Antigen-binding domain and method of use thereof

Abstract

Antibodies specific to V-set immunoglobulin domain 2 (VSIG2) and their antigen-binding fragments are provided herein. Also provided herein are cells, nucleic acids, vectors, compositions, and methods relating to VSIG2-specific antibodies or their antigen-binding domains.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 496,640, filed on 17 April 2023, and U.S. Provisional Application No. 63 / 590,361, filed on 13 October 2023, each of which is incorporated herein by reference in whole for all purposes.

[0002] Sequence List This application includes a sequence listing filed via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy thereof was created in XX / XX / 20XX, named XXXXXUS_sequencelisting.xml, and has a size of X,XXX,XXX bytes.

[0003] Chimeric antigen receptor (CAR) adoptive cell therapy, used to redirect the specificity and function of immune-responsive cells such as T cells, has shown efficacy in patients with lymphoid malignancies (Pule et al., Nat. Med. (14): 1264-1270 (2008); Maude et al., N Engl J Med. (371): 1507-17 (2014); Brentjens et al., Sci Transl Med. (5): 177ra38 (2013)). CAR T cells have been shown to induce complete remission in patients with CD19-expressing malignancies that have resulted in drug resistance and tumor progression due to chemotherapy. The success of CD19 CAR therapy offers optimism for the treatment of other malignancies, such as solid tumors.

[0004] One challenge in developing CAR therapies for solid tumors is the lack of suitable targets. The ability to identify appropriate CAR targets is crucial for effectively targeting and treating tumors without damaging normal cells that express the same target antigen. Therefore, there remains a need for CAR solid tumor therapies that target tumor cells without targeting normal cells or tissues, such as therapies for the treatment of colorectal cancer. [Overview of the project]

[0005] This specification provides an isolated antibody or its antigen-binding fragment that specifically binds to human V-set immunoglobulin domain 2 (VSIG2), which includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein VH comprises VH complementation region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1 and VH complementation region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3, and VL comprises VL complementation region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7 The molecule comprises a VL complementary region 2 (CDRL2) having an amino acid sequence, and (i) VH comprises a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and VL comprises a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) VH comprises a VH complementary region 3 (CDRH3) having one of the amino acid sequences of SEQ ID NOs. 67 to 87, and VL comprises a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0006] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human V-set immunoglobulin domain-containing 2 (VSIG2), which includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein VH comprises VH complementation region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 2 and VH complementation region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4, and VL comprises VL complementation region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6 and SEQ ID NO: 7 The molecule contains a VL complementary region 2 (CDRL2) having an amino acid sequence, and (i) VH contains a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and VL contains a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) VH contains a VH complementary region 3 (CDRH3) having one of the amino acid sequences of SEQ ID NOs. 67 to 87, and VL contains a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0007] In some embodiments, VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 88-107. In some embodiments, VL has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, A) CDRH3 has the amino acid sequence of SEQ ID NO: 5 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or B) CDRH3 has the amino acid sequence of SEQ ID NO: 67 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or C) CDRH3 has the amino acid sequence of SEQ ID NO: 68 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or D) CDRH3 has the amino acid sequence of SEQ ID NO: 67 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or E) CDRH3 has the amino acid sequence of SEQ ID NO: 68 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or F) CDRH3 has the amino acid sequence of SEQ ID NO: 69 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or G) CDRH3 has the amino acid sequence of SEQ ID NO: 69 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or H) CDRH3 has the amino acid sequence of SEQ ID NO: 70 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or I) CDRH3 has the amino acid sequence of SEQ ID NO: 71 The sequence has a column, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or J) CDRH3 has the amino acid sequence of SEQ ID NO: 72, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or K) CDRH3 has the amino acid sequence of SEQ ID NO: 73, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or L) CDRH3 has the amino acid sequence of SEQ ID NO: 74, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or M) CDRH3 has the amino acid sequence of SEQ ID NO: 75, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or N) CDRH3 has the amino acid sequence of SEQ ID NO: 76, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or O) CDRH3 has the amino acid sequence of SEQ ID NO: 77, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or P) CDRH3 has the amino acid sequence of SEQ ID NO: 78, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Q) CDRH3 has the amino acid sequence of SEQ ID NO: 79, and CDRL3 has the amino acid sequence of SEQ ID NO: 8.Or R) CDRH3 has the amino acid sequence of SEQ ID NO: 80 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or S) CDRH3 has the amino acid sequence of SEQ ID NO: 81 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or T) CDRH3 has the amino acid sequence of SEQ ID NO: 82 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or U) CDRH3 has the amino acid sequence of SEQ ID NO: 83 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or V) CDRH3 has the amino acid sequence of SEQ ID NO: 84 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or W) CDRH3 has the amino acid sequence of SEQ ID NO: 85 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or X) CDRH3 has the amino acid sequence of SEQ ID NO: 86 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Y) CDRH3 has the amino acid sequence of SEQ ID NO: 87 and CDRL3 has the amino acid sequence of SEQ ID NO: 8. In some embodiments, CDRH3 has the amino acid sequence of SEQ ID NO: 69, and CDRL3 has the amino acid sequence of SEQ ID NO: 8.

[0008] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human V-set immunoglobulin domain-containing 2 (VSIG2), which includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein (i) the VH region comprises the amino acid sequence of SEQ ID NO: 16 and the VL region comprises a selected amino acid sequence of SEQ ID NO: 15, or (ii) the VH region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107 and the VL region comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0009] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human V-set immunoglobulin domain-containing 2 (VSIG2), which includes a variable heavy chain (VH) region and a variable light chain (VL) region, wherein VL has the amino acid sequence of SEQ ID NO: 15. In some embodiments, VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107.

[0010] Also provided herein are isolated antibodies or antigen-binding fragments thereof that specifically bind to human V-set immunoglobulin domain-containing 2 (VSIG2) comprising a variable heavy (VH) region and a variable light (VL) region, wherein VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107. In some embodiments, VL has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises an F(ab) fragment, an F(ab’) fragment, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding fragment comprises a single-chain variable fragment (scFv). In some embodiments, the VH and VL of the scFv are separated by a peptide linker. In some embodiments, the antigen-binding domain comprises a VH-L-VL or VL-L-VH structure, wherein VH is a heavy-chain variable domain, L is a peptide linker, and VL is a light-chain variable domain. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37.

[0012] In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 108-132.

[0013] This specification also provides chimeric proteins comprising one of the isolated antibodies or antigen-binding fragments described herein, and a heterologous molecule or moiety. In some embodiments, the chimeric protein is an antibody-drug conjugate, and the heterologous molecule or moiety comprises a therapeutic agent. In some embodiments, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR is activated. In some embodiments, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some embodiments, one or more intracellular inhibitory domains include ICDs derived from PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, SIGLEC-10, PECAM-1, CD72, IRTA2, IRTA4, NKIR, TLT1, PCDHGC3, MPZL1, FCGR2B, SIGLEC-6, MPIG6B, SIGLEC-12, LIR8, IRTA1, KIR2DL4, KIR2DL5, SIGLEC-7, or FCRH3.In some embodiments, the intracellularly inhibitory domain comprises the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139), or an amino acid sequence that is 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%, at least 99%, or 100% identical to one or more of the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139).

[0014] In some embodiments, the intracellularly inhibitory domain comprises an enzymatically inhibitory domain. In some embodiments, the intracellularly inhibitory domain comprises an intracellular co-signaling inhibitory domain. In some embodiments, the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52.

[0015] Also provided herein is a composition comprising any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the chimeric proteins described herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0016] Also provided herein is an engineered nucleic acid encoding any one of the antibodies or antigen-binding fragments thereof described herein, any one of the chimeric proteins described herein, or any one of the engineered expression systems described herein.

[0017] This specification also provides an expression vector comprising one of the manipulated nucleic acids described herein or one of the manipulated expression systems described herein.

[0018] This specification also provides compositions comprising one of the engineered nucleic acids or expression vectors described herein, or one of the engineered expression systems described herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0019] This specification also provides a method for producing engineered cells, the method comprising transducing isolated cells using one of the engineered nucleic acids or expression vectors described herein, or one of the engineered expression systems described herein.

[0020] This specification also provides isolated cells containing one of the manipulated nucleic acids or expression vectors described herein.

[0021] This specification also provides a population of manipulated cells expressing any one of the manipulated nucleic acids or expression vectors described herein, or any one of the manipulated expression systems described herein.

[0022] Isolated cells containing one of the antibodies or antigen-binding fragments described herein, one of the chimeric proteins described herein, or one of the manipulated expression systems described herein.

[0023] This specification also provides a population of manipulated cells expressing any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the chimeric proteins described herein.

[0024] In some embodiments, the chimeric protein is recombinantly expressed. In some embodiments, the chimeric protein is expressed from a locus selected from a vector or the cell's genome. In some embodiments, a cell or population of cells further comprises one or more tumor-targeted chimeric receptors expressed on the cell surface. In some embodiments, each of the one or more tumor-targeted chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0025] In some embodiments, the cells or population of cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0026] In some embodiments, cells are autologous. In other embodiments, cells are allogeneic.

[0027] This specification also provides pharmaceutical compositions comprising an effective amount of any one of the cells or manipulated cell populations described herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0028] This specification also provides a pharmaceutical composition comprising a gene-modified cell expressing any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the chimeric proteins described herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. In some embodiments, the pharmaceutical composition is for the treatment and / or prevention of tumors.

[0029] This specification also provides a method for treating a subject in need of treatment, the method comprising administering a therapeutically effective dose of one of the compositions, cells, or pharmaceutical compositions described herein.

[0030] This specification also provides a method for stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of one of the compositions, cells, or pharmaceutical compositions described herein.

[0031] In some embodiments, the method comprises administering one of the cells described herein to a target, wherein the isolated cells or population of cells express a chimeric protein comprising one of the activated CARs described herein.

[0032] This specification also provides a method for inhibiting a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of one of the compositions, cells, or pharmaceutical compositions described herein.

[0033] In some embodiments, the method comprises administering to one of the cells described herein, the isolated cells or population of cells expressing a chimeric protein comprising one of the inhibitory CARs described herein.

[0034] This specification also provides a method for treating a subject having a tumor, the method comprising administering a therapeutically effective dose of one of the compositions, cells, or pharmaceutical compositions described herein.

[0035] This specification also provides kits for treating and / or preventing tumors, each kit comprising one of the chimeric proteins described herein. In some embodiments, the kit further includes written instructions for using the chimeric proteins to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0036] This specification also provides kits for treating and / or preventing tumors, each kit comprising one of the cells or populations of cells described herein. In some embodiments, the kit further includes written instructions for using the cells to treat and / or prevent tumors in a subject.

[0037] This specification also provides kits for treating and / or preventing tumors, each kit comprising one of the engineered nucleic acids described herein. In some embodiments, the kit further includes written instructions for using the nucleic acids to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0038] This specification also provides kits for treating and / or preventing tumors, each kit comprising one of the vectors described herein. In some embodiments, the kit further includes written instructions for using the vectors to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0039] This specification also provides kits for treating and / or preventing tumors, each kit comprising one of the compositions described herein. In some embodiments, the kit further includes written instructions for using the compositions for treating and / or preventing tumors in a subject.

[0040] This specification also provides an engineered expression system comprising: a first nucleic acid sequence encoding a first CAR, wherein the first CAR comprises a first extracellular antigen-binding domain that binds to an antigen selected from the group consisting of CEACAM5, CEA, CEACAM1, and CEACAM6; a first transmembrane domain; and one or more intracellular signaling domains; and a second nucleic acid sequence encoding a second CAR, wherein the second CAR comprises one of the antibodies or antigen-binding fragments described herein, or one of the chimeric proteins described herein.

[0041] In some embodiments of the manipulated expression system described herein, the first CAR includes a first spacer between the first extracellular antigen-binding domain and the first transmembrane domain. In some embodiments of the manipulated expression system described herein, the first spacer includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 52. In some embodiments of the manipulated expression system described herein, the first spacer includes the amino acid sequence of SEQ ID NO. 50. In some embodiments of the manipulated expression system described herein, the second CAR includes a second spacer between the second extracellular antigen-binding domain and the second transmembrane domain. In some embodiments of the manipulated expression system described herein, the second spacer includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 52. In some embodiments of the manipulated expression system described herein, the second spacer includes the amino acid sequence of SEQ ID NO. 50. In some embodiments of the manipulated expression systems described herein, one or more intracellular signaling domains of the first CAR are selected from the group consisting of CD3 zeta chain intracellular signaling domains, CD3 epsilon chain intracellular signaling domains, CD97 intracellular signaling domains, CD11a-CD18 intracellular signaling domains, CD2 intracellular signaling domains, ICOS intracellular signaling domains, CD27 intracellular signaling domains, CD154 intracellular signaling domains, CD8 intracellular signaling domains, OX40 intracellular signaling domains, 4-1BB intracellular signaling domains, CD28 intracellular signaling domains, ZAP40 intracellular signaling domains, CD30 intracellular signaling domains, GITR intracellular signaling domains, HVEM intracellular signaling domains, DAP10 intracellular signaling domains, DAP12 intracellular signaling domains, MyD88 intracellular signaling domains, 2B4 intracellular signaling domains, NKp46 intracellular signaling domains, NKp30 intracellular signaling domains, NKp44 intracellular signaling domains, NKG2D intracellular signaling domains, CD226 intracellular signaling domains, and CD160 intracellular signaling domains.In some embodiments of the manipulated expression systems described herein, the first CAR includes a CD28 intracellular signaling domain and a CD3 zeta chain intracellular signaling domain. In some embodiments of the manipulated expression systems described herein, the first transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a LAX transmembrane domain, a LAT transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a TIM3 transmembrane domain, a KIR3DS1 transmembrane domain, a KIR3DL1 transmembrane domain, an NKG2D transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain. In some embodiments of the manipulated expression systems described herein, the first CAR includes a CD28 transmembrane domain. In some embodiments of the engineered expression system described herein, the first and second nucleic acid sequences are contained within a single expression vector. In some embodiments of the engineered expression system described herein, the first nucleic acid sequence is contained within a first expression vector, and the second nucleic acid sequence is contained within a second expression vector. In some embodiments of the engineered expression system described herein, the first antigen-binding domain binds to CEACAM5. In some embodiments of the engineered expression system described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), where VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of hMN14 VH, and VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of hMN14 VL, and the antibody or its antigen-binding fragment is humanized.In some embodiments of the manipulated expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of hMN14 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of hMN14 VL. In some embodiments of the manipulated expression system described herein, VH comprises the amino acid sequence of hMN14 VH, and VL comprises the amino acid sequence of hMN14 VL. In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of BW431 / 26 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of BW431 / 26 VL, and the antibody or its antigen-binding fragment is humanized. In some embodiments, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of BW431 / 26 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of BW431 / 26 VL. In some embodiments of the CAR or manipulated expression system described herein, VH comprises the amino acid sequence of BW431 / 26 VH, and VL comprises the amino acid sequence of BW431 / 26 VL.In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of A5B7 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of A5B7 VL, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of A5B7 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of A5B7 VL. In some embodiments of the manipulated expression system described herein, VH comprises the amino acid sequence of A5B7 VH, and VL comprises the amino acid sequence of A5B7 VL. In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of MFE23 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of MFE23 VL, and the antibody or its antigen-binding fragment is humanized.In some embodiments of the CAR or engineered expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of MFE23 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of MFE23 VL. In some embodiments of the engineered expression system described herein, VH comprises the amino acid sequence of MFE23 VH, and VL comprises the amino acid sequence of MFE23 VL. In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of hMFE23 VH, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of hMFE23 VL, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression systems described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of hMFE23 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of hMFE23 VL. In some embodiments, VH comprises the amino acid sequence of hMFE23 VH, and VL comprises the amino acid sequence of hMFE23 VL.In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of FM4 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of FM4 VL, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of FM4 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of FM4 VL. In some embodiments of the manipulated expression system described herein, VH comprises the amino acid sequence of FM4 VH, and VL comprises the amino acid sequence of FM4 VL. In some embodiments of the manipulated expression system described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Furthermore, VH comprises the VH complementation region 1 (CDRH1), VH complementation region 2 (CDRH2), and VH complementation region 3 (CDRH3) of cibisatamab HC, and VL comprises the VL complementation region 1 (CDRL1), VL complementation region 2 (CDRL2), and VL complementation region 3 (CDRL3) of cibisatamab LC, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression systems described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of cibisatamab HC, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of cibisatamab LC. In some embodiments of the manipulated expression systems described herein, HC comprises the amino acid sequence of cibisatamab HC, and LC comprises the amino acid sequence of VL of cibisatamab LC. In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of tusamitamab HC, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of tusamitamab LC, and the antibody or its antigen-binding fragment is humanized.In some embodiments of the manipulated expression system described herein, HC comprises an amino acid sequence having 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% identity with the amino acid sequence of tusamitamab HC, and LC comprises an amino acid sequence having 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% identity with the amino acid sequence of tusamitamab LC. In some embodiments of the manipulated expression system described herein, VH comprises the amino acid sequence of VH of tusamitamab HC, and VL comprises the amino acid sequence of VL of tusamitamab LC. In some embodiments of the manipulated expression system described herein, the first antigen-binding domain binds to CEACAM1. In some embodiments of the manipulated expression systems described herein, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of MRG1 HC, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of MRG1 LC, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of MRG1 VH, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of MRG1 VL. In some embodiments of the manipulated expression system described herein, VH comprises the amino acid sequence of MRG1 VH, and VL comprises the amino acid sequence of MRG1 VL.In some embodiments of the engineered expression system described herein, the first antigen-binding domain binds to CEACAM6. In some embodiments of the engineered expression system described herein, the first antigen-binding domain comprises a heavy chain variable domain (VL) and a light chain variable domain (VL), where VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of tinurilimab HC, and VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of tinurilimab LC, and the antibody or its antigen-binding fragment is humanized. In some embodiments of the manipulated expression system described herein, VH comprises an amino acid sequence having 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% identity with the amino acid sequence of VH of tinuririmab HC, and VL comprises an amino acid sequence having 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% identity with the amino acid sequence of VL of tinuririmab LC. In some embodiments of the manipulated expression system described herein, VL comprises the amino acid sequence of VH of tinuririmab HC, and VL comprises the amino acid sequence of VL of tinuririmab LC. In some embodiments, the manipulated expression system further comprises a fourth nucleotide sequence encoding a first cytokine and a fifth nucleotide sequence encoding a second cytokine. In some embodiments of the engineered expression systems described herein, at least one of the first and second cytokines is a regulatory-release cytokine. In some embodiments of the engineered expression systems described herein, the regulatory-release cytokine has the following formula: SC-MT or MT-CS, where S comprises a secretible effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane anchoring domain.Optionally, the protease cleavage site is cleaved by ADAM10 and / or ADAM17, and optionally, the protease cleavage site contains the amino acid sequence PRAEALKGG or VTPEPIFSLI, and optionally, the cell membrane anchoring domain contains a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA, and optionally, the cell membrane anchoring domain contains a B7-1 transmembrane domain containing the amino acid sequence described in Table 14. In some embodiments of the manipulated expression systems described herein, the first cytokine is IL15, and optionally, IL15 contains the amino acid sequence of IL15 described in Table 10. In some embodiments of the engineered expression systems described herein, IL15 is controlled-release IL15 (crIL15). In some embodiments of the engineered expression systems described herein, the second cytokine is IL21, and optionally IL21 comprises the amino acid sequence listed in Table 10, and optionally IL21 is controlled-release IL21 (crIL21). In some embodiments of the engineered expression systems described herein, the first or second cytokine comprises the amino acid sequence listed in Table 10. In some embodiments of the engineered expression systems described herein, the first or second cytokine is encoded by the nucleic acid sequence listed in any one of the nucleic acid sequences listed in Table 10. [Brief explanation of the drawing]

