHHHHHHHHHHRIRRSGSGATNNFSLLKQAGDVEENPGPMWWRLWWLLLLLLLLWPMVWA AGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSQGLAGAGELFFGEGSRLTVLE GS DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAK It includes GSGSAWSHPQFEK (SEQ No. 637). In some embodiments, this scaffold may include a sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to SEQ No. 637. Within the sequence, the TRAV sequence is shown in bold, and the TRBV sequence is shown underlined. Within this sequence, the sequence DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAK (Bold, italic) forms an Fv clasp (sequence number 638).

[0421] In some embodiments, one exemplary scaffold design is 도 3, 스캐폴드 14 It includes an Fc fusion domain linked to a TCR chain as shown in [figure]. The Fc domains interlock through a knop-in-hole structure to provide structural stability to the fused molecule. An exemplary scaffold 14 has chains A and B. In some embodiments, this scaffold is the sequence MMGVKVLFALICIAVAEAD LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSCIt may include chain A having GGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSHHHHHHHHHH (Sequence No. 639). In some embodiments, these scaffolds may comprise a chain A sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to SEQ ID NO. 639. Bold alphabet indicates a TRAV:TRAC fusion polypeptide. In some embodiments, these scaffolds comprise the sequence MMGVKVLFALICIAVAEADD AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVVEDLKNVFPP EVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCIt may include chain B having GGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLEASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGLNDIFEAQKIEWHE (Sequence No. 640). In some embodiments, these scaffolds may include chain B sequences that are at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to SEQ ID NO. 640. Underlined sequences are TRBV sequences, and underlined and italicized sequences are immunoglobulin sequences.

[0422] In some embodiments, any of the exemplary scaffold designs described herein may be used as a modular platform for a person skilled in the art who wishes to create a available multispecific linkage construction. In some embodiments, the scaffold is SEQ ID NO. 641 ( Ros9a Scaffold 19 It includes a linked molecule that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence described in ). Here, the TCR alpha and beta chain variable domains are taken from a TCR that binds to a RAS having a TRAV sequence such as SEQ ID NO. 619 and a TRBV sequence such as SEQ ID NO. 620.

[0423] In some embodiments, the scaffold is the sequence described in SEQ ID NO. 642 LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGK GTTLSVSSDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSCGSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMMGVKVLFALICIAVAEAAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVVEDLKNVFPP EVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GGGGSGLNDIFEAQKIEWHEGGGGSGGGGSLPETGG( Ros9a Scaffold 19, signal peptide excluded It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules.

[0424] In some embodiments, the scaffold is SEQ ID NO. 643 ( Ros9a Scaffold 20b It includes a linker molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in ).

[0425] In some embodiments, the scaffold is sequence*( Ros9a scaffold 20b, signal peptide excluded It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules as )(SEQ ID NO. 644).

[0426] In some embodiments, the scaffold is a polypeptide chain ( Ros9a Scaffold 21 Chain A)It includes a linkage molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence having (sequence number 645).

[0427] In some embodiments, the scaffold is a polypeptide chain ( Ros9a Scaffold 21 Chain B It includes a linkage molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence having )(SEQ ID NO. 646).

[0428] In some embodiments, the scaffold is SEQ ID NO. 647 ( Ros9d Scaffold 19 It includes a linker molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in ).

[0429] In some embodiments, the scaffold is sequence LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGK GTTLSVSSDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSC GSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMMGVKVLFALICIAVAEAAIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGGLVQP GGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGQTNYGYTFGSGTRLTVVEDLKNVFPP EVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC ( Ros9d scaffold 19 excluding signal peptides It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules as )(SEQ No. 648).

[0430] In some embodiments, the scaffold is SEQ ID NO. 649 ( Ros9d Scaffold 21 Chain A It includes a linker molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence having the polypeptide chain described in ).

[0431] In some embodiments, the scaffold is SEQ ID NO. 650 ( Ros9d Scaffold 21 Chain B It includes a linker molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence having the polypeptide chain described in ).

[0432] In some embodiments, the scaffold is SEQ ID NO. 651 ( Ros10 Scaffold 19It includes a linker molecule that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in ).

[0433] In some embodiments, the scaffold is sequence QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLLIRDSQPSDSATYLCAAAMDSSYKLIFGS GTRLLVRPDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSC GSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMMGVKVLFALICIAVAEAAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS AGITQSPRYKITETGRQVTLMCHQTWSHSYMFWYRQDLGHGLRLIYYSAAADITDKGEVPDGYVVSRSKTENFPLTLESATRSQTSVYFCASSDPGTEAFFGQGTRLTVVEDLKNVFPPE VAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GGGGSGLNDIFEAQKIEWHEGGGGSGGGGSLPETGG( Ros10 Scaffold 19, signal peptide excluded It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules as )(SEQ ID NO. 652).

[0434] In some embodiments, the scaffold is the sequence MMGVKVLFALICIAVAEAD LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGK GTTLSVSSDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSCGSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMMGVKVLFALICIAVAEAQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDT SKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDY WGQGTTLTVSSGGGGS AGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVVEDLKNVFPP EVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GGGGSGLNDIFEAQKIEWHEGGGGSGGGGSLPETGG*( Ros9a Scaffold 22 It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules.

[0435] In some embodiments, the scaffold is sequence LAKTTQPISVDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGK GTTLSVSSDYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFTCANAFQNSIIPEDTFFPSPESSC GSGHHHHHHHHHHRIRRSGSGVKQTLNFDLLKLAGDVESNPGPMMGVKVLFALICIAVAEAQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDT SKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFT RYTMH WVKQRPGQGLEWIG YINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDYWGQGTTLTVSSGGGGSAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVV EDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GGGGSGLNDIFEAQKIEWHEGGGGSGGGGSLPETGG*( Ros9a Scaffold 22, signal peptide excluded It contains at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical linkage molecules.

[0436] In some embodiments, a polypeptide chain comprising a third binding domain that specifically interacts with an extracellular protein of a T cell is provided herein. In some embodiments, the extracellular protein to which the third binding domain specifically interacts is different from the extracellular protein to which the second binding domain specifically interacts. In some embodiments, the extracellular protein to which the third domain binding specifically interacts is present on the same T cell. In some embodiments, the third binding domain is a CD8 molecule or a fragment thereof.

[0437] In one embodiment, the recombinant polynucleic acid is mRNA.

[0438] In one aspect, a cell comprising any one of the polynucleic acids disclosed and described herein is provided herein.

[0439] In one aspect, a pharmaceutical composition for use in treating cancer is provided herein, comprising any one of a recombinant polynucleic acid, a recombinant polypeptide, or a cell of any one of the embodiments disclosed and described herein.

[0440] In some embodiments, the antigen of the target cell is a cancer antigen peptide, a pathogenic antigen peptide, or an autoimmune antigen peptide.

[0441] In one embodiment, the first binding domain, the second binding domain, the third binding domain, the additional binding domain of the first polypeptide chain, the additional binding domain of the second polypeptide chain and / or the additional binding domain of the third polypeptide chain comprise a TRAV / TRBV domain, a VH domain, a VL domain, a VNAR domain, a VHH domain, a short-chain variable fragment [scFv], Fab, a single-domain antibody (sdAb), a nanobody, a bispecific antibody, or a dimer antibody.

[0442] In any of the above embodiments, the BiTE or TRiTE comprises any one or more of (i) an immunoglobulin Fc region connecting two binding domains, and (ii) a modified immunoglobulin Fc region connecting two binding domains (e.g., having a knop and hole configuration) connected to a binding domain that binds to a different heterogeneous protein, e.g., a T cell expression protein.

[0443] Method for manufacturing a multispecific T cell linkage

[0444] The linkage described herein is produced as a recombinant protein. Generally, a polynucleotide sequence encoding the recombinant protein is constructed and inserted into an expression vector, such as a plasmid, with appropriate orientation and an accurate reading frame for expression; if necessary, the DNA may be linked to appropriate transcriptional and translational control nucleotide sequences recognized by the desired host (e.g., bacteria), although such regulation is generally available in the expression vector. The vector is then introduced into host bacteria for cloning using standard techniques (see, for example, the literature [Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]).

[0445] Recombinant polynucleotides are synthesized using molecular cloning techniques well known to a person skilled in the art, for example, by linking DNA encoding a first binding domain, a linker, and a second binding domain within the same open decoding frame. In some embodiments, for the generation of a single polypeptide, one or more polynucleotide sequences are arranged under the same promoter or regulatory element. In some embodiments, a short spacer may be inserted between two adjacent polynucleotides encoding two polypeptides, wherein the spacer may encode a post-translation cleavage site. The two polypeptides may be separated after translation by inducing cleavage at a specific cleavage site. In some embodiments, the construct may be a monocystron or a dasistron. In some embodiments, more than one polypeptide is generated, which is subsequently reassembled after translation. For example, the light and heavy chain domains of an antibody or part thereof may be generated by translation from two independent polynucleotide sequences, which allow them to be freely assembled together after translation. In another example, TRAV and TRBV of the TCR or part thereof can be generated by translation from two independent polynucleotide sequences. Alternatively, multiple polypeptide chains containing TRAV and TRBV variable domains that bind to each other can be transcribed, translated from a single polynucleotide, cleaved into their respective peptide chains after translation, and then reassembled. The polypeptide having the lead sequence is a precursor protein, and the lead sequence can be cleaved by a host cell to form a mature form of the polypeptide.

[0446] In some embodiments, the polynucleotide construct encodes an N-terminal signal sequence upstream of the polypeptide for the secretion of the peptide. In some embodiments, the N-terminal signal sequence includes a secretion sequence. The resulting translation protein product having the N-terminal signal sequence for secretion will be secreted by the cell. In some embodiments, the mature protein lacks the signal peptide. Thus, the amino acid sequence of the protein designed to have the signal peptide at the N-terminus initiates the sequence of the mature protein, because the mature protein will take the sequence of the described protein excluding the signal protein sequence.

[0447] In some embodiments, the signal peptide may have any one of the amino acid sequences of Table 7.

[0448] Exemplary N-terminal signal peptide sequence. order Sequence number METDTLLLWVLLLWVPGSTGD 653 METLLGVSLVILWLQLARVNS 654 MGTRLLCWAALCLLGAELTEA 655 MWWRLWWLLLLLLLLWPMVWAA 666 MMGVKVLFALICIAVAEAD 667 MMGVKVLFALICIAVAEA 668

[0449] In some embodiments, the plasmid vector is introduced into or contained in a cell by a known transfection method, such as the use of lipofectamine or calcium phosphate, or through physical means, such as electroporation or nucleofection. In some embodiments, the viral vector is introduced into or contained in a cell by infection, which is a process commonly known as viral transduction.

[0450] In some embodiments, the recombinant nucleic acid is incorporated into or included in an expression vector. The vector includes one or more promoter and enhancer binding sequences, initiation and terminal codons, a 5'UTR, a 3'UTR including a transcript stabilization element, any conserved regulatory protein binding sequence, and other regulatory components.

[0451] In some embodiments, the vectors used in this application are specifically enhanced for expression. Other exemplary vectors used throughout the process include phages, cosmids, or artificial chromosomes.

[0452] It is understood that any one of the first binding domains (domains that bind to target cells, such as cancer cells or affected cells, or pathogens) may be designed in combination with a second binding domain that binds to T cells, or a third binding domain described anywhere in this specification.

[0453] Virus vector : In some embodiments, the vector for the expression of the recombinant protein is of viral origin, namely a lentiviral vector or an adenoviral vector. In some embodiments, the nucleic acid encoding the recombinant nucleic acid is encoded by a lentiviral vector. In some embodiments, the lentiviral vector is manufactured in-house for the purpose and manufactured on a large scale. In some embodiments, as is known to a person skilled in the art, a commercially available lentiviral vector is used.

[0454] In some embodiments, the virus vector is an adeno-associated virus (AAV) vector.

[0455] Lipid nanoparticle-mediated delivery Lipid nanoparticles (LNPs) may contain polar and / or nonpolar lipids. In some embodiments, cholesterol is present in the LNP for efficient delivery. LNPs with a diameter of 100 to 300 nm provide an efficient means of mRNA delivery to various cell types, including monocytes or macrophages. In some embodiments, LNPs may be used to introduce recombinant nucleic acids into cells in in vitro cell cultures. In some embodiments, the LNP encapsulates a nucleic acid, wherein the nucleic acid is an exposed DNA molecule. In some embodiments, the LNP encapsulates a nucleic acid, wherein the nucleic acid is an mRNA molecule. In some embodiments, the LNP encapsulates a nucleic acid, wherein the nucleic acid is inserted into a vector, such as a plasmid vector. In some embodiments, the LNP encapsulates a nucleic acid, wherein the nucleic acid is a circRNA molecule.

[0456] In some embodiments, LNP is used to deliver nucleic acids to a target. LNP may be used to deliver nucleic acids from a target to the body. This may be delivered by injection. In some embodiments, LNP containing nucleic acids is injected via an intravenous route. In some embodiments, LNP is injected subcutaneously.

[0457] Microbubble-mediated delivery: In some embodiments, microbubbles may be used to deliver compositions, for example, containing nucleic acids, to a target. Microbubbles filled with perfluorocarbon are stable for circulating within vascular structures as blood pool contrast agents, and they act as carriers of these contrast agents until they reach the site of interest. Ultrasound applied to the skin surface may be used to rupture the microbubbles at this site to induce local release of the drug. Various other forms of microbubbles include Sonazoid Optison, gas-filled albumin microbubbles, and PESDA. Along with the intended delivery site, optimization of the microbubble composition relative to the composition of the delivered therapeutic agent is required.

[0458] In some embodiments, a recombinant protein, e.g., a linker, or a co-expressed inflammatory protein, or any related protein designed to be expressed in T cells, may be encoded by a recombinant nucleic acid, wherein the recombinant nucleic acid is RNA. In some embodiments, the recombinant nucleic acid is mRNA. In some embodiments, the mRNA includes one or more modifications for enhanced expression and stability. In some embodiments, the mRNA may be circularized. In some embodiments, modifications may include, but are not limited to, replacing a nucleobase with a base analog or a modified nucleotide; inserting one or more motifs into the mRNA; and introducing modifications to the 5' and 3' UTRs. In some embodiments, the recombinant nucleic acid may be administered directly to a subject who requires it.