[0042] This patent or application document includes at least one drawing made in color. Copies of this patent or patent application publication including the color drawing are available from the Patent Office upon request and payment of the necessary fees.

[0043] These and other features, aspects, and advantages of this disclosure will be better understood in relation to the following description and accompanying drawings.

[0044] [Figure 1]Figure 1 shows the killing of CEA+ / VSIG2+ target cells (left column) compared to killing of CEA+ target cells that do not express VSIG2 (right column), as evaluated by the rate of suppression of killing, and anti-VSIG2-mediated protection. [Figure 2] Figure 2 shows an overview of the results of the VSIG2 iCAR protection assay in Figure 1. [Figure 3] Figure 3 shows the expression of iCAR using various inhibitory ICDs. [Figure 4] Figure 4 shows anti-VSIG2 iCAR-mediated protection using various inhibitory ICDs. [Figure 5A] Figures 5A and 5B show the expression of aCAR, iCAR, and membrane-related IL-15 in the quad cistron payload in different orientations. [Figure 5B] Figures 5A and 5B show the expression of aCAR, iCAR, and membrane-related IL-15 in the quad cistron payload in different orientations. [Figure 6A] Figures 6A-6D show the expression of both CARs (aCAR and iCAR), IL15 and IL21, and iCAR NOT gate function in manipulated NK cells. Figure 6A shows the expression of both aCAR and iCAR, as well as membrane-associated IL15. [Figure 6B] Figure 6B shows the expression of soluble IL15 in transduced NK cells. [Figure 6C] Figure 6C shows the expression of soluble IL21 in transduced NK cells. [Figure 6D] Figure 6D shows the results of an in vitro cytotoxicity assay to demonstrate protection of VSIG2+ cells. [Figure 7A] Figures 7A–7F show the results of an in vivo co-culture assay in which VSIG2-positive cells, VSIG2-negative cells, and NOT-gated CAR-NK cells are provided in a subcutaneous solid tumor model. Figure 7A shows an exemplary schematic diagram of the NOT-gated gene circuit introduced into NK cells. [Figure 7B] Figure 7B shows the expression of VSIG2 inhibitory CARs on manipulated NK cells. [Figure 7C]Figure 7C shows the expression of membrane-associated mRNA on manipulated NK cells. [Figure 7D] Figure 7D shows the experimental scheme for an in vivo subcutaneous solid tumor model. [Figure 7E] Figure 7E shows exemplary flow cytometry results for no treatment and CAR NK treatment. [Figure 7F] Figure 7F shows the results of the in vivo co-culture assay shown in Figure 7D. [Modes for carrying out the invention]

[0045] The practices described herein, unless otherwise indicated, employ conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the scope of the art. Such methods are fully described in the literature, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006); Fundamental Virology, 3 rd Edition,vol.I&II(BNFields and DMKnipe,eds.);Handbook of Experimental Immunology,Vols.I-IV (DMWeir and CCBlackwell eds.,Blackwell Scientific Publications);ALLehninger,Biochemistry(Worth Publishers,Inc.,current addition);Sambrook,et al.,Molecular Cloning:A Laboratory Manual(3 rd See Edition, 2001; Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0046] definition Unless otherwise defined, all terms, notations, and other scientific terms used herein are intended to have meanings generally understood by those skilled in the art. In some cases, terms having generally understood meanings are defined herein for clarity and / or immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from what is generally understood in the art. The techniques and procedures described or referenced herein are generally well known and commonly used by those skilled in the art, for example, using conventional methodologies such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and conditions defined by the manufacturers, unless otherwise stated.

[0047] As used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise. Terms such as "includes" and "etc." are intended to convey inclusion without limitation unless otherwise indicated.

[0048] As used herein, the term “including” also specifically includes embodiments that “consist of” and “essentially consist of” the listed elements, unless otherwise indicated.

[0049] The term "approximately" indicates and encompasses both the value and the range above and below that value. In certain embodiments, the term "approximately" indicates a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "approximately" indicates a specified value ± one standard deviation of that value.

[0050] As used herein, the term “activating immune-responsive cells” refers to the induction of signaling or changes in protein expression in cells that result in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immune receptor tyrosine system inhibitory motifs (ITAMs), a signaling cascade is generated. In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, immunological synapse formation occurs, involving the clustering of many molecules in the vicinity of the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motif contained within the CD3 chain to be phosphorylated. This phosphorylation then initiates a T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce overall T cell gene expression by increasing IL-2 production for proliferation and inducing the expression of key regulatory T cell proteins, in order to initiate a T cell-mediated immune response.

[0051] As used herein, the terms “stimulating a cell-mediated immune response” or “stimulating an immune response” refer to the generation of signals by one or more cell types or cell populations that result in an immune response. Immune stimulating activity may include pro-inflammatory activity. In various embodiments, the immune response may occur after the activation of immune cells (e.g., T cells or NK cells) or, non-limited, simultaneously via receptors, including CD28, CD137(4-1BB), OX40, CD40 and ICOS, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides may be present in the tumor microenvironment and may activate an immune response against tumor cells. In various embodiments, promoting, stimulating, or otherwise harming pro-inflammatory polypeptides and / or their ligands may enhance the immune response of immune-responsive cells. While not bound by any particular theory, receiving multiple stimulating signals (e.g., co-stimulation) is crucial for carrying out robust and long-lasting cell-mediated immune responses, such as T-cell-mediated immune responses, in which T cells may be inhibited and unresponsive to antigens (also called "T-cell anergy") in the absence of co-stimulating signals. Without receiving these stimulating signals, T cells are quickly inhibited and become unresponsive to antigens. The various effects of co-stimulating signals, especially when combined with each other, may be altered and remain only partially understood, but co-stimulation generally results in increased gene expression to generate long-lived, proliferative, and anti-apoptotic resistant cells, such as T cells or NK cells, that respond strongly to antigens, for example, by mediating the complete and / or sustained elimination of target cells expressing congenital antigens.

[0052] As used herein, the terms “chimeric antigen receptor” or, alternatively, “CAR” refer to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) which includes a functional signaling domain.

[0053] As used herein, the terms “activated CAR” or “aCAR” refer to a CAR construct / structure that has the ability to induce changes in signaling or protein expression in activated CAR-expressing cells that initiate, activate, stimulate, or increase an immune response upon binding to a congeneral aCAR ligand.

[0054] As used herein, the terms “inhibitory CAR” or “iCAR” refer to a CAR construct / structure that has the ability to induce alterations in signaling or protein expression in inhibitory CAR-expressing cells that prevent, reduce, inhibit, decrease, suppress, or inhibit an immune response when binding to a congeneral iCAR ligand, such as reducing the activation of one or more stimulating signals, including co-stimulatory signals, or receiving immune-responsive cells.

[0055] As used herein, the term “enzyme inhibitory domain” refers to a protein domain that inhibits an intracellular signaling cascade, such as the innate T cell activation cascade. In some embodiments, the enzyme inhibitory domain of the chimeric inhibitory receptor of this disclosure includes at least a portion of an extracellular domain, a transmembrane domain, and / or an intracellular domain. In some embodiments, the enzyme inhibitory domain includes at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP (see, for example, Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19). In some embodiments, the portion of the enzyme includes an enzyme domain, an enzyme fragment, or a variant thereof. In some embodiments, a portion of the enzyme is the catalytic domain of the enzyme. In some embodiments, the enzyme domain, enzyme fragment, or variant thereof is selected to maximize efficacy and minimize basal inhibition.

[0056] As used herein, the term “intracellular signaling domain” refers to a functional portion of a protein that acts by transmitting information within a cell to modulate cellular activity via a defined signaling pathway, either by generating a second messenger or by acting as an effector in response to such a messenger.

[0057] As used herein, the terms “extracellular antigen-binding domain” or “antigen-binding domain” (ABD) refer to a polypeptide sequence or polypeptide complex that specifically recognizes or binds to a given antigen or epitope, such as, for example, a polypeptide sequence or polypeptide complex portion of a chimeric protein described herein that provides VSIG2-specific binding. An ABD (or antibody, antigen-binding fragment, and / or a chimeric protein containing it) is said to “recognize” an epitope (or more commonly, an antigen) to which the ABD specifically binds, and the epitope is said to be the “recognition specificity” or “binding specificity” of the ABD. An ABD is said to bind to its specific antigen or epitope with a particular affinity. As described herein, “affinity” refers to the strength of the non-covalent intermolecular force interaction between one molecule and another. Affinity, i.e., the strength of the interaction, can be expressed as the equilibrium dissociation constant (KD), where a lower KD value indicates a stronger intermolecular interaction. The KD value of an antibody construct is measured by methods well known in the art, including, but not limited to, biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays (e.g., flow cytometry). Specific binding, evaluated by affinity, may refer to binding molecules that have affinity between ABD and its congener antigen or epitope, and the KD value is 10 ―6 M, 10 ―7 M, 10 ―8 M, 10 ―9 M, or 10 ―10M is less than M. Specific binding may also include the recognition and binding of a biological molecule of interest (e.g., polypeptide), while not specifically recognizing or binding to other molecules in a sample, e.g., a biological sample naturally containing the polypeptide of this disclosure. In certain embodiments, specific binding refers to the binding of an epitope or antigen or antigenic determinant of an ABD, antibody, or antigen-binding fragment in such a manner that the binding may be substituted for or compete with a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0058] ABD can be an antibody. As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0059] An ABD can be an antigen-binding fragment of an antibody. As used herein, the term “antigen-binding fragment” refers to at least one portion of an intact antibody or a recombinant variant thereof that is sufficient to confer recognition and specific binding to a target such as an antigen or epitope. Examples of antigen-binding fragments include, but are not limited to, single-domain antibodies such as Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, sdAb(VL or VH), a camel VHH domain, and a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at a hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1 136, 2005). Antigen-binding fragments can also be transplanted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies).

[0060] The number of ABDs in a binding molecule such as a chimeric protein described herein defines the "valence" of the binding molecule. A binding molecule having a single ABD is "monovalent". A binding molecule having multiple ABDs is said to be "multivalent". A multivalent binding molecule having two ABDs is "divalent". A multivalent binding molecule having three ABDs is "trivalent". A multivalent binding molecule having four ABDs is "tetravalent". In various multivalent embodiments, all of the multiple ABDs have the same recognition specificity and can be referred to as "monospecific multivalent" binding molecules. In other multivalent embodiments, at least two of the multiple ABDs have different recognition specificities. Such a binding molecule is multivalent and "bispecific". In a multivalent embodiment where an ABD has two recognition specificities together, the binding molecule is "bispecific". In a multivalent embodiment where an ABD has three recognition specificities together, the binding molecule is "trispecific". In a multivalent embodiment where an ABD has multiple recognition specificities for different epitopes present on the same antigen, the binding molecule is "multiparatopic". A multivalent embodiment where an ABD recognizes two epitopes on the same antigen together is "biparatopic".

[0061] In various multivalent embodiments, the multivalent binding molecule improves the binding activity of the binding molecule to a particular target. As described herein, "binding activity" refers to the overall strength of the interaction between two or more molecules, e.g., a multivalent binding molecule to a particular target, and the binding activity is the cumulative strength of the interactions provided by the affinities of the multiple ABDs. The binding activity can be measured by the same methods used to determine affinity as described above. In certain embodiments, the binding activity of a binding molecule to a specific target is such that the interaction is a specific binding interaction and the binding activity between two molecules is 10 ―6 M, 10 ―7 M, 10 ―8 M, 10 ―9 M, or 10 ―10It has a KD value less than M. In certain embodiments, the binding activity of a binding molecule to a specific target has a KD value such that the interaction is a specific binding interaction, and one or more affinities of individual ABDs do not have a KD value such that they recognize the specific binding of themselves to their respective antigens or epitopes. In certain embodiments, binding activity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to a shared specific target or distinct antigens on a complex, such as distinct antigens found on individual cells. In certain embodiments, binding activity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to distinct epitopes on shared individual antigens.