[0459] Pharmaceutical composition

[0460] A pharmaceutical composition comprising at least a first therapeutic agent comprising a multispecific molecule is provided herein. The multispecific molecule in the composition may be in the form of a peptide or polypeptide or a complex of multiple peptides. The multispecific molecule may be provided in the composition as a purified recombinant protein. The multispecific molecule may be provided in the composition as a conjugated recombinant protein, a Vhh complex, a scFv complex, or a nanobody. The multispecific molecule may be in the form of a polynucleotide encoding the recombinant multispecific molecule. In some embodiments, the polynucleotide encoding the multispecific molecule may comprise DNA, mRNA or circRNA, or a liposomal composition of any one of these. The liposome is an LNP.

[0461] In addition to the active ingredient, the pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well known to a person skilled in the art. These substances must be non-toxic and must not impair the efficacy of the active ingredient. The specific properties of the carrier or other substances will vary depending on the route of administration.

[0462] Acceptable carriers, excipients, or stabilizers are those that are non-toxic to the recipient at the doses and concentrations used, and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN ® , PLURONICS ® , or includes polyethylene glycol (PEG).

[0463] Acceptable carriers are physiologically acceptable to the patient receiving the treatment and maintain the therapeutic properties of the compound administered with or within the carrier. Acceptable carriers and their formulations are generally, for example, from the literature [Remington's Pharmaceutical Sciences (18 thIt is described in [ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990]. An example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in transporting or delivering the target compound from a site of administration in one organ or part of the body to another organ or part of the body, or to an in vitro analytical system. An acceptable carrier is compatible with other components of the formulation and is not harmful to the subject to whom it is administered. An acceptable carrier must not alter the specific activity of the neoantigen.

[0464] In one embodiment, pharmaceutically acceptable or physiologically acceptable compositions are provided herein, comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating agents, isotonics, and absorption promoters or retarders that are compatible with pharmaceutical administration. Accordingly, a pharmaceutical composition or pharmaceutical formulation refers to a composition suitable for pharmaceutical use in the subject. The composition may be formulated to be compatible with a specific route of administration (i.e., systemic or topical). Accordingly, the composition includes a carrier, diluent, or excipient suitable for administration by various routes.

[0465] In some embodiments, the composition may further include acceptable additives to enhance the stability of immune cells in the composition. The acceptable additives may not alter the inactivity of the immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. The acceptable additives may be combined with acceptable carriers and / or excipients, such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants, such as polysorbate 20 or polysorbate 80, to increase the stability of the peptide and reduce gelation of the solution. The surfactant may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0466] The pharmaceutical composition may be administered, for example, by injection. The composition for injection may include an aqueous solution (if water-soluble) or a dispersion and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, a suitable carrier comprises physiological saline, bacteriostatic water, or phosphate buffered saline [PBS]. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Fluidity may be maintained, for example, by coating, e.g., by the use of lecithin; by maintaining the required particle size in the case of a dispersion; and by the use of a surfactant. Antimicrobial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, e.g., sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, may be included in the composition. The resulting aqueous solution may be packaged for immediate use or freeze-dried, and the freeze-dried formulation may subsequently be combined with a sterile solution prior to administration. For intravenous, injection, or wound injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and possesses suitable pH, isotonicity, and stability. A person skilled in the art can properly prepare a suitable solution using, for example, an isotonic vehicle, such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. A sterile injection solution may be prepared by introducing the required amount of the active ingredient into a suitable solvent with one or a combination thereof of the ingredients listed above as needed, followed by filtration sterilization. Generally, a dispersion is prepared by including an active ingredient in a sterile vehicle containing a basic dispersion medium and other necessary components among those listed above.In the case of sterile powder for the preparation of sterile injectable solutions, preferred manufacturing methods may be vacuum drying and freeze-drying, which produce a powder of the active ingredient + any desired additional ingredient from a pre-sterile filtered solution.

[0467] The composition may typically be administered intravenously, for example, by injection of a unit dose. In the case of injection, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is substantially free of pyrogens and possesses suitable pH, isotonicity, and stability. A person skilled in the art can properly prepare a suitable solution using, for example, an isotonic vehicle, such as sodium chloride injection, Ringer injection, or lactated Ringer injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Additionally, the composition may be administered via aerosolization.

[0468] When the composition is considered to be used in a medicine or any method provided herein, the composition is considered to be substantially free of pyrogens so as not to cause an inflammatory or unsafe allergic reaction when administered to a human patient. Testing f...

Claims

Claim 1 A composition comprising a recombinant nucleic acid for expression in mammalian cells, wherein the recombinant nucleic acid comprises a sequence encoding a multispecific molecule, wherein the multispecific molecule is (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TCR alpha variable, TRAV) domain and (b) a TCR beta variable (TCR beta variable, TRBV) domain; and (ii) a T cell linkage comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the T cell linkage comprises an anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGIYSDSSDFIGNL (SEQ ID NO. 713) or GIYSDSSDFIGNL (SEQ ID NO. 707), and is a composition comprising an engineered T cell receptor (TCR). Claim 2 A composition comprising a recombinant nucleic acid having a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a TCR alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain, wherein the peptide of the MHC-peptide complex comprises a RAS peptide sequence; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the T cell conjugate comprises an anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGIYSDSSDFIGNL (SEQ ID No. 713) or GIYSDSSDFIGNL (SEQ ID No. 707). Claim 3 In paragraph 2, the composition wherein the multispecific molecule comprises a post-translational modification. Claim 4 A composition according to any one of claims 1 to 3, wherein the multispecific molecule comprises a mammalian glycosylation signature. Claim 5 A composition according to any one of claims 1 to 4, wherein the multispecific molecule is soluble. Claim 6 A composition according to any one of claims 1 to 5, wherein the multispecific molecule lacks a membrane-penetrating domain. Claim 7 A composition according to any one of claims 1 to 6, wherein the multispecific molecule comprises two polypeptides, namely a first polypeptide and a second polypeptide. Claim 8 A composition according to claim 7, wherein the first polypeptide and the second polypeptide comprise one or more disulfide bonds between the two polypeptides. Claim 9 A composition according to any one of claims 1 to 8, wherein the multispecific molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the TRAV domain and the second polypeptide comprises the TRBV domain. Claim 10 A composition according to any one of claims 1 to 9, wherein the peptide of the MHC-peptide complex is a peptide derived from a cancer antigen. Claim 11 A composition according to any one of claims 2 to 10, wherein the peptide of the MHC-peptide complex is a mutant peptide, and the MHC of the MHC-peptide complex binds to the mutant peptide with a higher affinity compared to the corresponding wild-type peptide. Claim 12 A composition according to any one of claims 1 to 11, wherein the peptide of the MHC-peptide complex does not include the amino acid sequence YLEPGPVTA. Claim 13 A composition according to any one of claims 1 to 12, wherein the MHC of the MHC-peptide complex comprises a class I MHC. Claim 14 A composition according to any one of claims 1 to 13, wherein the MHC of the MHC-peptide complex is a class I MHC polypeptide. Claim 15 A composition according to any one of claims 1 to 14, wherein the MHC of the MHC-peptide complex is encoded by HLA that appears in less than 1% of the human population. Claim 16 A composition according to any one of claims 1 to 15, wherein the MHC of the MHC-peptide complex has a peptide binding affinity greater than at least 50 nM. Claim 17 A composition according to any one of claims 1 to 16, wherein one or more binding domains of the T cell linkage bind to an extracellular domain of an endogenous receptor expressed by a T cell. Claim 18 A composition according to any one of claims 1 to 17, wherein one or more binding domains of the T cell linkage bind to an extracellular domain of a receptor selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICAM-1, and CD8. Claim 19 A composition according to any one of claims 1 to 18, wherein one or more binding domains of the T cell linkage bind to CD3 delta, CD3 gamma, or CD3 epsilon. Claim 20 A composition according to any one of claims 1 to 19, wherein one or more binding domains of the T cell linkage comprise an antibody domain or an antigen-binding fragment thereof. Claim 21 A composition according to any one of claims 1 to 20, wherein the binding domain of the T cell linkage comprises scFv or sdAb. Claim 22 A composition according to claim 21, wherein one or more binding domains of the T cell linkage comprise VHH. Claim 23 A composition according to any one of claims 1 to 22, wherein the one or more binding domains of the T cell linkage comprise a first binding domain that binds to an extracellular domain of a first receptor expressed by a T cell and a second binding domain that binds to an extracellular domain of a second receptor expressed by a T cell. Claim 24 A composition according to claim 23, wherein the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by the T cell comprises a first VHH, and the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by the T cell comprises a second VHH. Claim 25 A composition according to claim 23 or 24, wherein (i) the C-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the C-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. Claim 26 A composition according to claim 23 or 24, wherein (i) the N-terminus of the first binding domain of the T cell linkage is connected to the N-terminus of the second binding domain of the T cell linkage, or (ii) the N-terminus of the first binding domain of the T cell linkage is connected to the C-terminus of the second binding domain of the T cell linkage. Claim 27 A composition according to any one of claims 23 to 26, wherein the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by the T cell comprises scFv, and the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by the T cell comprises VHH. Claim 28 A composition according to any one of claims 23 to 27, wherein the first binding domain that binds to the extracellular domain of the first receptor expressed by a T cell and the second binding domain that binds to the extracellular domain of the second receptor expressed by a T cell are connected by a peptide linker. Claim 29 A composition according to any one of claims 23 to 28, wherein the first binding domain binds to an extracellular domain of a first receptor expressed by a T cell selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8, and the second binding domain binds to an extracellular domain of a second receptor expressed by a T cell selected from the group consisting of CD3, CD2, CD7, CD5, CD4, CD28, ICOS, and CD8. Claim 30 A composition according to any one of claims 23 to 29, wherein the first receptor expressed by T cells and the second receptor expressed by T cells are different. Claim 31 A composition according to any one of claims 23 to 30, wherein the first receptor expressed by the T cell is CD3 and the second receptor expressed by the T cell is CD2. Claim 32 A composition according to any one of claims 23 to 30, wherein the first receptor expressed by the T cell is CD2 and the second receptor expressed by the T cell is CD3. Claim 33 A composition according to any one of claims 23 to 29, wherein the first binding domain and the second binding domain of the T cell linkage are configured to bind to the same receptor expressed by the T cell. Claim 34 A composition according to any one of claims 1 to 33, wherein the VH of the anti-CD3 binding domain comprises the HC CDR2 sequence of IYTNNIGST (SEQ No. 712) or CIYTNNIGSTWYASWAKG (SEQ No. 706). Claim 35 A composition according to claim 34, wherein the VH of the anti-CD3 binding domain comprises the HC CDR1 sequence of GFSFSNNYY (SEQ No. 711) or NNYYMC (SEQ No. 705). Claim 36 A composition according to claim 34 or 35, wherein the anti-CD3 binding domain comprises a light chain variable region [VL] comprising the sequence of light chain complementarity determining region 3 [LC CDR3] of QQYNTIINVDRT (SEQ No. 713). Claim 37 A composition according to claim 36, wherein the VL of the anti-CD3 binding domain comprises the LC CDR2 sequence of KAS (SEQ No. 715) or KASTLAS (SEQ No. 709). Claim 38 A composition according to claim 36 or 37, wherein the VL of the anti-CD3 binding domain comprises the LC CDR1 sequence of QTIYNY (SEQ No. 714) or QASQTIYNYLA (SEQ No. 708). Claim 39 A composition according to any one of claims 34 to 38, wherein the claim-CD3(IMGT) binding domain comprises a. the HC CDR1 sequence of GFSFSNNYY (SEQ No. 711), b. the HC CDR2 sequence of IYTNNIGST (SEQ No. 712), c. the HC CDR3 sequence of ARGIYSDSSDFIGNL (SEQ No. 713), d. the LC CDR1 sequence of QTIYNY (SEQ No. 714), e. the LC CDR2 sequence of KAS (SEQ No. 715), and f. the LC CDR3 sequence of QQYNTIINVDRT (SEQ No. 716). Claim 40 A composition according to any one of claims 34 to 38, wherein the claim-CD3 (kavart) binding domain comprises a. the HC CDR1 sequence of NNYYMC (SEQ No. 705), b. the HC CDR2 sequence of CIYTNNIGSTWYASWAKG (SEQ No. 706), c. the HC CDR3 sequence of GIYSDSSDFIGNL (SEQ No. 707), d. the LC CDR1 sequence of QASQTIYNYLA (SEQ No. 708), e. the LC CDR2 sequence of KASTLAS (SEQ No. 709), and f. the LC CDR3 sequence of QQYNTIINVDRT (SEQ No. 710). Claim 41 A composition according to any one of claims 34 to 40, wherein the VH of the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS (Sequence No. 703). Claim 42 A composition according to any one of claims 36 to 41, wherein the VL of the anti-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIK (Sequence No. 704). Claim 43 A composition according to any one of claims 34 to 42, wherein the claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSAIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIK (Sequence No. 701). Claim 44 A composition according to any one of claims 34 to 42, wherein the claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQAPGKGLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS (Sequence No. 702). Claim 45 A composition according to any one of claims 1 to 44, wherein the T cell linkage comprises an additional anti-CD3 binding domain. Claim 46 A composition according to any one of claims 1 to 45, wherein the T cell linkage further comprises an anti-TRBC1 binding domain. Claim 47 A composition according to any one of claims 1 to 46, wherein the T cell linkage further comprises an anti-CD2 binding domain. Claim 48 A composition according to any one of claims 1 to 47, wherein the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising a heavy chain complementarity determining region 3 [HC CDR3] sequence of ARYYDDHYCLDY (SEQ No. 720), ARYYDDHYSLDY (SEQ No. 721), YYDDHYCLDY (SEQ No. 675), or YYDDHYSLDY (SEQ No. 717). Claim 49 A composition according to claim 48, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR2 sequence of