[0062] As used herein, the terms “single-chain variable fragment” or “scFv” refer to a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are linked via a short, mobile polypeptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless otherwise specified, an scFv may have VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0063] As used herein, “variable region” refers to a variable region arising from recombination, for example, in an immunoglobulin gene in a B cell or a T cell receptor (TCR) gene in a T cell, after V, J, and / or D segment recombination. In immunoglobulin genes, variable regions are typically defined from the antibody chain from which they are induced; for example, VH refers to the variable region of the antibody heavy chain, and VL refers to the variable region of the antibody light chain. Selective VH and VL can bind together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0064] As used herein, the terms “complementarity-determining region” or “CDR” refer to sequences within the antibody variable regions VH and VL that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using one of several well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids of VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of VH, e.g., mammalian VH, e.g., human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of VL, e.g., mammalian VL, e.g., human VL. In various embodiments, the CDR is a mammalian sequence, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CDR is a human sequence. In various embodiments, the CDR is a naturally occurring sequence.

[0065] As used herein, the term “framework region” or “FR” typically refers to a generally conserved sequence within the antibody variable regions VH and VL, which functions as a scaffold for scattered CDRs in the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (N-terminus to C-terminus). In various embodiments, the FR is a mammalian sequence, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In certain embodiments, the FR is a human sequence. In various embodiments, the FR is a naturally occurring sequence. In various embodiments, the FR is a synthetic sequence, including, but not limited to, a reasonably designed sequence. In some embodiments, the human FR sequence is a naturally occurring sequence (e.g., a human germline antibody sequence such as IGHV3-21 for the heavy chain and IGKV1-5 for the light chain). In some embodiments, the human FR sequence is a naturally occurring sequence containing one or more mutations, such as “reverse” mutations, to match the species of origin of the CDRs transplanted into the human framework.

[0066] As used herein, the term “antibody heavy chain” refers to the larger of two types of polypeptide chains present in antibody molecules in their naturally occurring three-dimensional structure, which typically determine the class to which the antibody belongs.

[0067] As used herein, the term “antibody light chain” refers to the smaller of two types of polypeptide chains present in antibody molecules in their naturally occurring three-dimensional structures. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0068] As used herein, the term “recombinant antibody” refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as meaning an antibody produced by the synthesis of a DNA molecule encoding the antibody, in which the DNA molecule expresses an antibody protein, or an amino acid sequence that identifies the antibody, and the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technologies that are available and well known in the art.

[0069] As used herein, the terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may involve antibody production, activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen.

[0070] As used herein, the terms “antitumor effect” or “antitumor activity” refer to a biological effect that can be manifested by a variety of means, including, but not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in lifespan, a decrease in tumor cell proliferation, a decrease in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. The “antitumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of this disclosure to prevent tumor development in the first place, such as through prophylactic therapy or treatment.

[0071] As used herein, the term “autologous” refers to any substance derived from the same subject that is later reintroduced into the subject.

[0072] As used herein, the term “homogeneous” refers to any material originating from different animals of the same species as the subject into which the material is introduced. Two or more subjects are said to be homogeneous if their genes at one or more loci are not identical. In some embodiments, homogeneous materials from individuals of the same species may be sufficiently genetically different to interact antigenically at certain genes, such as MHC alleles. In some embodiments, homogeneous materials from individuals of the same species may be sufficiently genetically identical to not interact antigenically at certain genes, such as MHC alleles.

[0073] The isolated nucleic acid molecules of this disclosure include any nucleic acid molecules encoding a polypeptide or fragment thereof of this disclosure. Such nucleic acid molecules do not need to be 100% homologous or identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. Nucleic acids having “substantial identity” or “substantial homology” to the endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. As used herein, “hybridization” refers to pairing to form a double-stranded molecule with a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof, under conditions of varying strictness. For example, the exact salt concentrations may typically be about 750 mM NaCl and less than 75 mM trisodium citrate, about 500 mM NaCl and less than 50 mM trisodium citrate, or about 250 mM NaCl and less than 25 mM trisodium citrate. Low-tight hybridization can be obtained in the absence of organic solvents, such as formamide, while high-tight hybridization can be obtained in the presence of at least about 35% or at least about 50% formamide. Tight temperature conditions 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, the concentration of detergent, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various levels of tightness can be achieved.

[0074] "Substantially identical" or "substantially homologous" means that a polypeptide or nucleic acid molecule exhibits at least 50% homology or identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical with the sequence used for comparison at the amino acid level or at the nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, 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 typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine and tyrosine. An exemplary method for measuring the degree of identity may involve using the BLAST program, where probability scores from e-3 to e-100 indicate closely related sequences.

[0075] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, or the biological properties derived therefrom, that serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a prescribed sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a prescribed sequence of amino acids. Thus, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, where the nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, used as a template for the transcription of a gene or cDNA, may be said to code for a protein or other product of that gene or cDNA. Unless otherwise indicated, “nucleotide sequence coding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. The phrase “nucleotide sequence coding a protein or RNA” may also include introns to the extent that a nucleotide sequence coding a protein may include introns in some versions.

[0076] As used herein, the term “ligand” refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, enabling intercellular recognition and / or interaction.

[0077] The terms “effective dose” and “therapeutic dose” are used interchangeably herein and refer to the amount of a compound, formulation, substance, or composition described herein that is effective in achieving a particular biological outcome. In some embodiments, “effective dose” or “therapeutic dose” is an amount sufficient to block, improve, or inhibit the continued proliferation, growth, or metastasis of a disease or disorder of interest, such as a bone marrow disorder.

[0078] As used herein, the term “immunely responsive cell” refers to a cell that functions in an immune response (e.g., an immune effector response), its precursor, or its offspring. Examples of immune effector cells include, but are not limited to, alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.

[0079] As used herein, the terms “immune effector response” or “immune effector function” refer to the function or response of an immune-responsive cell, for example, that enhances or promotes the immune attack of a target cell. For example, an immune effector function or response may refer to the properties of a T cell or NK cell that promote the death or inhibition of growth or proliferation of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0080] As used herein, the terms “mobile polypeptide linker” or “linker” refer to peptide linkers consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link variable heavy chain and variable light chain regions together. In one embodiment, the mobile polypeptide linker is a Gly / Ser linker with an amino acid sequence (Gly-Gly-Gly-Gly-Ser) n Or (Gly-Gly-Gly-Ser) n This includes n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10. In some embodiments, the mobile polypeptide linker includes, but is not limited to, Gly4Ser or (Gly4Ser)3. In other embodiments, the linker includes multiple iterations of (Gly2Ser), (GlySer), or (Gly3Ser). In some embodiments, the mobile polypeptide linker includes a Whitlow linker (e.g., GTSTGSGKPGSGEGSTKG [SEQ ID NO: 36]). Within the scope of this disclosure, for example, the linker described in International Publication No. 2012 / 138475 is also included.

[0081] As used herein, the terms “treat,” “treatment,” and “treating” refer to the reduction or mitigation of the progression, severity, and / or duration of a proliferative disorder (e.g., cancer), or the mitigation of one or more symptoms (preferably one or more identifiable symptoms) of a proliferative disorder by the administration of one or more therapies (e.g., one or more therapeutic agents such as CARs of this disclosure). In some embodiments, reduction or improvement refers to the improvement of at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms “treat,” “treatment,” and “treating” refer to the inhibition of the progression of a proliferative disorder, physically, for example, by stabilization of an identifiable symptom, physiologically, for example, by stabilization of a physical parameter, or both. In some embodiments, reduction or improvement includes a reduction or stabilization of tumor size or cancer cell count.

[0082] As used herein, the term “subject” is intended to include living organisms (e.g., mammals, humans) from which an immune response may be induced.

[0083] Other aspects of this disclosure are described in the following sections and are within the claims.

[0084] Other Interpretation Rules

[0085] The ranges listed herein are understood to be all abbreviations of values ​​within a range that include the listed endpoints. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subranges of 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, and 50.

[0086] Unless otherwise specified, references to compounds having one or more stereocenters refer to each stereoisomer and all combinations of those stereoisomers.

[0087] Solid tumor antigen Certain aspects of this disclosure relate to chimeric receptors and cells such as immune-responsive cells genetically modified to express one or more such chimeric receptors that bind to an antigen of interest, as well as methods for using such receptors and cells to treat and / or prevent solid malignancies, such as lung cancer, pancreatic cancer, gastrointestinal cancer, colon cancer, brain cancer, nerve tissue cancer, endocrine tumors, bone cancer, bone marrow cancer, immune system cancer, muscle cancer, liver cancer, gallbladder cancer, kidney cancer, bladder cancer, male reproductive organ cancer, female reproductive organ cancer, adipose cancer, soft tissue cancer, skin cancer, and other medical conditions requiring an antigen-specific immune response. Malignant cells have developed a set of mechanisms to protect themselves from immune recognition and elimination. This disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0088] Certain aspects of this disclosure relate to chimeric receptors that specifically bind to one or more antigens expressed on bone marrow cells useful for the treatment of solid malignancies, and to immune-responsive cells genetically modified to express such chimeric receptors. Solid tumors are clonal diseases caused by genetic and epigenetic changes that disrupt important processes such as cell proliferation and differentiation. Solid malignancies can be chronic or acute.

[0089] Certain aspects of this disclosure generally relate to chimeric receptors, engineered expression systems, cells, and therapeutic methods that target a combination of a first solid tumor antigen, which is a member of the CEA family (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6), and a second antigen, which is VSIG2. In various embodiments, this disclosure relates to NOT logic gates for controlling, regulating, or inhibiting the activity of one or more activated chimeric receptors on healthy cells (e.g., cells expressing VSIG2).

[0090] In some embodiments, the disclosure relates to an engineered expression system, cells, and therapeutic method comprising a bivalent chimeric receptor comprising a first antigen-binding domain that binds to a CEA family member (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6) and a second antigen-binding domain that binds to VSIG2. In some embodiments, the disclosure relates to an engineered expression system, cells, and therapeutic method comprising a first chimeric receptor comprising an antigen-binding domain that binds to a CEA family member (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6) and a second chimeric receptor comprising an antigen-binding domain that binds to VSIG2.

[0091] In certain embodiments, this disclosure relates to solid tumor antigens and combinations of solid tumor antigens that are suitable for use with chimeric receptors (e.g., chimeric TCRs or CRAs) to increase efficacy in the treatment of solid tumors and / or reduce extratumor toxicity. In certain embodiments, the first solid tumor antigen is a member of the CEA family. In certain embodiments, the first solid tumor antigen is a member of the CEA family selected from the group consisting of CEA, CEACAM1, CEACAM5, and CEACAM6. As used herein, “CEA” refers to a family of highly relevant proteins (CD66 proteins) including, but not limited to, CEACAM1 (CD66a), CEACAM5 (CD66e), and CEACAM6 (CD66c). In certain embodiments, the antibody or antigen-binding fragment that binds to CEA binds to two or more CD66 proteins.

[0092] Table 1 provides CEA family antigens suitable for use with chimeric receptors as described in the methods and compositions presented herein.

[0093] (Table 1) TIFF2026514757000002.tif76149

[0094] In some embodiments, the first solid tumor antigen is the CEACAM1 antigen. CEACAM1 is also known in the art as BGP, BGP1, BGPI, or CD66a. In some embodiments, the first solid tumor antigen is the CEACAM5 antigen. CEACAM5 was previously known in the art as CEA. Currently, CEACAM5 is also known as meconium antigen 100, carcinoembryonic antigen, or CD66e. In some embodiments, the first solid tumor antigen is the CEACAM6 antigen. CEACAM6 is also known in the art as CEAL, NCA, normal cross-reactive antigen, nonspecific cross-reactive antigen, or CD66c.

[0095] In certain embodiments, the second antigen is the VSIG2 antigen. VSIG2 is encoded by the VSIG2 gene and is known in the art as V-Set and immunoglobulin domain-containing 2, CTXL, CTH, corticothymocyte-like protein, CT-like protein, and corticothymocyte receptor (X. Laevis CTX)-like, and is represented by UniProt accession number Q96IQ7.

[0096] Chimeric receptor Certain aspects of this disclosure relate to chimeric receptors and nucleic acids encoding such chimeric receptors that bind to an antigen of interest. In certain embodiments, the chimeric receptor of the present invention comprises a first antigen-binding domain and a second antigen-binding domain (i.e., a “bivalent” chimeric receptor). In some embodiments, the chimeric receptor of the present invention comprises a single antigen-binding domain.

[0097] Antibody and antigen-binding fragments In some embodiments, the chimeric receptor comprises one or more antigen-binding domains capable of binding to solid tumor antigens, such as CEA family member antigens (listed in Table 1). The antigen-binding domain of the chimeric receptor may include an antibody sequence or antigen-binding fragment of a representative anti-CEA antibody provided in Table 2. In some embodiments, the antigen-binding domain comprises a CDR sequence of the antibody or antigen-binding fragment of Table 2.

[0098] (Table 2) TIFF2026514757000003.tif241170TIFF2026514757000004.tif255164

[0099] In some embodiments, commercially available antibodies may be used to bind to solid tumor antigens. The CDRs of commercially available antibodies are readily accessible to those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art can construct nucleic acids encoding scFv and chimeric receptors (e.g., CAR and TCR) based on the CDRs of such commercially available antibodies.

[0100] In some embodiments, the chimeric receptor includes an antigen-binding domain that specifically binds to CEA.

[0101] In some embodiments, the chimeric receptor includes an antigen-binding domain that specifically binds to CEACAM1. In some embodiments, the CEACAM1-specific antigen-binding domain is derived from an anti-CEACAM1 antibody, such as an MRG1 antibody or its antigen-binding fragment. In certain embodiments, the CEACAM1-specific antigen-binding domain includes a heavy chain variable domain (VH) having an amino acid sequence identical to at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the VH of MRG1 disclosed in Table 2, and a light chain variable domain (VL) having an amino acid sequence identical to at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the VL of MRG1 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of MRG1 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM1 and one or more additional antigens. In some embodiments, the chimeric receptor may be specific to CEACAM1 and CEACAM5. In some embodiments, the chimeric receptor may be specific to CEACAM1 and CEACAM6. In some embodiments, the chimeric receptor may be specific to CEACAM5 and CEACAM6.

[0102] In some embodiments, the chimeric receptor includes an antigen-binding domain that specifically binds to CEACAM5. In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, e.g., rabetsizimab (i.e., hMN14) or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of hMN14 disclosed in Table 2, and a light chain variable domain (VL) having an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of hMN14 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of hMN14 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0103] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as sibisatamab or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain comprises a heavy chain (HC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the HC of sibisatamab disclosed in Table 2, and a light chain (LC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the LC of sibisatamab disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of the HC and LC sequences of sibisatamab disclosed in Table 2, respectively. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3, of the HC and LC sequences of sibisatamab disclosed in Table 2, respectively, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv containing the light chain variable domain and the heavy chain variable domain. In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0104] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as tusamitamab or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain comprises a heavy chain (HC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the HC of tusamitamab disclosed in Table 2, and a light chain (LC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the LC of tusamitamab disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of the HC and LC sequences of tusamitamab disclosed in Table 2, respectively. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3, of the HC and LC sequences of tusamitamab disclosed in Table 2, respectively, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising the light chain variable domain and the heavy chain variable domain. In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0105] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, e.g., BW431 / 26 or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of BW431 / 26 disclosed in Table 2, and a light chain variable domain (VL) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of BW431 / 26 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of BW431 / 26 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv containing a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0106] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, e.g., A5B7 or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of A5B7 disclosed in Table 2, and a light chain variable domain (VL) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of A5B7 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of A5B7 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0107] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as MFE23 or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of MFE23 disclosed in Table 2, and a light chain variable domain (VL) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of MFE23 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of MFE23 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0108] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as hMFE23 or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of hMFE23 disclosed in Table 2, and a light chain variable domain (VL) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of hMFE23 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of hMFE23 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0109] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody that can specifically bind to glycosylated CEACAM5. In some embodiments, the glycosylated CEACAM5-specific antigen-binding domain is derived from an anti-glycosylated CEACAM5 antibody, such as FM4 (also referred to herein as "MG7") or its antigen-binding fragment. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a heavy chain variable domain (VH) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of FM4 disclosed in Table 2, and a light chain variable domain (VL) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of FM4 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, respectively, of the VH and VL sequences of FM4 disclosed in Table 2, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM5 and one or more additional antigens.

[0110] In some embodiments, the chimeric receptor includes an antigen-binding domain that specifically binds to CEACAM6. In some embodiments, the CEACAM6-specific antigen-binding domain is derived from an anti-CEACAM6 antibody, such as tinuririmab or its antigen-binding fragment. In certain embodiments, the CEACAM6-specific antigen-binding domain includes a heavy chain (HC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the HC of tinuririmab disclosed in Table 2, and a light chain (LC) having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the LC of tinuririmab disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of the HC and LC sequences of tinuririmab disclosed in Table 2, respectively. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3, of the HC and LC sequences of tinuririmab disclosed in Table 2, respectively, as determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the Art. The antigen-binding domain may be an scFv containing the light chain variable domain and the heavy chain variable domain. In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific to CEACAM6 and one or more additional antigens.

[0111] Certain aspects of this disclosure relate to chimeric receptors (e.g., CARs or chimeric TCRs) comprising an extracellular antigen-binding domain that binds to one or more antigens of this disclosure. In some embodiments, the antigen-binding domain is derived from an antibody or its antigen-binding fragment, and CDR sequences and known systems for defining them, such as Kabat, are discussed in detail above.

[0112] Preferred antibodies of this disclosure include any antibody, whether natural or synthetic, full-length or fragmented, monoclonal or polyclonal, that binds sufficiently strongly and specifically to solid tumor antigens, such as CEA, CEACAM1, CEACAM5, or CEACAM6. In some embodiments, the antibody may be up to approximately 10 -6 M, maximum about 10 -7 M, maximum about 10 -8 M, maximum about 10 -9 M, maximum about 10 -10 M, maximum about 10 -11 M, or up to approximately 10 -12 M's K D It may have.

[0113] V set and immunoglobulin domain-containing 2 (VSIG2) specific antigen-binding domain This disclosure provides chimeric proteins that bind to V-set and immunoglobulin domain-containing protein 2 (VSIG2), as well as polynucleotides encoding such chimeric proteins. In some embodiments, the VSIG2-specific chimeric protein binds to human VSIG2 (e.g., Uniprot Q96IQ7, incorporated herein by reference for all purposes) or its epitope fragment. VSIG2 may be expressed on epithelial cells. VSIG2 may be expressed on cells generally considered healthy, such as healthy epithelial cells. Examples of VSIG2-specific antibodies include OTI2D8 (also known as “2D8” and referred to herein as Ab) and OTI5A10 (also known as “5A10”).

[0114] This disclosure provides VSIG2-specific antigen-binding domains comprising one or more complementarity-determining region (CDR) amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation from the parent CDR amino acid sequence shown in Table 3. Generally, the VSIG2-specific antigen-binding domains described herein comprise the CDRL3 light chain amino acid sequence QHHAVIPWT (SEQ ID NO: 9) and / or one of the CDRH3 heavy chain amino acid variant sequences from SEQ ID NOs: 67 to 87 shown in Table 22.

[0115] (Table 3) Parent VSIG2-specific antigen-binding domain sequences TIFF2026514757000005.tif71170

[0116] In some embodiments, the VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein VH includes a VH complementary region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1 and a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3, and VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6 and a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and (i) VH includes a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5 and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) VH includes a VH complementary region 3 (CDRH3) having one of the amino acid sequences of SEQ ID NOs from 67 to 87, and VL includes a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0117] In some embodiments, the VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein VH includes a VH complementary region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 2 and a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4, and VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6 and a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and (i) VH includes a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5 and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) VH includes a VH complementary region 3 (CDRH3) having the amino acid sequence of any one of SEQ ID NOs from 67 to 87, and VL includes a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0118] In some embodiments, VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 88-107. In some embodiments, VL has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, A) CDRH3 has the amino acid sequence of SEQ ID NO: 5 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or B) CDRH3 has the amino acid sequence of SEQ ID NO: 67 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or C) CDRH3 has the amino acid sequence of SEQ ID NO: 68 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or D) CDRH3 has the amino acid sequence of SEQ ID NO: 67 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or E) CDRH3 has the amino acid sequence of SEQ ID NO: 68 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or F) CDRH3 has the amino acid sequence of SEQ ID NO: 69 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or G) CDRH3 has the amino acid sequence of SEQ ID NO: 69 and CDRL3 has the amino acid sequence of SEQ ID NO: 9, or H) CDRH3 has the amino acid sequence of SEQ ID NO: 70 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or I) CDRH3 has the amino acid sequence of SEQ ID NO: 71 J) CDRH3 has the amino acid sequence of SEQ ID NO: 8, or K) CDRH3 has the amino acid sequence of SEQ ID NO: 73, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or L) CDRH3 has the amino acid sequence of SEQ ID NO: 74, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or M) CDRH3 has the amino acid sequence of SEQ ID NO: 75, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or N) CDRH3 has the amino acid sequence of SEQ ID NO: 76, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or O) CDRH3 has the amino acid sequence of SEQ ID NO: 77, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or P) CDRH3 has the amino acid sequence of SEQ ID NO: 78, and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Q) CDRH3 has the amino acid sequence of SEQ ID NO: 79, and CDRL3 has the amino acid sequence of SEQ ID NO: 8.Or R) CDRH3 has the amino acid sequence of SEQ ID NO: 80 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or S) CDRH3 has the amino acid sequence of SEQ ID NO: 81 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or T) CDRH3 has the amino acid sequence of SEQ ID NO: 82 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or U) CDRH3 has the amino acid sequence of SEQ ID NO: 83 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or V) CDRH3 has the amino acid sequence of SEQ ID NO: 84 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or W) CDRH3 has the amino acid sequence of SEQ ID NO: 85 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or X) CDRH3 has the amino acid sequence of SEQ ID NO: 86 and CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Y) CDRH3 has the amino acid sequence of SEQ ID NO: 87 and CDRL3 has the amino acid sequence of SEQ ID NO: 8. In some embodiments, CDRH3 has the amino acid sequence of SEQ ID NO: 69, and CDRL3 has the amino acid sequence of SEQ ID NO: 8.

[0119] In some embodiments, the VSIG2-specific antigen-binding domain has a heavy-chain variable (VH) region and a light-chain variable (VL) region, where (i) the VH region includes the amino acid sequence of SEQ ID NO: 16 and the VL region includes an amino acid sequence selected from SEQ ID NO: 15, or (ii) the VH region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 88 to 107 and the VL region includes the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0120] In some embodiments, the VSIG2-specific antigen-binding domain has a variable heavy chain (VH) region and a variable light chain (VL) region, where VL has the amino acid sequence of SEQ ID NO: 15. In some embodiments, VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88 to 107.

[0121] In some embodiments, the VSIG2-specific antigen-binding domain has a variable heavy chain (VH) region and a variable light chain (VL) region, where VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88 to 107. In some embodiments, VL has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0122] In some embodiments, the VSIG2-specific antigen-binding domain has a VH region comprising an amino acid sequence having 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%, at least 99%, or 100% identity with the amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation with respect to the parental CDR amino acid sequence shown in Table 3.

[0123] In some embodiments, the VSIG2-specific antigen-binding domain has a VL region containing an amino acid sequence that has 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%, at least 99%, or 100% identity with the amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation with respect to the parent CDR amino acid sequence shown in Table 3.

[0124] The VSIG2-specific antigen-binding domain can be any of the formats described herein, such as Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH, and multispecific formats. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab) format. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab') format.

[0125] In some embodiments, the VSIG2-specific antigen-binding domain is a single-stranded variable fragment (scFv) format comprising an scFv format having one of the peptide linkers described herein (see, for example, Table 4). In some embodiments, the VSIG2-specific antigen-binding domain has the structure VH-L-VL or VL-L-VH, where L is the peptide linker.

[0126] In some embodiments, scFV has an amino acid sequence selected from SEQ ID NOs: 108-132.

[0127] In some embodiments, the VSIG2-specific antigen-binding domain is humanized, and the CDRs are non-human mammalian sequences including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, which are transplanted onto a scaffold having human framework region (FR) sequences, and typically the CDRs are scattered in an FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus). The FR sequences of the light chain may include the FR1 sequence DIQMTQSPSTLSASVGDRVTITC, the FR2 sequence WYQQKPGKAPKLLIY, the FR3 sequence GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC, and / or the FR4 sequence FGQGTKVEIK. The heavy chain FR sequences may include the FR1 sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFS, the FR2 sequence WVRQAPGKGLEWVA, the FR3 sequence RFTISRDNAKSSLYLQMNSLRAEDTAVYYCAR, and / or the FR4 sequence WGQGTLVTVSS.

[0128] This disclosure also provides chimeric proteins comprising a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation from the parent CDR amino acid sequence shown in Table 3, and nucleic acids encoding such chimeric proteins. The chimeric proteins may comprise any of the aforementioned VSIG2-specific antigen-binding domains.

[0129] Chimeric antigen receptor (CAR) Certain aspects of this disclosure relate to chimeric receptors having one of the VSIG2-specific antigen-binding domains described herein and having the ability to specifically bind to the VSIG2 protein, a VSIG2-derived antigen, or a VSIG2-derived epitope. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). Generally, a CAR is a chimeric protein comprising an antigen-binding domain and a polypeptide molecule heterogeneous to the antigen-binding domain, such as a peptide heterogeneous to an antibody in which the antigen-binding domain can be induced. Polypeptide molecules heterogeneous to the antigen-binding domain include, but are not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or a combination thereof.

[0130] In some embodiments, a CAR is an engineered receptor that implants or confers a desired specificity (e.g., VSIG2) to immune effector cells. In certain embodiments, a CAR can be used to implant antibody specificity into immune-responsive cells such as T cells. In some embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0131] In some embodiments, the chimeric antigen receptor is an activated chimeric antigen receptor (aCAR, and also commonly referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its homologous ligand is sufficient to induce activation of immune-responsive cells. In some embodiments, binding of the chimeric antigen receptor to its homologous ligand is sufficient to induce stimulation of immune-responsive cells. In some embodiments, activation of immune-responsive cells results in the death of target cells. In some embodiments, activation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces differentiation of immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces proliferation of immune-responsive cells. In some embodiments, activation and / or stimulation of immune-responsive cells can be a combination of the above responses.

[0132] The CARs of this disclosure may be first, second, or third-generation CARs. "First-generation" CARs generally contain a single intracellular signaling domain derived from the T cell receptor chain. "First-generation" CARs generally have an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary signaling factor from the endogenous TCR. "First-generation" CARs give de novo antigen recognition and, independently of HLA-mediated antigen presentation, transmit signals via the CD3ζ chain signaling domain within a single fusion molecule to CD4 + and CD8 +It induces activation of both T cells. Second-generation CARs provide additional signaling to T cells by adding a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR. Second-generation CARs provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that second-generation CARs can enhance the antitumor activity of immune-responsive cells such as T cells. Third-generation CARs have multiple intracellular co-stimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0133] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, one or more chimeric inhibitory receptors bind to antigens expressed on non-tumor cells derived from tissues selected from the group consisting of brain, nerve tissue, endocrine system, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

[0134] In some embodiments, a chimeric inhibitory receptor (e.g., a VSIG2-specific chimeric inhibitory receptor) may be used in conjunction with one or more activated chimeric receptors (e.g., activated chimeric TCRs or CARs) expressed on cells of the Disclosure (e.g., immune-responsive cells) as a NOT logic gate to control, modulate, or otherwise inhibit the activity of one or more activated chimeric receptors. For example, if healthy cells express both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by a chimeric inhibitory receptor, immune-responsive cells expressing the tumor antigen may bind to the healthy cells. In such cases, the inhibitory chimeric antigen also binds to its homologous ligand on the healthy cells, and the inhibitory function of the chimeric inhibitory receptor reduces, mitigates, prevents, or inhibits the activation of immune-responsive cells via the tumor-targeting chimeric receptor ("NOT logic gating"). In some embodiments, the chimeric inhibitory receptors of the Disclosure may inhibit the activity of one or more cells of the Disclosure (e.g., immune-responsive cells). In some embodiments, immune-responsive cells may include one or more tumor-targeted chimeric receptors and one or more chimeric inhibitory receptors that target antigens not expressed on tumors or not generally considered to be expressed on tumors (e.g., VSIG2). Combinations of tumor-targeted chimeric receptors and chimeric inhibitory receptors in the same immune-responsive cells can be used to reduce extratumor toxicity on the target.

[0135] In some embodiments, the extracellular antigen-binding domain of the CAR disclosed herein is approximately 2 × 10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 M or less, approximately 2×10 -9 M or less, or approximately 1 × 10 -9 Dissociation constants less than or equal to M (K d ) binds to one or more antigens (e.g., VSIG2). In some embodiments, K d It is approximately 2 x 10-7 M ~ approx. 1×10 -9 This is in the range of M. In some embodiments, the VSIG2-specific antigen-binding domain in aCAR format can be selected based on affinity, including selection based on having a higher or lower affinity compared to other VSIG2-specific antigen-binding domains. In some embodiments, the VSIG2-specific antigen-binding domain in iCAR format can be selected based on affinity, including selection based on having a higher or lower affinity compared to other VSIG2-specific antigen-binding domains.

[0136] The binding of the extracellular antigen-binding domain of the CARs of this disclosure can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or Western blot assay. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using the complex of interest and a specific labeling reagent (e.g., antibody or scFv). For example, scFv may be radiolabeled and used in RIA assays. Radioisotopes can be detected by means such as the use of a γ counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific to the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., by radiation or with a fluorescent marker).

[0137] In some embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to VSIG2 (e.g., VSIG2 protein, VSIG2-derived antigen, or VSIG2-derived epitope), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a crosslinkable Fab fragment. In certain embodiments, the extracellular binding domain is an F(ab)2 fragment.

[0138] Extracellular antigen-binding domain The extracellular antigen-binding domain of the CARs of this disclosure specifically binds to VSIG2 (e.g., VSIG2 protein, VSIG2-derived antigen, or VSIG2-derived epitope). In certain embodiments, the extracellular antigen-binding domain binds to VSIG2 expressed in hematopoietic stem cells. In certain embodiments, the extracellular antigen-binding domain binds to VSIG2 expressed in cells generally considered healthy, such as healthy HSCPs. In some embodiments, VSIG2 is human VSIG2.

[0139] The antigen-binding domains of this disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, complex antibodies, human antibodies, and humanized antibodies, as well as functional fragments thereof, including, but not limited to, single-domain antibodies (sdAbs) such as heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-derived nanobody variable domains (VHH), and any domains to which they bind, as well as any domains to which they bind to alternative scaffolds known in the art that can function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCRs), recombinant TCRs with enhanced affinity, or fragments thereof, such as single-chain TCRs. In some cases, it is beneficial that the antigen-binding domains originate from the same species in which the CARs are ultimately used. For example, for use in humans, it may be beneficial that the antigen-binding domains of the CARs include human or humanized residues for the antigen-binding domains of the antibody fragments.

[0140] In some embodiments, the extracellular antigen-binding domain includes an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the extracellular antigen-binding domain includes an antigen-binding fragment of the antibody.

[0141] In some embodiments, the extracellular antigen-binding domain includes the F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain includes the F(ab') fragment.

[0142] In some embodiments, the extracellular antigen-binding domain includes scFv. In some embodiments, the extracellular antigen-binding domain includes two single-stranded variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain includes a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to distinct epitopes on the same antigen. In certain embodiments, the scFv is a mammalian scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

[0143] In certain embodiments, VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises one of the amino acid sequences shown in Table 4. In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, each of one or more scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFv are linked together, each scFv can be linked to the next scFv to which a peptide is linked. In some embodiments, each of one or more scFv is separated by a peptide linker. In some embodiments, the peptide linker separating each of the scFv comprises one of the amino acid sequences shown in Table 4.