INPSRGYT (SEQ No. 719) or YINPSRGYTNYNQKFKD (SEQ No. 674). Claim 50 A composition according to claim 48 or 49, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR1 sequence of GYTFTRYT (SEQ No. 718) or RYTMH (SEQ No. 673). Claim 51 A composition according to any one of claims 48 to 50, wherein the additional claim-CD3 binding domain comprises a light chain variable region [VL] comprising the sequence of the light chain complementarity determining region 3 [LC CDR3] of QQWSSNPLT (SEQ No. 678). Claim 52 A composition according to claim 51, wherein the VL of the additional claim-CD3 binding domain comprises the LC CDR2 sequence of DTS (SEQ No. 723) or DTSKVAS (SEQ No. 677). Claim 53 A composition according to claim 51 or 52, wherein the VL of the additional claim-CD3 binding domain comprises the LC CDR1 sequence of SSVSY (SEQ No. 722) or RASSSVSYMN (SEQ No. 676). Claim 54 A composition according to any one of claims 48 to 53, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of GYTFTRYT (SEQ No. 718), b. the HC CDR2 sequence of INPSRGYT (SEQ No. 719), c. the HC CDR3 sequence of ARYYDDHYCLDY (SEQ No. 720) or ARYYDDHYSLDY (SEQ No. 721), d. the LC CDR1 sequence of SSVSY (SEQ No. 722), e. the LC CDR2 sequence of DTS (SEQ No. 723), and f. the LC CDR3 sequence of QQWSSNPLT (SEQ No. 724). Claim 55 A composition according to any one of claims 48 to 53, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of RYTMH (SEQ No. 673), b. the HC CDR2 sequence of YINPSRGYTNYNQKFKD (SEQ No. 674), c. the HC CDR3 sequence of YYDDHYCLDY (SEQ No. 675) or YYDDHYSLDY (SEQ No. 717), d. the LC CDR1 sequence of RASSSVSYMN (SEQ No. 676), e. the LC CDR2 sequence of DTSKVAS (SEQ No. 677), and f. the LC CDR3 sequence of QQWSSNPLT (SEQ No. 678). Claim 56 A composition according to any one of claims 48 to 55, wherein the VH of the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS (Sequence No. 671) or QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 726). Claim 57 A composition according to any one of claims 51 to 56, wherein the VL of the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (Sequence No. 672). Claim 58 In any one of claims 48 to 57, the additional claim-CD3 binding domain is QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQD No. 727) or A composition comprising a sequence having at least 80% sequence identity with QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS (Sequence No. 728). Claim 59 In any one of claims 48 to 57, the additional claim-CD3 binding domain is QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQD No. 725) or A composition comprising a sequence having at least 80% sequence identity with QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (Sequence No. 728). Claim 60 A composition according to any one of claims 1 to 59, wherein the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARSGYYGDSDWYFDV (SEQ No. 731) or SGYYGDSDWYFDV (SEQ No. 607). Claim 61 A composition according to claim 60, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR2 sequence of INPYKGVS (SEQ No. 730) or LINPYKGVSTYNQKFKD (SEQ No. 608). Claim 62 A composition according to claim 60 or 61, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR1 sequence of GYSFTGYT (SEQ No. 729) or GYTMN (SEQ No. 605). Claim 63 A composition according to any one of claims 60 to 62, wherein the additional claim-CD3 binding domain comprises a light chain variable region [VL] comprising the sequence of light chain complementarity determining region 3 [LC CDR3] of QQGNTLPWT (SEQ No. 610). Claim 64 A composition according to claim 63, wherein the VL of the additional claim-CD3 binding domain comprises the LC CDR2 sequence of YTS (SEQ No. 733) or YTSRLES (SEQ No. 609). Claim 65 A composition according to claim 63 or 64, wherein the VL of the additional claim-CD3 binding domain comprises the LC CDR1 sequence of QDIRNY (SEQ No. 732) or RASQDIRNYLN (SEQ No. 608). Claim 66 A composition according to any one of claims 60 to 65, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of GYSFTGYT (SEQ No. 729), b. the HC CDR2 sequence of INPYKGVS (SEQ No. 730), c. the HC CDR3 sequence of ARSGYYGDSDWYFDV (SEQ No. 731), d. the LC CDR1 sequence of QDIRNY (SEQ No. 732), e. the LC CDR2 sequence of YTS (SEQ No. 733), and f. the LC CDR3 sequence of QQGNTLPWT (SEQ No. 610). Claim 67 A composition according to any one of claims 60 to 65, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of GYTMN (SEQ No. 605), b. the HC CDR2 sequence of LINPYKGVSTYNQKFKD (SEQ No. 606), c. the HC CDR3 sequence of SGYYGDSDWYFDV (SEQ No. 607), d. the LC CDR1 sequence of RASQDIRNYLN (SEQ No. 608), e. the LC CDR2 sequence of YTSRLES (SEQ No. 609), and f. the LC CDR3 sequence of QQGNTLPWT (SEQ No. 610). Claim 68 A composition according to any one of claims 60 to 67, wherein the VH of the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (Sequence No. 603). Claim 69 A composition according to any one of claims 63 to 68, wherein the VL of the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (Sequence No. 604). Claim 70 A composition according to any one of claims 60 to 69, wherein the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGSAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID No. 602). Claim 71 A composition according to any one of claims 60 to 69, wherein the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (Sequence No. 601). Claim 72 A composition according to any one of claims 1 to 71, wherein the T cell linkage comprises an additional anti-CD3 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of AAKIRPYIFKIAGQYDY (SEQ No. 736) or KIRPYIFKIAGQYDY (SEQ No. 618). Claim 73 A composition according to claim 72, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR2 sequence of IVWSDGNT (SEQ No. 735) or AIVWSDGNTYYEDFVKG (SEQ No. 617). Claim 74 A composition according to claim 72 or 73, wherein the VH of the additional claim-CD3 binding domain comprises the HC CDR1 sequence of GRTYRGYS (SEQ No. 734) or GYSMA (SEQ No. 616). Claim 75 A composition according to any one of claims 72 to 74, wherein the additional claim-CD3 binding domain is a VHH domain. Claim 76 A composition according to any one of claims 72 to 75, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of GRTYRGYS (SEQ No. 734), b. the HC CDR2 sequence of IVWSDGNT (SEQ No. 735), and c. the HC CDR3 sequence of AAKIRPYIFKIAGQYDY (SEQ No. 736). Claim 77 A composition according to any one of claims 72 to 75, wherein the additional claim-CD3 binding domain comprises a. the HC CDR1 sequence of GYSMA (SEQ No. 616), b. the HC CDR2 sequence of AIVWSDGNTYYEDFVKG (SEQ No. 617), and c. the HC CDR3 sequence of KIRPYIFKIAGQYDY (SEQ No. 618). Claim 78 A composition according to any one of claims 72 to 77, wherein the VH of the additional claim-CD3 binding domain comprises a sequence having at least 80% sequence identity with the sequence EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS (Sequence No. 615). Claim 79 A composition according to any one of claims 1 to 78, wherein the T cell linkage further comprises an anti-TRBC1 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of ARGAGYNFDGAYRFEDF (SEQ ID No. 742) or GAGYNFDGAYRFEDF (SEQ ID No. 739). Claim 80 A composition according to claim 79, wherein the VH of the claim-TRBC1 binding domain comprises the HC CDR2 sequence of INPYNDDI (SEQ No. 741) or FINPYNDDIQSNERFRG (SEQ No. 738). Claim 81 A composition according to claim 79 or 80, wherein the VH of the claim-TRBC1 binding domain comprises the HC CDR1 sequence of GYTFTGYV (SEQ No. 740) or GYVMH (SEQ No. 737). Claim 82 A composition according to any one of claims 79 to 81, wherein the claim-TRBC1 binding domain comprises a light chain variable region [VL] comprising the sequence of light chain complementarity determining region 3 [LC CDR3] of SQSTHVPYT (SEQ No. 745). Claim 83 A composition according to claim 82, wherein the VL of the above-mentioned claim-TRBC1 binding domain comprises the LC CDR2 sequence of RVS (SEQ No. 747) or RVSNRFP (SEQ No. 744). Claim 84 A composition according to claim 82 or 83, wherein the VL of the claim-TRBC1 binding domain comprises the LC CDR1 sequence of QRLVHSNGNTY (SEQN 746) or RSSQRLVHSNGNTYLH (SEQN 743). Claim 85 A composition according to any one of claims 79 to 84, wherein the claim-TRBC1 binding domain comprises a. the HC CDR1 sequence of GYTFTGYV (SEQ No. 740), b. the HC CDR2 sequence of INPYNDDI (SEQ No. 741), c. the HC CDR3 sequence of ARGAGYNFDGAYRFEDF (SEQ No. 742), d. the LC CDR1 sequence of QRLVHSNGNTY (SEQ No. 746), e. the LC CDR2 sequence of RVS (SEQ No. 747), and f. the LC CDR3 sequence of SQSTHVPYT (SEQ No. 745). Claim 86 A composition according to any one of claims 79 to 84, wherein the claim-TRBC1 binding domain comprises a. the HC CDR1 sequence of GYVMH (SEQ No. 737), b. the HC CDR2 sequence of FINPYNDDIQSNERFRG (SEQ No. 738), c. the HC CDR3 sequence of GAGYNFDGAYRFEDF (SEQ No. 739), d. the LC CDR1 sequence of RSSQRLVHSNGNTYLH (SEQ No. 743), e. the LC CDR2 sequence of RVSNRFP (SEQ No. 744), and f. the LC CDR3 sequence of SQSTHVPYT (SEQ No. 745). Claim 87 A composition according to any one of claims 79 to 86, wherein the VH of the claim-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with the sequence EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (Sequence No. 625). Claim 88 A composition according to any one of claims 82 to 87, wherein the VL of the claim-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with the sequence DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (Sequence No. 626). Claim 89 A composition according to any one of claims 79 to 88, wherein the claim-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR (SEQ ID No. 624). Claim 90 A composition according to any one of claims 79 to 88, wherein the claim-TRBC1 binding domain comprises a sequence having at least 80% sequence identity with DVVMTQSPLSLPYSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVYKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFEDFWGQGTTLTVSS (SEQ ID No. 748). Claim 91 A composition according to any one of claims 1 to 90, wherein the T cell linkage further comprises an anti-CD2 binding domain comprising a heavy chain variable region [VH] comprising the heavy chain complementarity determining region 3 [HC CDR3] sequence of AAVRDYVGMPYYSGSAYEY (SEQ No. 749) or VRDYVGMPYYSGSAYEY (SEQ No. 750). Claim 92 A composition according to claim 91, wherein the VH of the anti-CD2 binding domain comprises the HC CDR2 sequence of IRPGTIP (SEQ No. 751) or AIRPGTIPYYSESVKG (SEQ No. 752). Claim 93 A composition according to claim 91 or 92, wherein the VH of the anti-CD2 binding domain comprises the HC CDR1 sequence of GLTFSSYG (SEQ No. 753) or SYGMA (SEQ No. 754). Claim 94 A composition according to any one of claims 91 to 93, wherein