[0144] (Table 4) Peptide linkers TIFF2026514757000006.tif105170

[0145] In some embodiments, the peptide linker includes the amino acid sequence GGS. In some embodiments, the peptide linker includes the amino acid sequence GGSGGS. In some embodiments, the peptide linker includes the amino acid sequence GGSGGSGGS. In some embodiments, the peptide linker includes the amino acid sequence GGSGGSGGSGGS. In some embodiments, the peptide linker includes the amino acid sequence GGSGGSGGSGGSGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGSGGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGSGGGSGGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGSGGGSGGGSGGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGGSGGGGS. In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGS. In some embodiments, the peptide linker includes the amino acid sequence GTSTGSGKPGSGEGSTKG. In some embodiments, the peptide linker includes the amino acid sequence EAAAKEAAAKEAAAKEAAAK. In some embodiments, the peptide linker includes the amino acid sequence GGSGSGGSGGGSGS.

[0146] In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domains of the first and second chimeric receptors may be suitable antigen-binding domains described herein or known in the art. For example, the first or second antigen-binding domain may be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-stranded variable fragments (scFv), or single-domain antibodies (sdAb). In some embodiments, the antigen-binding domains of the first and / or second chimeric receptors comprise two single-stranded variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first chimeric receptor may be specific to VSIG2, and the chimeric receptor may be specific to a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on CRC tumor cells).

[0147] In some embodiments, the extracellular antigen-binding domain includes a single-domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0148] In some embodiments, the CAR of this disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds to the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different antigen.

[0149] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a mobile linker. In some embodiments, each of the two antigen-binding domains may be independently selected from an antibody, an antigen-binding fragment of an antibody, scFv, sdAb, recombinant fibronectin domain, T cell receptor (TCR), a recombinant TCR with enhanced affinity, and a single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0150] In certain embodiments, a bispecific CAR or tanCAR includes an antigen-binding domain containing a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH may be upstream or downstream of VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VL (VL1) with its VH (VH1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VH (VH2) with its VL (VL2), resulting in the overall bispecific antibody molecule having the configuration VH1-VL1-VL2-VH2. In other embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VH(VH1) with its VL(VL1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VL(VL2) with its VH(VH2), so that the overall bispecific antibody molecule has the configuration VL1VH1-VH2-VL2. In some embodiments, a linker is positioned between two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2 if the construct is positioned as VH1-VL1-VL2-VH2, or between VH1 and VH2 if the construct is positioned as VL1-VH1-VH2-VL2. The linker may be a linker described herein, for example, a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. In general, the linker between two scFvs must be long enough to avoid mismatching between the domains of the two scFvs. In some embodiments, the linker is located between the VL and VH of the first scFv. In some embodiments, the linker is located between the VL and VH of the second scFv. In a structure having multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR includes VL, VH and may further include one or more linkers in the arrangements described herein.

[0151] In some embodiments, the chimeric receptor includes a divalent CAR. In some embodiments, the divalent CAR is a VSIG2 divalent CAR. In some embodiments, the divalent VSIG2 CAR includes one or more anti-VSIG2 sequences shown in Tables 3, 21, 22, and / or 23, in which at least one CDR amino acid sequence contains a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3. In some embodiments, each ABD of the divalent VSIG2 CAR contains the same ABD.

[0152] In some embodiments of bicistronic, the chimeric receptor comprises a bicistron chimeric antigen receptor. In some embodiments, the bicistron chimeric antigen receptor comprises a VSIG2 CAR. In some embodiments, the bicistron VSIG2 CAR comprises one or more anti-VSIG2 sequences shown in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation relative to the parent CDR amino acid sequence shown in Table 3.

[0153] transmembrane domain In some embodiments, the transmembrane domain of the CARs of this disclosure (e.g., the VSIG2-specific CAR described herein) includes a hydrophobic alpha-helix spanning at least a portion of the cell membrane. It has been shown that different transmembrane domains can result in different receptor stabilization. After antigen recognition, the receptors cluster, and the signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CARs of this disclosure may include the transmembrane domains of CD8 polypeptide, CD28 polypeptide, SIRPα polypeptide, CD25 polypeptide, CD7 polypeptide, CD3-zeta polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, LAX polypeptide, LAT polypeptide, PD-1 polypeptide, LAG-3 polypeptide, TIM3 polypeptide, KIR3DS1 polypeptide, KIR3DL1 polypeptide, NKG2D polypeptide, NKG2A polypeptide, TIGIT polypeptide, 2B4 polypeptide, BTLA polypeptide, LIR-1 (LILRB1) polypeptide, or synthetic peptides, or any combination thereof.

[0154] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide can be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide can be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide can be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from the CTLA-4 polypeptide.Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Examples of 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Examples of BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Examples of LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0155] In some embodiments, the transmembrane domains are NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009 The polypeptide comprises an amino acid sequence or fragment thereof that is 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%, at least 99%, or 100% homologous to the sequence of NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions.In some embodiments, the polypeptide has an amino acid length of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240, as indicated by NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_03 It may have an amino acid sequence that is a contiguous portion of 1668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0156] Further examples of suitable polypeptides from which the transmembrane domain may be derived include T cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, I This includes, but is not limited to, the transmembrane regions of the alpha, beta, or zeta strands of TGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NG2C.

[0157] In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 38). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 39). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 40).

[0158] Spacer area In some embodiments, the CARs of this disclosure (e.g., VSIG2-specific CARs as described herein) may also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge derived from a human protein. For example, the spacer (also referred to herein as “hinge”) may be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may include an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may include any of the amino acid sequences listed in Table 5, or any amino acid sequence that is 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 any of the amino acid sequences listed in Table 5. In some embodiments, the nucleic acid encoding any of the spacer regions of this disclosure may include any of the nucleic acid sequences listed in Table 6, or any nucleic acid sequence that is 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 any of the nucleic acid sequences listed in Table 6.

[0159] (Table 5) Spacer amino acid sequence TIFF2026514757000007.tif128170

[0160] (Table 6) Spacer nucleic acid sequences TIFF2026514757000008.tif108170TIFF2026514757000009.tif95170

[0161] In some embodiments, the spacer region includes the sequence for the CD28 hinge shown in SEQ ID NO: 41. In some embodiments, the spacer region includes the sequence for the IgG4 minimal hinge shown in SEQ ID NO: 42. In some embodiments, the spacer region includes the sequence for the IgG4 minimal hinge, without disfield, shown in SEQ ID NO: 43. In some embodiments, the spacer region includes the sequence for the IgG4 S228P minimal hinge, enhanced disfield formation, shown in SEQ ID NO: 44. In some embodiments, the spacer region includes the sequence shown for the IgG1 minimal hinge in SEQ ID NO: 45. In some embodiments, the spacer region includes the sequence shown for the extended CD8a hinge in SEQ ID NO: 46. In some embodiments, the spacer region includes the sequence shown for the LNGFR hinge in SEQ ID NO: 47. In some embodiments, the spacer region includes the sequence shown for the cleaved LNGFR hinge (TNFR-Cys1) in SEQ ID NO: 48. In some embodiments, the spacer region includes the sequence shown for the PDGFRβ extracellular linker in SEQ ID NO: 49. In some embodiments, the spacer region includes the arrangement shown for the exemplary spacer (CD8 hinge) of Sequence ID 50. In some embodiments, the spacer region includes the arrangement shown for the exemplary spacer of Sequence ID 51. In some embodiments, the spacer region includes the arrangement shown for the exemplary spacer of Sequence ID 52.

[0162] In some embodiments, the CAR of this disclosure may further include a short oligopeptide or polypeptide linker having a length of 2 to 10 amino acid residues that can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a preferred linker is a glycine-serine double chain. In some embodiments, the linker includes the amino acid sequence GGCKJSGGCKJS (SEQ ID NO: 62).

[0163] In some embodiments, the transmembrane domain further comprises at least a portion of the extracellular domain of the same protein.

[0164] Intracellular signal transduction domains In some embodiments, the CARs of this disclosure (e.g., the VSIG2-specific CARs described herein) include one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of the CAR may include intracellular signaling domains.

[0165] Examples of suitable intracellular signaling domains that may be used in the CARs of this disclosure include, but are not limited to, cytoplasmic sequences of T cell receptors (TCRs), co-receptors that act cooperatively to modulate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capacity.

[0166] While we do not wish to be bound by theory, the signals generated through the TCR alone are insufficient for complete T cell activation, and therefore, typically, secondary and / or co-stimulatory signals are also considered necessary for complete activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains), and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains). Furthermore, T cell signaling and function (e.g., activation signaling cascades) can be negatively regulated by inhibitory receptors present on T cells via intracellular inhibitory co-signaling domains.

[0167] In some embodiments, the intracellular signaling domain of the CARs of this disclosure may include an inhibitory intracellular signaling domain. Examples of inhibitory intracellular domains (ICDs) that may be used include one or more intracellular domains from the following proteins: PD-1, CTLA4, TIGIT, BTLA, LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, SIGLEC-10, PECAM-1, CD72, IRTA2, IRTA4, NKIR, TLT1, PCDHGC3, MPZL1, FCGR2B, SIGLEC-6, MPIG6B, SIGLEC-12, LIR8, IRTA1, KIR2DL4, KIR2DL5, SIGLEC-7, and FCRH3. Exemplary inhibitory ICD domain sequences are shown in Table 25. In some embodiments, the inhibitory intracellular signaling domain includes an amino acid sequence having 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%, at least 99%, or 100% identity with one or more of the amino acid sequences shown in Table 25. In some embodiments, the inhibitory intracellular signaling domain includes the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139).In some embodiments, the inhibitory intracellular signaling domain includes an amino acid sequence having 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%, at least 99%, or 100% identity with one or more amino acid sequences of VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139). In some embodiments, the inhibitory intracellular signaling domain includes one or more intracellular inhibitory co-signaling domains. In some embodiments, one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., transmembrane domains) via peptide linkers (e.g., see Table 4) or spacer or hinge sequences (e.g., see Table 5). In some embodiments, if two or more intracellular inhibitory co-signaling domains are present, they may be linked via a peptide linker (e.g., see Table 4) or a spacer or hinge sequence (e.g., see Table 5). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, one or more intracellular inhibitory co-signaling domains of a chimeric protein include one or more ITIM-containing proteins or fragments thereof. ITIM is a conserved amino acid sequence found at the cytoplasmic terminals of many inhibitory immune receptors. In some embodiments, one or more intracellular inhibitory co-signaling domains include one or more non-ITIM scaffold proteins or fragments thereof. The inhibitory intracellular signaling domain may further include an enzyme-inhibitory domain. In some embodiments, the enzyme-inhibitory domain includes an enzyme-catalyzed domain. In some embodiments, the enzyme-catalyzed domain is derived from an enzyme selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP.Examples of enzymatic regulation of signal transduction are described in detail by Pavel Otahal et al. (Biochim Biophys Acta. 2011 Feb;1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 Jun;14(6):669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19), each of which is incorporated herein by reference for all purposes.

[0168] In some embodiments, the intracellular signaling domain of the CARs of this disclosure may include a primary signaling domain that controls primary activation of the TCR complex either stimulatively or inhibitorily. A primary intracellular signaling domain acting stimulatively may include a known signaling motif as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of suitable ITAM-containing primary intracellular signaling domains that may be used in the CARs of this disclosure include, but are not limited to, those of CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0169] In some embodiments, the CARs of this disclosure (e.g., VSIG2-specific CARs as described herein) include an intracellular signaling domain, such as the primary signaling domain of a CD3-zetapolypeptide. The CD3-zetapolypeptide of this disclosure may have an amino acid sequence or fragment thereof that is 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%, at least 99%, or 100% homologous to the sequence of NCBI reference number NP_932170 or NP_001106864.2. In some embodiments, the CD3-zetapolypeptide may contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide may have an amino acid sequence that is a contiguous portion of NCBI reference number NP_932170 or NP_001106864.2, having a length of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids.

[0170] In other embodiments, the primary signaling domain includes a modified ITAM domain, such as a mutant ITAM domain, whose activity has been altered (e.g., increased or decreased) compared to a native ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four, or more ITAM motifs.

[0171] In some embodiments, the intracellular signaling domain of the CAR of this disclosure may include a CD3-zeta signaling domain by itself, or it may be combined with any other desired intracellular signaling domain useful in the context of the CAR of this disclosure. For example, the intracellular signaling domain of the CAR may include a portion of the CD3 zeta chain and a costimulatory signaling domain. The costimulatory signaling domain may refer to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. The costimulatory molecule of this disclosure is a cell surface molecule other than an antigen receptor or its ligand that may be required for an efficient lymphocyte response to an antigen.Examples of suitable co-stimulatory molecules include ligands that specifically bind to CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, CD83, MHC class I molecules, TNF receptor proteins, and immunoglobulins. Phosphorus-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRD3S1, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, I TGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, I TGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84 This includes, but is not limited to, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, etc.

[0172] Table 7 provides non-exclusive examples of intracellular signaling domains (ICDs).

[0173] (Table 7) TIFF2026514757000010.tif111170TIFF2026514757000011.tif255167TIFF2026514757000012.tif68170

[0174] In some embodiments, intracellular signaling sequences within a portion of the cytoplasm of the CARs of this disclosure may be linked to one another in a random or specific order. In some embodiments, for example, short oligopeptides or polypeptide linkers of 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may form links with the intracellular signaling sequences. In one embodiment, a glycine-serine double chain may be used as a suitable linker. In one embodiment, a single amino acid, such as alanine or glycine, may be used as a suitable linker.

[0175] In some embodiments, the intracellular signaling domain includes two or more co-stimulatory signaling domains, for example, two co-stimulatory signaling domains, three co-stimulatory signaling domains, four co-stimulatory signaling domains, five co-stimulatory signaling domains, six co-stimulatory signaling domains, seven co-stimulatory signaling domains, eight co-stimulatory signaling domains, nine co-stimulatory signaling domains, ten co-stimulatory signaling domains, or more co-stimulatory signaling domains. In one embodiment, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, two or more co-stimulatory signaling domains are separated by a linker of the Disclosure (for example, any of the linkers described in Table 4). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0176] In some embodiments, the CAR of the present disclosure further includes an epitope tag. The epitope tag is a polypeptide sequence included within the polypeptide as a label detectable, for example, by a monoclonal antibody. Examples of epitope tags include FLAG tags, strep tags, HA tags, V5 tags, and myc tags. An exemplary epitope tag is the myc tag of the amino acid sequence EQKLISEEDLNGAA (SEQ ID NO: 20).

[0177] In some embodiments, the cells of the Disclosure express a CAR comprising an antigen-binding domain that binds to VSIG2, a transmembrane domain of the Disclosure, a primary signaling domain, and one or more co-stimulatory signaling domains.

[0178] In some embodiments, the cells of the Disclosure express an iCAR comprising an antigen-binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3), the transmembrane domain of the Disclosure, and one or more intracellular inhibitory co-signaling domains. In some embodiments, the VSIG2-specific antigen-binding domain in iCAR format may be selected based on a higher affinity for VSIG2 compared to other VSIG2-specific antigen-binding domains. In some embodiments, the VSIG2-specific antigen-binding domain in iCAR format may be selected based on a lower affinity for VSIG2 compared to other VSIG2-specific antigen-binding domains. In some embodiments, the cells of the Disclosure express a CAR comprising a VSIG2-binding antigen-binding domain (e.g., a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, wherein at least one CDR amino acid sequence contains a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3), a transmembrane domain of the Disclosure, a primary signaling domain, and one or more co-stimulatory signaling domains.

[0179] In some embodiments, the transmembrane domain is derived from the same protein as one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR and comprises a transmembrane domain derived from PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, SIGLEC-10, PECAM-1, CD72, IRTA2, IRTA4, NKIR, TLT1, PCDHGC3, MPZL1, FCGR2B, SIGLEC-6, MPIG6B, SIGLEC-12, LIR8, IRTA1, KIR2DL4, KIR2DL5, SIGLEC-7, or FCRH3, and at least one intracellular inhibitory co-signaling domain.

[0180] In some embodiments, the transmembrane domain originates from a first protein, and one or more intracellular signaling domains originate from a second protein distinct from the first protein.

[0181] Natural Killer Car (NK CAR) In some embodiments, the CARs of the Disclosure comprise one or more components of natural killer (NK) cells, thereby forming an NK CAR. The NK components include KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS The transmembrane domain, hinge domain, or cytoplasmic domain may be from any suitable natural killer cell receptor, including, but not limited to, the following: killer cell immunoglobulin-like receptors (KIRs) such as DPI; innate cytotoxic receptors (NCRs) such as NKp30, NKp44, and NKp46; the signaling lymphocyte-activating molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NK-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. The above-described structural components of the CAR can also be applied to the structure of the NK CAR.

[0182] Exemplary configurations and sequences of CARs containing NK receptor components are described in International Patent Publication 2014 / 145252, published on September 18, 2014.

[0183] Further chimeric receptor targets Certain aspects of this disclosure relate to chimeric receptors that bind to an antigen of interest in addition to VSIG2, and nucleic acids encoding such chimeric receptors. Furthermore, certain aspects of this disclosure relate to chimeric receptors and cells, such as immune-responsive cells genetically modified to express one or more such chimeric receptors that bind to an antigen of interest in addition to VSIG2, and to methods for treating and / or preventing myeloid malignancies, such as CRC, and other medical conditions where an antigen-specific immune response is desired, using such receptors and cells. Malignant cells have developed a set of mechanisms to protect themselves from immune recognition and elimination. This disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0184] In some embodiments, the first chimeric receptor includes an antigen-binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, with at least one CDR amino acid sequence having a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3), and the second chimeric receptor includes an additional antigen-binding domain that binds to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, cells can express a first chimeric receptor specific to VSIG2 (e.g., a CAR including a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, with at least one CDR amino acid sequence having a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3), and a second chimeric receptor specific to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, cells can express a first chimeric inhibitory receptor specific to VSIG2 (e.g., an inhibitory CAR comprising a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23) and a second chimeric receptor specific to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). For example, cells (e.g., immune-responsive cells) can be configured to co-express, or be engineered to co-express, an iCAR comprising an antigen-binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen-binding domain having one or more amino acid sequences listed in Tables 3, 21, 22, and / or 23, where at least one CDR amino acid sequence contains a sequence mutation relative to the parental CDR amino acid sequence shown in Table 3) and an aCAR that targets a tumor-associated antigen (e.g., a CRC-associated antigen). In addition to VSIG2, suitable antibodies for binding to the antigen include any antibody, whether natural or synthetic, full-length or fragmented, monoclonal or polyclonal, that binds sufficiently strongly and specifically to a second antigen, such as a tumor-associated antigen (e.g., CRC-associated antigen). In some embodiments, commercially available antibodies that bind to a second antigen, such as a tumor-associated antigen (e.g., CRC-associated antigen), may be used.The CDRs of commercially available antibodies are readily accessible to those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art can construct nucleic acids encoding scFv and chimeric receptors (e.g., CAR and TCR) based on the CDRs of such commercially available antibodies.

[0185] T cell receptor (TCR) Certain embodiments of this disclosure relate to a chimeric receptor that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen), where the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). The TCR of this disclosure is a disulfide-bonded heterodimer protein comprising two variable chains expressed as part of a complex with an invariant CD3 chain molecule. The TCR is found on the surface of T cells and plays a role in recognizing the antigen as a peptide bound to a major histocompatibility complex (MHC) molecule. In certain embodiments, the TCR of this disclosure comprises an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0186] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to peptide / MHC complexes. Each variable region has three complementarity-determining regions (CDRs).

[0187] In certain embodiments, the TCR can form receptor complexes with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. When the TCR complex engages with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0188] In some embodiments, the TCRs of this disclosure are recombinant TCRs. In certain embodiments, the TCRs are non-naturally derived TCRs. In certain embodiments, the TCRs differ from naturally derived TCRs by at least one amino acid residue. In some embodiments, the TCRs differ from naturally derived TCRs by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least eleven amino acid residues, at least twelve amino acid residues, at least thirteen amino acid residues, at least fourteen amino acid residues, at least fifteen amino acid residues, at least twenty amino acid residues, at least twenty-five amino acid residues, at least thirty amino acid residues, at least forty amino acid residues, at least fifty amino acid residues, at least sixty amino acid residues, at least seventy amino acid residues, at least eighty amino acid residues, at least ninety amino acid residues, at least one hundred amino acid residues, or more amino acid residues. In certain embodiments, the TCR is modified from a naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR is modified from a naturally occurring TCR by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least one hundred, or more amino acid residues.

[0189] Chimera TCR In some embodiments, the TCRs of this disclosure include one or more antigen-binding domains that can be transplanted onto one or more constant domains of a TCR chain, e.g., a TCR alpha chain or a TCR beta chain, in order to create a chimeric TCR that specifically binds to a second antigen of interest, such as a tumor-associated antigen (e.g., a CRC-associated antigen). While we do not wish to be bound by theory, it is thought that chimeric TCRs can signal through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., scFv) can be transplanted onto at least a portion of the constant domains of a TCR chain, such as the TCR alpha chain and / or TCR beta chain, e.g., the extracellular constant domain, the transmembrane domain, and the cytoplasmic domain. As another example, a CDR of an antibody or antibody fragment can be transplanted onto the TCR alpha chain and / or beta chain to create a chimeric TCR that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). Such chimeric TCRs can be prepared by methods well known in the art (see, for example, Willemsen RA et al., Gene Therapy 2000;7:1369-1377; Zhang T et al., Cancer Gene Ther 2004 11:487-496; and Aggen et al., Gene Ther. 2012 Apr;19(4):365-74).

[0190] VSIG2-specific protein-coding nucleic acid constructs Certain aspects of this disclosure relate to nucleic acids (e.g., isolated nucleic acids) encoding one or more VSIG2-specific proteins (e.g., VSIG2-specific CARs as described herein). In some embodiments, the nucleic acid is an RNA construct, such as a messenger RNA (mRNA) transcript or modified RNA. In some embodiments, the nucleic acid is a DNA construct.

[0191] In some embodiments, the nucleic acids of this disclosure encode a chimeric receptor comprising one or more antigen-binding domains, each domain binding to a target antigen (e.g., VSIG2), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the nucleic acids encode a chimeric receptor comprising an antigen-binding domain, a transmembrane domain, a primary signaling domain (e.g., a CD3-zeta domain), and one or more costimulatory signaling domains. In some embodiments, the nucleic acids further comprise a nucleotide sequence encoding a spacer region. In some embodiments, the antigen-binding domain is connected to the transmembrane domain by the spacer region. In some embodiments, the spacer region comprises a nucleic acid sequence selected from any of the nucleic acid sequences listed in Table 5. In some embodiments, the nucleic acids further comprise a nucleotide sequence encoding a leader sequence.

[0192] The nucleic acids of this disclosure can be obtained using any suitable recombinant methods known in the art, including, but not limited to, screening a library from cells expressing the gene of interest, inducing the gene of interest from a vector known to contain the gene, or directly isolating the gene of interest from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

[0193] In some embodiments, the nucleic acids of the Disclosure are contained within a vector. In some embodiments, the nucleic acids of the Disclosure are expressed intracellularly via transposons, CRISPR / Cas9 systems, TALENs, or zinc finger nucleases.

[0194] In some embodiments, the expression of nucleic acids encoding the chimeric receptors of this disclosure can be achieved by operably ligating the nucleic acid with a promoter and incorporating the construct into an expression vector. Suitable vectors can be replicated and incorporated in eukaryotic cells. Typical cloning vectors include transcriptional and translational terminators, start sequences, and promoters useful for regulating the expression of the desired nucleic acid.

[0195] In some embodiments, the expression constructs of the Disclosure may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols (e.g., U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, and U.S. Patent No. 5,589,466). In some embodiments, the vectors of the Disclosure are gene therapy vectors.

[0196] The nucleic acids of this disclosure may be cloned into several types of vectors. For example, the nucleic acids may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, or cosmids. In some embodiments, the vector may be an expression vector, a replication vector, a probe-generating vector, or a sequencing vector.

[0197] In some embodiments, the plasmid vector includes a transposon / transposase system for incorporating the nucleic acid of this disclosure into the host cell genome. Methods for expressing proteins in immune cells using transposon and transposase plasmid systems are generally described in Chicaybam L, Hum Gene Ther. 2019 Apr;30(4):511-522.doi:10.1089 / hum.2018.218; and Ptackova P, Cytotherapy. 2018 Apr;20(4):507-520.doi:10.1016 / j.jcyt.2017.10.001, each of which is incorporated herein by reference in its entirety. In some embodiments, the transposon system is the Sleeping Beauty transposon / transposase or the piggyBac transposon / transposase.

[0198] In some embodiments, the expression vectors of the Disclosure may be delivered to cells in the form of viral vectors. Suitable viral vector systems are well known in the Art. For example, viral vectors may be derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In some embodiments, the vectors of the Disclosure are lentiviral vectors. Lentiviral vectors are suitable for long-term gene transfer because such vectors allow for the long-term and stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors also have an advantage over vectors derived from onchoretroviruses (e.g., mouse leukemia virus) in that they can be transduced into non-proliferating cells. In some embodiments, the vectors of the Disclosure are adenovirus vectors (A5 / 35). In some embodiments, the vectors of this disclosure include a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., International Publication No. 01 / 96584; International Publication No. 01 / 29058; and U.S. Patent No. 6326193). Numerous virus-based systems have been developed for gene transfer into mammalian cells. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to mammalian cells either in vivo or ex vivo. Numerous retroviral systems are known in the art.

[0199] In some embodiments, the vectors of this disclosure include additional promoter elements, such as enhancers that modulate the frequency of transcription initiation. Enhancers are typically located 30 bp to 110 bp upstream of the initiation site, although many promoters have been shown to also include functional elements downstream of the initiation site. The spacing between promoter elements can be flexible so that promoter function is maintained when elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decrease. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Exemplary promoters may include, but are not limited to, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EF1a promoter, the ubiquitin C promoter, and the phosphoglycerokinase (PGK) promoter.

[0200] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells such as the immune-responsive cells of the Disclosure is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in promoting chimeric receptor expression from nucleic acids cloned into lentiviral vectors.

[0201] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells such as immune-responsive cells of the Disclosure is a constitutive promoter. For example, a preferred constitutive promoter is the pre-early cytomegalovirus (CMV) promoter. The CMV promoter is a potent constitutive promoter capable of driving high levels of expression of any polynucleotide sequence operably linked to the promoter. Other preferred constitutive promoters include, but are not limited to, the ubiquitin C (UbiC) promoter, the Simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus pre-early promoter, the Roussarcoma virus promoter, the actin promoter, the myosin promoter, the elongation factor 1a promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0202] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells such as immune-responsive cells of the Disclosure is an inducible promoter. The use of an inducible promoter may provide a molecular switch that can induce or suppress the expression of the nucleic acids of the Disclosure when the promoter is operably linked to the nucleic acid. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0203] In some embodiments, the vectors of the present disclosure may further include signal sequences for promoting secretion, polyadenylation signals and transcription terminators, factors enabling episomal replication, and / or factors enabling selection.

[0204] In some embodiments, the vectors of this disclosure may further include selectable marker genes and / or reporter genes to facilitate the identification and selection of chimeric receptor-expressing cells from a population of cells transduced with the vector. In some embodiments, the selectable markers may be encoded by nucleic acids that are isolated from the vector and used in a simultaneous translocation procedure. Either the selectable marker or reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Examples of selectable markers include, but are not limited to, antibiotic resistance genes, e.g., neo and similar.

[0205] In some embodiments, a reporter gene may be used to identify transduced cells and to evaluate the functionality of a regulatory sequence. As disclosed herein, a reporter gene is a gene that encodes a polypeptide that is not present or expressed in the recipient organism or tissue, and whose expression imparts readily detectable properties such as enzymatic activity. The expression of the reporter gene can be assayed at a suitable time after the nucleic acid has been introduced into the recipient cells. Examples of reporter genes include, but are not limited to, genes encoding luciferase, β-galactosidase, chloranphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein. Suitable expression systems are known in the art and can be prepared using known techniques or are commercially available. In some embodiments, a construct having the smallest 5' adjacent region exhibiting the highest level of expression of the reporter gene is identified as a promoter. Such a promoter region may be ligated to the reporter gene and used to evaluate a drug for its ability to regulate promoter-driven transcription.

[0206] In some embodiments, a vector comprising a nucleic acid sequence encoding a VSIG2-specific protein of the present disclosure (e.g., a chimeric receptor) further comprises a second nucleic acid encoding a polypeptide that increases the activity of the chimeric receptor.

[0207] In embodiments where VSIG2-specific protein-expressing cells include two or more heterogeneous proteins (e.g., two or more chimeric receptors), a single nucleic acid may encode two or more proteins under a single regulatory element (e.g., a promoter) or under separate regulatory elements for each nucleotide sequence encoding each protein contained in the nucleic acid. In some embodiments where VSIG2-specific protein-expressing cells include two or more heterogeneous proteins, each heterogeneous protein may be encoded by a separate nucleic acid. In some embodiments, each separate nucleic acid includes its own regulatory element (e.g., a promoter). In some embodiments, a single nucleic acid encodes two or more chimeric receptors, and the nucleotide sequences encoding the chimeric receptors are in the same reading frame and expressed as a single polypeptide chain. In such embodiments, two or more chimeric receptors may be separated by one or more peptide cleavage sites, e.g., autocleavage sites or substrates of intracellular proteases. Suitable peptide cleavage sites may include, but are not limited to, T2A peptide cleavage sites, P2A peptide cleavage sites, E2A peptide cleavage sites, and F2A peptide cleavage sites. In some embodiments, two or more chimeric receptors include T2A peptide cleavage sites. In some embodiments, two or more chimeric receptors include an E2A peptide cleavage site. In some embodiments, two or more chimeric receptors include a T2A and an E2A peptide cleavage site.

[0208] Methods for introducing and expressing genes in cells are known in the art. For example, in some embodiments, expression vectors can be introduced into host cells by physical, chemical, or biological means. Examples of physical means for introducing nucleic acids into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Examples of chemical means for introducing nucleic acids into host cells include, but are not limited to, colloidal dispersions, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of biological means for introducing nucleic acids into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0209] In some embodiments, liposomes may be used as a nonviral delivery system for introducing nucleic acids or vectors of this disclosure into host cells in vitro, ex vivo, or in vivo. In some embodiments, nucleic acids may associate with lipids by, for example, being encapsulated within an aqueous liposome, being dispersed within the lipid bilayer of a liposome, being attached to a liposome via a linking molecule that associates with both the liposome and the nucleic acid, being confined within a liposome, being complexed with a liposome, being dispersed in a lipid-containing solution, being mixed with lipids, being combined with lipids, being contained as a suspension in lipids, being contained in or complexed in micelles, or being associated with lipids in other ways. As disclosed herein, lipid-associated nucleic acid or vector compositions are not limited to any particular structure in solution. In some embodiments, such compositions may exist as micelles or in bilayer structures having a “broken-down” structure. Such compositions may also be dispersed in solution to form aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that may occur naturally or be synthesized. In some embodiments, lipids may include naturally occurring lipid droplets in the cytoplasm, or a class of compounds including long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids may be obtained from commercial sources and include, but are not limited to, dimyristylphosphatidylcholine ("DMPC"), dicetyl phosphate ("DCP"), cholesterol, and dimyristylphosphatidylglycerol ("DMPG"). Storage solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the solvent because it evaporates more readily than methanol. As used herein, "liposomes" may encompass a variety of single and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. In some embodiments, liposomes may be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium.In some embodiments, multilayer liposomes may have multiple lipid layers separated by an aqueous medium. Multilayer liposomes can spontaneously form when phospholipids are suspended in an excess aqueous solution. In some embodiments, lipid components may undergo self-rearrangement before the formation of a closed structure, allowing for the trapping of water and dissolved solutes between the lipid bilayers. In some embodiments, lipids may exist in micelle structures or only as heterogeneous aggregates of lipid molecules.

[0210] In some embodiments, the nucleic acids or vectors of the Disclosure are introduced into mammalian host cells, such as immune-responsive cells of the Disclosure. In some embodiments, the presence of the nucleic acids or vectors of the Disclosure in host cells can be confirmed by any suitable assay known in the Art, including but not limited to Southern blot assays, Northern blot assays, RT-PCR, PCR, ELISA assays, and Western blot assays.