the claim-CD2 binding domain is a VHH domain. Claim 95 A composition according to any one of claims 91 to 94, wherein the anti-CD2 binding domain comprises a. the HC CDR1 sequence of GLTFSSYG (SEQ No. 753), b. the HC CDR2 sequence of IRPGTIP (SEQ No. 751), and c. the HC CDR3 sequence of AAVRDYVGMPYYSGSAYEY (SEQ No. 749). Claim 96 A composition according to any one of claims 91 to 94, wherein the anti-CD2 binding domain comprises a. the HC CDR1 sequence of SYGMA (SEQ No. 754), b. the HC CDR2 sequence of AIRPGTIPYYSESVKG (SEQ No. 752), and c. the HC CDR3 sequence of VRDYVGMPYYSGSAYEY (SEQ No. 750). Claim 97 A composition according to any one of claims 91 to 96, wherein the VH of the anti-CD2 binding domain comprises a sequence having at least 80% sequence identity with the sequence QVQLVESGGGLVQAGGSLRLSCAASGLTFSSYGMAWFRRALGREREFVGAIRPGTIPYYSESVKGRFTVSKDNAKNTVSLQMNSLKPEDTAVYYCAAVRDYVGMPYYSGSAYEYWGQGTQVTVSS (Sequence No. 623). Claim 98 A composition according to any one of claims 7 to 97, wherein one or more binding domains of the T cell linkage are connected to the first polypeptide or the second polypeptide. Claim 99 A composition according to any one of claims 7 to 98, wherein the first polypeptide further comprises a dimerization domain fused to the TRAV domain, and the second polypeptide comprises a dimerization domain fused to the TRBV domain. Claim 100 A composition according to claim 99, wherein the first polypeptide comprises the dimerization domain, peptide linker, and TRAV domain of the first polypeptide in the direction from the N-terminus to the C-terminus, and the second polypeptide comprises the dimerization domain, peptide linker, and TRBV domain of the second polypeptide in the direction from the N-terminus to the C-terminus. Claim 101 A composition according to claim 99 or 100, wherein the dimerization domain of the first polypeptide comprises a TCR alpha constant (TRAC) domain or a part thereof, and the dimerization domain of the second polypeptide comprises a TCR beta constant (TRBC) domain or a part thereof. Claim 102 A composition according to any one of claims 99 to 101, wherein the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by one or more disulfide bridges. Claim 103 A composition according to claim 102, wherein the dimerization domain of the first polypeptide and the dimerization domain of the second polypeptide are connected by a single disulfide bridge. Claim 104 A composition according to any one of claims 7 to 103, wherein the first polypeptide comprises the T cell linkage. Claim 105 A composition according to claim 104, wherein the T cell linkage is connected to the dimerization domain of the first polypeptide. Claim 106 In claim 104, the composition wherein the first polypeptide comprises the T cell linkage, the dimerization domain, and the TRAV domain in the direction from the N-terminus to the C-terminus. Claim 107 A composition according to claim 104, wherein the first polypeptide comprises the TRAV domain and the T cell linkage in the direction from the N-terminus to the C-terminus. Claim 108 A composition according to any one of claims 7 to 103, wherein the second polypeptide comprises the T cell linkage. Claim 109 A composition according to claim 108, wherein the second polypeptide comprises the T cell linkage, the dimerization domain, and the TRBV domain in the direction from the N-terminus to the C-terminus. Claim 110 In claim 108, the composition wherein the second polypeptide comprises the TRBV domain and the T cell linkage in the direction from the N-terminus to the C-terminus. Claim 111 A composition according to claim 104 or 108, wherein the T cell linkage comprises scFv that binds to the receptor expressed by the T cell, and the polypeptide comprises the scFv, the dimerization domain, and the TRAV domain in the direction from the N-terminus to the C-terminus. Claim 112 A composition according to claim 104 or 108, wherein the T cell linkage comprises scFv that binds to the receptor expressed by the T cell, and the polypeptide comprises the dimerization domain, the TRAV domain, and the scFv in the direction from the N-terminus to the C-terminus. Claim 113 A composition according to claim 106 or 107, wherein the T cell linkage comprises scFv that binds to the receptor expressed by the T cell, and the polypeptide comprises the scFv, the dimerization domain, and the TRBV domain in the direction from the N-terminus to the C-terminus. Claim 114 A composition according to claim 104 or 108, wherein the T cell linkage comprises scFv that binds to a receptor expressed by a T cell, and the polypeptide comprises the dimerization domain, the TRBV domain, and the scFv in the direction from the N-terminus to the C-terminus. Claim 115 A composition according to claim 104 or 108, wherein the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises VHH, the dimerization domain, and the TRAV domain in the direction from the N-terminus to the C-terminus. Claim 116 A composition according to claim 104 or 108, wherein the T cell linkage comprises VHH that binds to a receptor expressed by a T cell, and the polypeptide comprises the dimerization domain, the TRAV domain, and the VHH in the direction from the N-terminus to the C-terminus. Claim 117 A composition according to claim 104 or 108, wherein the T cell linkage comprises VHH that binds to the receptor expressed by the T cell, and the polypeptide comprises the VHH, the dimerization domain, and the TRBV domain in the direction from the N-terminus to the C-terminus. Claim 118 A composition according to claim 104 or 108, wherein the T cell linkage comprises VHH that binds to the receptor expressed by the T cell, and the polypeptide comprises the dimerization domain, the TRBV domain, and the VHH in the direction from the N-terminus to the C-terminus. Claim 119 A composition according to any one of claims 115 to 117, wherein the T cell linkage comprises a first VHH domain and a second VHH domain configured to bind to the same receptor expressed by the T cell. Claim 120 A composition according to any one of claims 115 to 117, wherein the T cell linkage comprises a first VHH domain that binds to the first receptor expressed by the T cell and the second VHH domain that binds to the second receptor expressed by the T cell. Claim 121 A composition according to any one of claims 21 to 120, wherein the T cell linker comprises scFv that binds to a first T cell receptor and VHH that binds to a second T cell receptor, wherein the scFv and the VHH are connected by a peptide linker. Claim 122 A composition according to claim 23, wherein the first polypeptide comprises the first binding domain of the T cell linkage that binds to the extracellular domain of the first receptor expressed by the T cell, and the second polypeptide comprises the second binding domain of the T cell linkage that binds to the extracellular domain of the second receptor expressed by the T cell. Claim 123 A composition according to claim 122, wherein the first binding domain of the T cell linkage is a first VHH fused to the first polypeptide and binding to CD3, and the second binding domain of the T cell linkage is a second VHH fused to the second polypeptide and binding to CD2. Claim 124 A composition according to claim 122, wherein the first binding domain of the T cell linkage is a first VHH that binds to CD2, and the second binding domain of the T cell linkage is a second VHH that binds to CD3. Claim 125 A composition according to any one of claims 7 to 124, wherein the T cell linker is fused with the first or second polypeptide through a peptide linker. Claim 126 A composition comprising the multispecific molecule encoded by the sequence of the recombinant nucleic acid of any one of claims 1 to 125. Claim 127 In claim 126, the composition wherein the multispecific molecule is isolated or purified. Claim 128 A pharmaceutical composition comprising the composition of any one of claims 1 to 127. Claim 129 A method for treating a subject requiring cancer treatment, comprising the step of administering a therapeutically effective amount of the medicine of claim 128. Claim 130 A method for producing a multispecific molecule, wherein the multispecific molecule is an MHC-peptide complex conjugate comprising (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the multispecific molecule comprises a T cell conjugate comprising a post-translational modification, and the method comprises the step of producing a multispecific molecule comprising a mammalian post-translational modification signature by expressing the multispecific molecule from a recombinant nucleic acid comprising a sequence encoding the multispecific molecule in a mammalian cell. Claim 131 In claim 130, the method further comprises the step of isolating or purifying the multispecific molecule containing the mammalian post-translational modification signature. Claim 132 A method according to claim 130, wherein the cell secretes the first polypeptide and the second polypeptide encoded by the sequence of the recombinant nucleic acid of the composition of any one of claims 7 to 125. Claim 133 A mammalian cell comprising the recombinant nucleic acid of the composition of any one of claims 1 to 125. Claim 134 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the one or more binding domains of the T cell conjugate, the TRBV domain, the TRBC domain, the TRAV domain, and the TRAC domain in the direction from the N-terminus to the C-terminus. Claim 135 In claim 134, the recombinant nucleic acid further comprises a linker connecting the TRBC domain and the TRAV domain, wherein the multispecific molecule further comprises the TRBC domain and the TRAV domain. Claim 136 In paragraph 134 or 135, the linker is a recombinant nucleic acid that is not a cleavage type linker. Claim 137 In any one of claims 134 to 136, the linker is a recombinant nucleic acid comprising a sequence of 20 to 38 amino acids. Claim 138 In any one of claims 134 to 137, the linker is a structure-directed flexible linker, a recombinant nucleic acid. Claim 139 In any one of claims 134 to 138, the linker is a recombinant nucleic acid comprising Gly, Ser, Ala and / or Glu residues. Claim 140 In claim 139, the linker is a recombinant nucleic acid comprising a G4S motif comprising 1, 4, 5, or 7 repeats of a GGGGS sequence. Claim 141 In paragraph 139, the linker comprises a linker represented by the formula GGSSGSG-X25-GSGSG, wherein X is an amino acid selected from A, G, S and T, a recombinant nucleic acid. Claim 142 In claim 139, the multispecific molecule is a recombinant nucleic acid comprising one or more sequences of SEQ ID NO. 604, SEQ ID NO. 682, SEQ ID NO. 603, SEQ ID NO. 620, SEQ ID NO. 863, SEQ ID NO. 864, SEQ ID NO. 619, SEQ ID NO. 865 and SEQ ID NO.