[0211] In some embodiments, the nucleic acids or vectors of the Disclosure are stably transduced into immune-responsive cells of the Disclosure. In some embodiments, cells exhibiting stable expression of the nucleic acids or vectors express the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months after transduction.

[0212] In embodiments in which the VSIG2-specific protein of the Disclosure (e.g., a chimeric receptor) is transiently expressed in cells, the nucleic acid or vector encoding the VSIG2-specific protein of the Disclosure is transfected into immune-responsive cells of the Disclosure. In some embodiments, the immune-responsive cells express the VSIG2-specific protein for approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after transfection.

[0213] In some embodiments, the nucleic acid construct encodes a bicistronically encoded chimeric antigen receptor. In some embodiments, the encoded bicistronic chimeric antigen receptor includes a VSIG2 CAR (e.g., a VSIG2 inhibitory CAR) and a CAR specific to a second antigen (e.g., a tumor-targeted chimeric receptor).

[0214] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a VSIG2 antigen-binding domain and a second antigen-binding domain.

[0215] Multi-cistron expression system In this specification, various embodiments provide multi-cistron expression systems. In some embodiments, the multi-cistron expression system comprises (a) an exogenous polynucleotide encoding a first cytokine, (b) an exogenous polynucleotide encoding a second cytokine, and (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the multi-cistron expression system comprises an activated CAR (aCAR) and an inhibitory CAR (iCAR).

[0216] In this specification, in various embodiments, immune-responsive cells are also provided that are engineered to have (a) an exogenous polynucleotide encoding a first cytokine, (b) an exogenous polynucleotide encoding a second cytokine, and (c) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

[0217] The multi-cistron expression systems or immune-responsive cells disclosed herein may include activation-regulating polypeptides. ACP may include synthetic transcription factors. These synthetic transcription factors are non-native proteins comprising a DNA-binding domain and a transcription effector domain, capable of regulating (i.e., activating or repressing) transcription through binding to a related promoter recognized by the DNA-binding domain (ACP-responsive promoter). In some embodiments, ACP is a transcriptional repressor. In some embodiments, ACP is a transcriptional activator.

[0218] Membrane-cleavable chimeric proteins can be manipulated so that the secretion of effector molecules can be regulated in a protease-dependent manner. Specifically, membrane-cleavable chimeric proteins can be manipulated so that the secretion of effector molecules can be regulated as part of a “membrane-cleavable” system, where the incorporation of a protease-cleavage site ("C") and a cell membrane anchoring domain ("MT") enables regulated secretion of effector molecules in a protease-dependent manner. While we do not wish to be constrained by theory, secretion is generally regulated through the following cellular processes by the components of the membrane-cleavable system present in membrane-cleavable chimeric proteins:

[0219] MT: The cell membrane anchoring domain contains a transmembrane domain (or transmembrane-intracellular domain) that directs the cellular transport of a chimeric protein so that the protein is inserted into the cell membrane or associated with (anchored) the cell membrane.

[0220] C: Following the expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, causing the effector molecule to be released (secreted) into the extracellular space. Generally, the protease cleavage site is protease-specific and includes sites that have been engineered to be protease-specific. The protease cleavage site may be selected or engineered to achieve optimal protein expression, cell type-specific cleavage, cell state-specific cleavage, and / or cleavage and release of the payload in the desired dynamics (e.g., ratio of membrane-bound chimeric protein to secreted chimeric protein levels).

[0221] In some embodiments, a membrane-cleavable chimeric protein (or an engineered nucleic acid encoding a membrane-cleavable chimeric protein) is provided herein, having a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane anchoring domain.

[0222] An "effector molecule" refers to a molecule that binds to another molecule and modulates the biological activity of that molecule (e.g., nucleic acids, such as DNA or RNA, or proteins (polypeptides) or peptides). For example, an effector molecule may act as a ligand to increase or decrease enzyme activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms. By directly binding to and modulating another molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0223] Generally, for all membrane-cleavable chimeric proteins described herein, the effector molecule is a cytokine or its active fragment, which is a secreted effector molecule (referred to as "S" in formula SC-MT or MT-CS) containing a cytokine or its active fragment.

[0224] The term “modulate” encompasses the maintenance of biological activity, the inhibition (partial or complete) of biological activity, and the stimulation / activation (partial or complete) of biological activity. The term also encompasses reducing or increasing (e.g., enhancing) biological activity. Two different effector molecules are considered to “modulate different tumor-mediated immunosuppressive mechanisms” if one effector molecule modulates a different tumor-mediated immunosuppressive mechanism (e.g., stimulating T cell signaling) that is modulated by the other effector molecule (e.g., stimulating antigen presentation and / or processing).

[0225] Regulation by effector molecules can be direct or indirect. Direct regulation occurs when an effector molecule binds to another molecule and modulates its activity. Indirect regulation occurs when an effector molecule binds to another molecule and modulates its activity, which in turn modulates the activity of yet another molecule (not to which an effector molecule is bound).

[0226] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%) in the immunostimulatory and / or antitumor immune response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in the immunostimulatory and / or antitumor immune response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase of 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200% in the immunostimulatory and / or antitumor immune response. It should be understood that the “increase” in immunostimulatory and / or antitumor immune responses is related to the immunostimulatory and / or antitumor immune responses that would otherwise occur, for example, systemically or within the tumor microenvironment, in the absence of effector molecules.

[0227] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase of at least twofold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100fold) in the immunostimulatory and / or antitumor immune response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in an increase of at least threefold, at least fivefold, at least tenfold, at least twentyfold, at least fiftyfold, or at least 100fold in the immunostimulatory and / or antitumor immune response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase of 2-10fold, 2-20fold, 2-30fold, 2-40fold, 2-50fold, 2-60fold, 2-70fold, 2-80fold, 2-90fold, or 2-100fold in the immunostimulatory and / or antitumor immune response.

[0228] Non-limiting examples of immunostimulatory and / or antitumor immune mechanisms include T cell signaling, activity and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activity and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activity and / or recruitment, stromal degradation, production of immunostimulatory metabolites, interferon gene stimulator (STING) signaling (which increases the secretion of IFN and Th1 polarization and promotes an antitumor immune response), and / or type I interferon signaling. Effector molecules may stimulate at least one (or more) of the aforementioned immunostimulatory mechanisms, thus potentially leading to an increase in the immunostimulatory response. Changes in the aforementioned immunostimulatory and / or antitumor immune mechanisms can be evaluated, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., of specific markers), and / or cell secretion assays (e.g., of cytokines).

[0229] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in a reduction of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%) in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in a reduction of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a reduction of 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200% in the immunosuppressive response. It should be understood that the “reduction” in the immunosuppressive response relates to the immunosuppressive response that would otherwise occur, for example, systemically or in the tumor microenvironment, in the absence of effector molecules.

[0230] In some embodiments, modulation of tumor-mediated immunosuppression by at least one effector molecule results in a reduction of at least twofold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100fold) in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppression may result in a reduction of at least threefold, at least fivefold, at least tenfold, at least twentyfold, at least fiftyfold, or at least 100fold in the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppression may result in a reduction of 2-10fold, 2-20fold, 2-30fold, 2-40fold, 2-50fold, 2-60fold, 2-70fold, 2-80fold, 2-90fold, or 2-100fold in the immunosuppressive response.

[0231] Non-limiting examples of immunosuppressive mechanisms include negative co-stimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), T-regulatory (Treg) cell signaling, production / maintenance of tumor checkpoint molecules, bone marrow-derived suppressor cell signaling, activation, and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. Effector molecules can inhibit at least one (or more) of the aforementioned immunosuppressive mechanisms, thus resulting in a reduction in the immunosuppressive response. Changes in the aforementioned immunosuppressive mechanisms may include, for example, an increase in T cell proliferation and / or an increase in IFNγ production (negative co-stimulatory signaling, T reg Cellular signaling and / or MDSCs); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (production / maintenance of tumor checkpoint molecules); RNA and enzyme assays via ELISA, LUMINEX®, qPCR, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and assays for phosphorylation of PI3K, Akt, and p38 (VEGF signaling).

[0232] In some embodiments, effector molecules function additively: the effect of two effector molecules may be equal to, for example, the sum of the effects of two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules may be greater than, for example, the combined function of two effector molecules.

[0233] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or modify the tumor microenvironment may be any of the cytokines described herein.

[0234] In some embodiments, at least one of the effector molecules stimulates an immunostimulatory mechanism in the tumor microenvironment and / or inhibits an immunosuppressive mechanism in the tumor microenvironment.

[0235] In some embodiments, at least one effector molecule (a) stimulates T cell signaling, activity, and / or recruitment; (b) stimulates antigen presentation and / or processing; (c) stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment; (d) stimulates dendritic cell differentiation and / or maturation; (e) stimulates immune cell recruitment; (f) stimulates pro-inflammatory macrophage signaling, activity, and / or recruitment, or inhibits anti-inflammatory macrophage signaling, activity, and / or recruitment; (g) stimulates stromal degradation; (h) stimulates immunostimulatory metabolite production; (i) stimulates type I interferon signaling; (j) inhibits negative co-stimulatory signaling; (k) inhibits pro-apoptotic signaling of anti-tumor immune cells; and (l) regulates T(T) reg (m) inhibit cell signaling, activity, and / or recruitment; (n) inhibit tumor checkpoint molecules; (o) stimulate interferon gene stimulator (STING) signaling; (p) inhibit bone marrow-derived suppressor cell signaling, activity, and / or recruitment; (q) degrade immunosuppressive factors / metabolites; (r) inhibit vascular endothelial growth factor signaling; and (c) directly kill tumor cells.

[0236] Table 8 lists non-exclusive examples of cytokines. Effector molecules may be human or human equivalents of the mammalian effector molecules listed in Table 8. Effector molecules may be of human origin, for example, endogenous human effector molecules or effector molecules modified and / or optimized for function, e.g., with codons optimized to improve expression, modified to improve stability, or modified in their signal sequence (see below). Various programs and algorithms for optimizing function are known to those skilled in the art and can be selected based on desired improvements, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).

[0237] (Table 8) Exemplary effector molecules TIFF2026514757000013.tif76170

[0238] (Table 10) Sequences encoding exemplary effector molecules TIFF2026514757000014.tif152170TIFF2026514757000015.tif255167TIFF20265147570 00016.tif255167TIFF2026514757000017.tif255167TIFF2026514757000018.tif255166 TIFF2026514757000019.tif255166TIFF2026514757000020.tif255166TIFF20265147570 00021.tif255166TIFF2026514757000022.tif255165TIFF2026514757000023.tif179166

[0239] The first manipulated nucleic acid may contain nucleotide sequences that are 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 sequences shown in Table 10. The first manipulated nucleic acid may contain nucleotide sequences having the sequences shown in Table 10.

[0240] The first manipulated nucleic acid may contain a nucleotide sequence that is 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 human IL-15 sequence. The first manipulated nucleic acid may contain a nucleotide sequence having the human IL-15 sequence.

[0241] The second manipulated nucleic acid may contain nucleotide sequences that are 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 sequences shown in Table 10.

[0242] The second manipulated nucleic acid may contain a nucleotide sequence that is 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 human IL21 sequence. The second manipulated nucleic acid may contain a nucleotide sequence having the human IL21 sequence.

[0243] (b) The first manipulated nucleic acid may contain a nucleotide sequence having the first sequence shown in Table 10, and (b) the second manipulated nucleic acid may contain a nucleotide sequence having the sequence shown in Table 10.

[0244] (b) The first engineered nucleic acid may contain a nucleotide sequence having the first sequence of human IL-15, and (b) the second engineered nucleic acid may contain a nucleotide sequence having the sequence of human IL-21.

[0245] The immune-responsive cells provided herein may comprise any one of the engineered nucleic acids described herein. The immune-responsive cells provided herein may comprise any one combination of the engineered nucleic acids described herein. The immune-responsive cells provided herein may comprise two or more of the engineered nucleic acids described herein.

[0246] The immune-responsive cells provided herein may contain nucleotide sequences that are 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 sequences listed in Table 10. The immune-responsive cells provided herein may contain nucleotide sequences having the sequences shown in Table 10.

[0247] The immune-responsive cells provided herein may contain nucleotide sequences that are 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 human IL-15 sequence. The immune-responsive cells provided herein may contain nucleotide sequences having the human IL-15 sequence.

[0248] The immune-responsive cells provided herein may contain nucleotide sequences that are 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 of human IL21. The immune-responsive cells provided herein may contain nucleotide sequences having the sequence of human IL21.

[0249] The immune-responsive cells provided herein may comprise a nucleotide sequence having a first sequence provided in Table 10, and a second engineered nucleic acid comprising a nucleotide sequence having a first sequence provided in Table 10.

[0250] The immune-responsive cells provided herein may comprise (b) a nucleotide sequence having a first sequence of human IL-15, and (b) a second engineered nucleic acid comprising a nucleotide sequence having a human IL-15 sequence.

[0251] The expression vectors provided herein may comprise one of the engineered nucleic acids described herein. The expression vectors provided herein may comprise one combination of the engineered nucleic acids described herein. The expression vectors provided herein may comprise two or more of the engineered nucleic acids described herein.

[0252] The expression vectors provided herein may contain nucleotide sequences that are 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 sequences listed in Table 10.

[0253] The expression vectors provided herein may contain a nucleotide sequence that is 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 human IL15 sequence.

[0254] The expression vectors provided herein may contain a nucleotide sequence that is 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 human IL21 sequence.

[0255] The expression vectors provided herein may include a nucleotide sequence having a first sequence provided in Table 10, and a second manipulated nucleic acid having a nucleotide sequence having a sequence provided in Table 10.

[0256] The expression vectors provided herein may comprise a first nucleotide sequence having the sequence of human IL-15, and a second engineered nucleic acid comprising a nucleotide sequence having the sequence of human IL-21.

[0257] In various embodiments, the first and / or second cytokines of the multicistron expression system disclosed herein are calibrated releasing cytokines. As used herein, the terms “membrane-cleavable,” “release-controlled,” and “release-calibrated” are interchangeable. In certain embodiments, the cytokine is membrane-cleavable. In certain embodiments, the cytokine is a release-calibrated (cr) cytokine. In certain embodiments, the release-calibrated cytokine comprises a B7-1 transmembrane domain. In certain embodiments, the B7-1 transmembrane domain comprises the amino acid sequence of the B7-1 transmembrane domain disclosed in Table 14. In certain embodiments, the release-calibrated cytokine comprises a “slow” protease cleavage site comprising the amino acid sequence of VTPEPIFSLI. In certain embodiments, the release-calibrated cytokine comprises a “fast” protease cleavage site comprising the amino acid sequence of PRAEALKGG.

[0258] In some embodiments, the cytokine is release-calibrated IL15 (crIL15). In some embodiments, crIL15 includes a “slow” protease cleavage site. In certain embodiments, crIL15 including a “slow” protease cleavage site includes the amino acid sequence of crIL15-“slow” protease cleavage site disclosed in Table 10. Exemplary nucleic acid sequences encoding crIL15 including a “slow” protease cleavage site are disclosed in Table 10. In certain embodiments, the nucleic acid encoding crIL15 including a “slow” protease cleavage site includes a sequence that is 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 nucleic acid sequence of crIL15 including a “slow” protease cleavage site disclosed in Table 10.

[0259] In some embodiments, crIL15 comprising a "slow" protease cleavage site also comprises a furin cleavage site. CrIL15 comprising a "slow" protease cleavage site and a furin cleavage site may comprise the amino acid sequences of the crIL15 "slow" protease cleavage site and the furin cleavage site disclosed in Table 10. Exemplary nucleic acid sequences encoding crIL15 comprising a "slow" protease cleavage site and a furin cleavage site are disclosed in Table 10. In certain embodiments, the nucleic acid encoding crIL15 comprising a "slow" protease cleavage site and a furin cleavage site comprises a sequence that is 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 nucleic acid sequences of the crIL15 "slow" protease cleavage site and the furin cleavage site disclosed in Table 10.