866. Claim 143 In claim 139, the multiple specific molecule is a recombinant nucleic acid comprising the sequence of SEQ ID NO.

862. Claim 144 In claim 139, the multiple specific molecule is a recombinant nucleic acid comprising the sequence of SEQ ID NO. 862 excluding the signal peptide of SEQ ID NO.

668. Claim 145 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the one or more binding domains of the T cell conjugate, the TRAV domain, the TRAC domain, the TRBV domain, and the TRBC domain in the direction from the N-terminus to the C-terminus. Claim 146 In paragraph 145, the recombinant nucleic acid further comprises a linker connecting the TRAC domain and the TRBV domain, wherein the multispecific molecule. Claim 147 In paragraph 146, the linker is a recombinant nucleic acid comprising 20 to 38 amino acids, including Gly, Ser, Ala and / or Glu amino acids. Claim 148 In claim 145 or 147, the one or more binding domains of the T cell linkage comprise a single chain variable fragment [scFv], recombinant nucleic acid. Claim 149 In claim 148, the scFv is a recombinant nucleic acid comprising a light chain variable domain (VL) followed by a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus. Claim 150 In claim 148 or 149, the scFv comprises a light chain variable domain (VL) following a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus, a recombinant nucleic acid. Claim 151 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRAV domain, the TRAC domain, the one or more binding domains of the T cell conjugate, the TRBV domain, and the TRBC domain in the direction from the N-terminus to the C-terminus. Claim 152 In claim 151, the recombinant nucleic acid further comprises a linker connecting the TRAC domain and one or more binding domains of the T cell linkage. Claim 153 In claim 151 or 152, the one or more binding domains of the T cell linkage comprise a short-chain variable fragment [scFv], recombinant nucleic acid. Claim 154 A recombinant nucleic acid according to any one of claims 151 to 153, wherein the scFv comprises a heavy chain variable domain (VH) following a light chain variable domain (VL) in the direction from the N-terminus to the C-terminus. Claim 155 A recombinant nucleic acid according to any one of claims 151 to 153, wherein the scFv comprises a light chain variable domain (VL) following a heavy chain variable domain (VH) in the direction from the N-terminus to the C-terminus. Claim 156 A recombinant nucleic acid according to any one of claims 151 to 155, wherein the multiple-specific molecule comprises one or more sequences selected from the group consisting of SEQ ID NO. 619, SEQ ID NO. 868, SEQ ID NO. 869, SEQ ID NO. 604, SEQ ID NO. 870, SEQ ID NO. 603, SEQ ID NO. 620, SEQ ID NO. 871, and SEQ ID NO.

866. Claim 157 In any one of claims 151 to 156, the multiple specific molecule is a recombinant nucleic acid comprising the sequence of SEQ ID NO.

867. Claim 158 In any one of claims 151 to 156, the multiple specific molecule comprises the sequence of SEQ ID NO. 867 excluding the signal peptide of SEQ ID NO. 667, a recombinant nucleic acid. Claim 159 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises: (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and a TCR alpha constant (TRAC) domain and (b) a TCR beta variable (TRBV) domain and a TCR beta constant (TRBC) domain; and (ii) a T cell conjugate comprising one or more binding domains that bind to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRBV domain, the TRBC domain, the one or more binding domains of the T cell conjugate, the TRAV domain, and the TRAC domain in the direction from the N-terminus to the C-terminus. Claim 160 In claim 159, the multispecific molecule further comprises a linker connecting the TRBC domain and one or more binding domains of the T cell linker, wherein the linker comprises 20 to 37 amino acids and comprises Gly, Ser, Ala and / or Glu amino acids, recombinant nucleic acid. Claim 161 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the scFv, the TRBV domain and the TRAV domain in the direction from the N-terminus to the C-terminus. Claim 162 In paragraph 161, a recombinant nucleic acid further comprising a TCR alpha-invariant (TRAC) domain. Claim 163 Recombinant nucleic acid according to claim 161 or 162, further comprising a TCR beta constant (TRBC) domain. Claim 164 A recombinant nucleic acid according to any one of claims 161 to 163, wherein the T cell linkage forms a continuous single polypeptide chain comprising the scFv, the TRBV domain, the TRBC domain, the TRAV domain and the TRAC domain in the direction from the N-terminus to the C-terminus. Claim 165 In any one of claims 161 to 164, the recombinant nucleic acid comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. Claim 166 In any one of claims 161 to 164, the recombinant nucleic acid comprises a VH domain and a VL domain in the direction from the N-terminus to the C-terminus. Claim 167 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the scFv, the TRAV domain and the TRBV domain in the direction from the N-terminus to the C-terminus. Claim 168 In paragraph 167, a recombinant nucleic acid further comprising a TCR alpha-constant (TRAC) domain. Claim 169 Recombinant nucleic acid according to claim 167 or 168, further comprising a TCR beta constant (TRBC) domain. Claim 170 A recombinant nucleic acid according to any one of claims 167 to 169, wherein the T cell linkage forms a continuous single polypeptide chain comprising the scFv, the TRBV domain, the TRBC domain, the TRAV domain and the TRAC domain in the direction from the N-terminus to the C-terminus. Claim 171 In any one of claims 167 to 170, the recombinant nucleic acid comprising a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. Claim 172 In any one of claims 167 to 170, the recombinant nucleic acid comprising a VH domain and a VL domain in the direction from the N-terminus to the C-terminus. Claim 173 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRAV domain, the scFv and the TRBV domain in the direction from the N-terminus to the C-terminus. Claim 174 In paragraph 173, a recombinant nucleic acid further comprising a TCR alpha constant (TRAC) domain. Claim 175 Recombinant nucleic acid according to claim 173 or 174, further comprising a TCR beta constant (TRBC) domain. Claim 176 A recombinant nucleic acid according to any one of claims 173 to 175, wherein the T cell linkage forms a continuous single polypeptide chain comprising the scFv, the TRAV domain, the TRAC domain, the TRBV domain and the TRBC domain in the direction from the N-terminus to the C-terminus. Claim 177 In any one of claims 173 to 176, the recombinant nucleic acid comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. Claim 178 In any one of claims 173 to 177, the recombinant nucleic acid comprising a VH domain and a VL domain in the direction from the N-terminus to the C-terminus. Claim 179 A recombinant nucleic acid comprising a sequence encoding a multispecific molecule, wherein the multispecific molecule comprises (i) an MHC-peptide complex conjugate comprising (a) a T cell receptor (TCR) alpha variable (TRAV) domain and (b) a TCR beta variable (TRBV) domain; and (ii) a T cell conjugate comprising a short-chain variable fragment [scFv] that binds to an extracellular domain of a receptor expressed by a T cell, wherein the MHC-peptide complex conjugate and the T cell conjugate form a continuous single polypeptide chain comprising the TRBV domain, the scFv and the TRAV domain in the direction from the N-terminus to the C-terminus. Claim 180 In paragraph 179, a recombinant nucleic acid further comprising a TCR alpha-invariant (TRAC) domain. Claim 181 Recombinant nucleic acid according to claim 179 or 180, further comprising a TCR beta constant (TRBC) domain. Claim 182 A recombinant nucleic acid according to any one of claims 179 to 181, wherein the T cell linkage forms a continuous single polypeptide chain comprising the scFv, the TRAV domain, the TRAC domain, the TRBV domain and the TRBC domain in the direction from the N-terminus to the C-terminus. Claim 183 In any one of claims 179 to 182, the recombinant nucleic acid comprises a VL domain and a VH domain in the direction from the N-terminus to the C-terminus. Claim 184 In any one of claims 179 to 183, the recombinant nucleic acid comprising a VH domain and a VL domain in the direction from the N-terminus to the C-terminus. Claim 185 A recombinant nucleic acid according to any one of claims 134 to 184, wherein the peptide linker is encoded, said linker comprises an amino acid sequence having 90% sequence identity with the sequence of SEQ ID NO. 864, or 872, or 873. Claim 186 In any one of claims 134 to 185, the TRAC domain comprises a wild-type mouse TRAC sequence or a wild-type human TRAC sequence, a recombinant nucleic acid. Claim 187 In any one of claims 134 to 186, the TRAC domain is a recombinant nucleic acid that does not contain mutations. Claim 188 In any one of claims 134 to 187, the TRAC domain is a recombinant nucleic acid comprising a mutation compared to a rat TRAC sequence or a wild-type human TRAC sequence. Claim 189 In any one of paragraphs 134 to 188, the mutation is a recombinant nucleic acid that is a stability-enhancing mutation. Claim 190 A recombinant nucleic acid according to any one of claims 134 to 189, wherein the mutation is selected from the group consisting of S139F, T150I, and A190T, and the residue positions are numbered based on the Kavat numbering system. Claim 191 In any one of claims 134 to 190, the TRBC domain comprises a wild-type mouse TRBC sequence or a wild-type human TRBC sequence, a recombinant nucleic acid. Claim 192 In any one of claims 134 to 191, the TRBC domain is a recombinant nucleic acid that does not contain mutations. Claim 193 In any one of claims 134 to 192, the TRBC domain comprises a mutation compared to a rat TRBC sequence or a wild-type human TRBC sequence, a recombinant nucleic acid. Claim 194 In any one of paragraphs 134 to 193, the mutation is a recombinant nucleic acid that is a stability-enhancing mutation. Claim 195 A recombinant nucleic acid according to any one of claims 134 to 194, wherein the mutation is selected from the group consisting of E134K, H139R, D155P and S170D, and the residue positions are numbered based on the Kavat numbering system.

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