[0260] In certain embodiments, crIL15 comprises a "fast" protease cleavage site comprising the amino acid sequence PRAEALKGG. In certain embodiments, crIL15 comprising a "fast" protease cleavage site comprises the amino acid sequence of the crIL15-"fast" protease cleavage site disclosed in Table 10. Exemplary nucleic acid sequences encoding crIL15 comprising a "fast" protease cleavage site are disclosed in Table 10. In certain embodiments, the nucleic acid encoding crIL15 comprising a "fast" protease cleavage site comprises a sequence that is 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 nucleic acid sequence of the crIL15-"fast" protease cleavage site disclosed in Table 10.

[0261] In certain embodiments, crIL15 comprises the amino acid sequence of crIL15 disclosed in Table 10. Exemplary nucleic acid sequences encoding crIL15 are disclosed in Table 10. In certain embodiments, the nucleic acid encoding crIL15 comprises a sequence that is 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 nucleic acid sequence of crIL15 disclosed in Table 10.

[0262] In certain embodiments, crIL15 includes a sucoid domain. In certain embodiments, crIL15 includes an IgE reader sequence. In certain embodiments, crIL15 includes a sucoid domain and an IgE reader sequence. In certain embodiments, crIL15 includes the amino acid sequences of the crIL15-sucoid domain and IgE reader sequence disclosed in Table 10. Exemplary nucleic acid sequences encoding crIL15 including the sucoid domain and IgE reader sequence are disclosed in Table 10. In certain embodiments, the nucleic acid encoding crIL15 including the sucoid domain and IgE reader sequence includes a sequence that is 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 nucleic acid sequences of crIL15 provided in Table 10.

[0263] In certain embodiments, the chimeric IL15 comprises a sushi domain. In certain embodiments, the chimeric IL15 comprises an IgE leader sequence. In certain embodiments, the chimeric IL15 comprises a sushi domain and an IgE leader sequence. In certain embodiments, the chimeric IL15 comprises the amino acid sequences of the chimeric IL15-sushi domain and IgE leader sequence disclosed in Table 10. An exemplary nucleic acid sequence encoding a chimeric IL15 comprising a sushi domain and an IgE leader sequence is disclosed in Table 10. In certain embodiments, the nucleic acid encoding a chimeric IL15 comprising a sushi domain and an IgE leader sequence comprises a sequence that is 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 nucleic acid sequence of the chimeric IL15-sushi domain and IgE leader sequence disclosed in Table 10.

[0264] In certain embodiments, IL15 is membrane-bound IL15 (mbIL15). In certain embodiments, mbIL15 comprises the amino acid sequence of mbIL15 disclosed in Table 10. An exemplary nucleic acid sequence encoding mbIL15 is disclosed in Table 10. In certain embodiments, the nucleic acid encoding mbIL15 comprises a sequence that is 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 nucleic acid sequence of mbIL15 disclosed in Table 10.

[0265] In certain embodiments, IL21 comprises the amino acid sequence of IL21 disclosed in Table 10. An exemplary nucleic acid sequence encoding IL21 is disclosed in Table 10. In certain embodiments, the nucleic acid encoding IL21 comprises a sequence that is 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 nucleic acid sequence of IL21 disclosed in Table 10.

[0266] In certain embodiments, IL21 includes a codon-optimized IL21 reader sequence. In certain embodiments, IL21 includes the amino acid sequence of the IL21 codon-optimized reader sequence disclosed in Table 10. Two exemplary nucleic acid sequences encoding IL21 including the codon-optimized IL21 reader sequence are provided in Table 10. In certain embodiments, the nucleic acid encoding IL21 including the codon-optimized IL21 reader sequence includes a sequence that is 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 nucleic acid sequence of IL21-codon-optimized reader sequence-1. In certain embodiments, the nucleic acid encoding IL21, including a codon-optimized IL21 reader sequence, includes a sequence that is 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 nucleic acid sequence of IL21-codon-optimized reader sequence-2.

[0267] In some embodiments, IL21 includes a furin cleavage site. In certain embodiments, IL21 includes the amino acid sequence of the IL21-furin cleavage sequence disclosed in Table 10. Table 10 provides exemplary nucleic acid sequences encoding IL21 including a furin cleavage site. In certain embodiments, the nucleic acid encoding IL21 including a furin cleavage site includes a sequence that is 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 nucleic acid sequence of the IL21-furin cleavage sequence disclosed in Table 10.

[0268] In certain embodiments, IL7 comprises the amino acid sequence of IL7 disclosed in Table 10. Exemplary nucleic acid sequences encoding IL7 are disclosed in Table 10. In certain embodiments, the nucleic acid encoding IL7 comprises a sequence that is 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 nucleic acid sequence of IL7 disclosed in Table 10.

[0269] In certain embodiments, IL12p70 comprises the amino acid sequence of IL12p70. Exemplary nucleic acid sequences encoding IL12p70 are disclosed in Table 10. In certain embodiments, the nucleic acid encoding IL12p70 comprises a sequence that is 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 nucleic acid sequences of IL12p70 disclosed in Table 10.

[0270] Secretory signals and signal anchors One or more effector molecules of membrane-cleavable chimeric proteins provided herein (e.g., any of the cytokines described herein) may generally be secretory effector molecules having a secretory signal peptide (also referred to as a signal peptide or signal sequence) at the N-terminus of the chimeric protein (e.g., the N-terminus of the effector molecule for SC-MT) that directs a newly synthesized protein, destined for secretion or membrane localization (also referred to as membrane insertion), to a suitable protein processing pathway. For chimeric proteins having the formula MT-CS, the membrane anchoring domain generally has a signal anchor sequence (e.g., a signal anchor sequence of a type II transmembrane protein) that directs a newly synthesized protein, destined for membrane localization, to a suitable protein processing pathway. For chimeric proteins having the formula SC-MT, a membrane anchoring domain with a reverse signal anchor sequence (e.g., the signal anchor sequence of a specific type III transmembrane protein) can be used, generally directing a newly synthesized protein destined for membrane localization to a suitable protein processing pathway, without the need for separate secretory signal peptides.

[0271] Generally, for all membrane-cleavable chimeric proteins described herein, one or more effector molecules are secretory effector molecules (referred to as "S" in formula SC-MT or MT-CS). In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretory signal. In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretory signal, but each effector molecule enables secretion from the manipulated cell after cleavage of the protease cleavage site.

[0272] Secretory signal peptides operablely associated with effector molecules may be intrinsic secretory signal peptides (e.g., endogenous secretory signal peptides of cytokines, which are generally endogenously associated with a given effector molecule). Secretory signal peptides operablely associated with effector molecules may be non-intrinsic secretory signal peptides or intrinsic secretory signal peptides. Non-intrinsic secretory signal peptides can promote improved expression and function, such as maintained secretion, in specific environments, such as the tumor microenvironment. A non-limiting list of non-intrinsic secretory signal peptides is shown in Table 11.

[0273] (Table 11) Exemplary signaling secretory peptides TIFF2026514757000024.tif98165TIFF2026514757000025.tif255165TIFF2026514757000026.tif42165

[0274] Protease cleavage site In general, all membrane-cleavable chimeric proteins described herein contain protease cleavage sites (referred to as "C" in formulas SC-MT or MT-CS). Generally, protease cleavage sites can be any amino acid sequence motif that can be cleaved by a protease. Examples of protease cleavage sites include type 1 transmembrane protease cleavage sites, type 2 transmembrane protease cleavage sites, GPI-anchored protease cleavage sites, ADAM8 protease cleavage sites, ADAM9 protease cleavage sites, ADAM10 protease cleavage sites, ADAM12 protease cleavage sites, ADAM15 protease cleavage sites, ADAM17 protease cleavage sites, ADAM19 protease cleavage sites, ADAM20 protease cleavage sites, ADAM21 protease cleavage sites, ADAM28 protease cleavage sites, ADAM30 protease cleavage sites, and ADAM30 protease cleavage sites. This includes, but is not limited to, thease cleavage sites, ADAM33 protease cleavage sites, BACE1 protease cleavage sites, BACE2 protease cleavage sites, SIP protease cleavage sites, MT1-MMP protease cleavage sites, MT3-MMP protease cleavage sites, MT5-MMP protease cleavage sites, furin protease cleavage sites, PCSK7 protease cleavage sites, matryptase protease cleavage sites, matryptase-2 protease cleavage sites, MMP9 protease cleavage sites, or NS3 protease cleavage sites.

[0275] One example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, which includes, but is not limited to, NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B cleavage sites. For a description of representative sequences of NS3 proteases and their cleavage sites for various strains of HCV, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; which is incorporated herein by reference in its entirety. For example, sequences of the HCV NS4A / 4B protease cleavage site; the HCV NS5A / 5B protease cleavage site; the C-terminal degron with the NS4A / 4B protease cleavage site; and the N-terminal degron with the HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. For example, NCBI entries: Accession numbers: YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, A See HH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of these sequences (as entered by the filing date of this application) are incorporated herein by reference.

[0276] Another example of a protease cleavage site is the ADAM17-specific protease (also referred to as tumor necrosis factor α-converting enzyme [TACE]) cleavage site. The ADAM17-specific protease cleavage site may be an endogenous sequence of a substrate spontaneously cleaved by ADAM17. The ADAM17-specific protease cleavage site may be an engineered sequence capable of being cleaved by ADAM17. Engineered ADAM17-specific protease cleavage sites may be engineered for specific desired properties, including, but are not limited to, optimal expression of the chimeric protein, specificity for ADAM17, cleavage rate by ADAM17, ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cellular states. Protease cleavage sites can be selected for specific cleavage by ADAM17. For example, a specific protease cleavage site capable of being cleaved by ADAM17 may also be capable of cleavage by additional ADAM family proteases, such as ADAM10. Therefore, ADAM17-specific protease cleavage sites can be selected and / or manipulated so that cleavage by other proteases, such as ADAM10, is reduced or eliminated. Protease cleavage sites can be selected for the cleavage rate by ADAM17. For example, it may be desirable to select a protease cleavage site that exhibits a specific cleavage rate by ADAM17, such as reduced cleavage kinetics with respect to the endogenous sequence of the substrate that is naturally cleaved by ADAM17. In such cases, a specific cleavage rate can generally be selected to regulate the processing rate of the chimeric protein, which in turn regulates the release / secretion rate of the payload effector molecule. Therefore, ADAM17-specific protease cleavage sites can be selected and / or manipulated so that the sequence exhibits a desired cleavage rate by ADAM17. Protease cleavage sites can be selected for both specific cleavage by ADAM17 and the cleavage rate by ADAM17. Exemplary ADAM17-specific protease cleavage sites, including those exhibiting specific specificity and cleavage velocity dynamics, are shown in Table 12 below, along with references to the cleavage sites (P5-P1: N-terminus; P1'-P5': C-terminus).Further details of ADAM17 and ADAM10, including their expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 199(8)2865-2872), Pham et al. (Anticancer Res. 2017 Oct; 37(10): 5507-5513), Caescu et al. (Biochem J. 2009 Oct 23; 424(1): 79-88), and Tucher et al. (J. Proteome Res. 2014, 13, 4, 2205-2214), each incorporated herein by reference for its intended purpose.

[0277] (Table 12) Potential ADAM17 protease cleavage site sequences TIFF2026514757000027.tif84128

[0278] In some embodiments, the protease cleavage site includes a first region having the amino acid sequence PRAE. In some embodiments, the protease cleavage site includes a second region having the amino acid sequence KGG. In some embodiments, the first region is N-terminal to the second region. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEX1X2KGG, where X1 is A, Y, P, S, or F, and X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAVKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEALKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEYSKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEPIKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAYKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAESSKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEFTKGG. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAAKGG. In some embodiments, the protease cleavage site includes the amino acid sequence DEPHYSQRR. In some embodiments, the protease cleavage site includes the amino acid sequence PPLGPIFNPG. In some embodiments, the protease cleavage site includes the amino acid sequence PLAQAYRSS. In some embodiments, the protease cleavage site includes the amino acid sequence TPIDSSFNPD. In some embodiments, the protease cleavage site includes the amino acid sequence VTPEPIFSLI.

[0279] In certain embodiments, the cleavage site includes a linker sequence. The cleavage site may be flanked by linker sequences on the N-terminal and / or C-terminal sides. For example, but not limited to, the cleavage site may be flanked on both the N-terminal and C-terminal sides by partial glycine-serine (GS) linker sequences. Upon cleavage, the N-terminal partial GS linker and the C-terminal partial GS linker combine to form a GS linker sequence such as the amino acid sequence number SGGGGSGGGGSGGGGSGGGGSGGGSLQ.

[0280] In certain embodiments, the cleavage site and linker contain the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ. An exemplary nucleic acid sequence encoding SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ is TCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTTCAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA. In some embodiments, the nucleic acid encoding SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ includes a nucleic acid sequence that is 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 thereto.

[0281] In certain embodiments, the protease cleavage site is N-terminus with respect to the linker. In certain embodiments, the protease cleavage site and linker contain the amino acid sequence PRAEALKGGSGGGGSGGGGSGGGGGSGGGSGGGSLQ. An exemplary nucleic acid sequence encoding PRAEALKGGSGGGGSGGGGSGGGSGGGSGGGSLQ is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT. In some embodiments, the nucleic acid encoding PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ may include a nucleic acid sequence that is 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 thereto.

[0282] In some embodiments, the protease cleavage site comprises the amino acid sequence ITQGLAVSTISSFF, which is a cleavage site that is native to CD16 and is cleavable by ADAM17. In certain embodiments, ITQGLAVSTISSFF is included within a linker. In certain embodiments, the linker comprises the amino acid sequence SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ. An exemplary nucleic acid sequence encoding SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ is AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCGTGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTGGATCTCTTCAA. In some embodiments, the nucleic acid encoding SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ comprises the nucleic acid sequence AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCGTGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTGGATCTCTTCAA, or a nucleic acid sequence that is 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 thereto.

[0283] The protease cleavage site may be at the C-terminus of the secreted effector molecule. The protease cleavage site may be at the N-terminus of the secreted effector molecule. Generally, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is one of the following: (1) the C-terminus of the secreted effector molecule and the N-terminus of the cell membrane anchoring domain (in other words, the protease cleavage site is between the secreted effector molecule and the cell membrane anchoring domain); or (2) the N-terminus of the secreted effector molecule and the C-terminus of the cell membrane anchoring domain (also between the secreted effector molecule and the cell membrane anchoring domain with reversed domain orientation). The protease cleavage site can be linked to the secreted effector molecule by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the effector molecule or the protease cleavage site. The protease cleavage site can be linked to the cell membrane anchoring domain by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the protease cleavage site. The polypeptide linker may be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker may be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG), A(EAAAK)3A, and Whitlow linkers (e.g., "KEGS" linkers, e.g., amino acid sequence KESGSVSSEQLAQFRSLD, eGK linkers, e.g., amino acid sequence EGKSSGSGSESKST, LR1 linkers, e.g., amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ, and linkers described in detail in published U.S. Patent No. 5,990,275 incorporated herein by reference). Additional exemplary polypeptide linkers include SGGGGSGGGGSG, TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, and GGGSGGGGSGGGSLQ.Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art. An example of a nucleic acid sequence encoding TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGAC. In certain embodiments, the nucleic acid encoding TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD includes a sequence that is 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 ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGAC.

[0284] In membrane-cleaving systems, the expression and localization of chimeric proteins to the cell membrane are followed by cleavage of the chimeric proteins by protease cleavage sites, and effector molecules are released (secreted) into the extracellular space of the cell.

[0285] Generally, a protease that cleaves a protease cleavage site is a protease specific to that particular protease cleavage site. For example, in the case of disintegrin and metalloproteinase (ADAM) family proteases, a protease that cleaves a specific ADAM protease cleavage site is generally limited to ADAM proteases that specifically recognize a particular ADAM protease cleavage site motif. Protease cleavage sites can be selected and / or manipulated to reduce or eliminate cleavage by undesirable proteases. Proteases can be membrane-bound or membrane-associated. Proteases can be secreted, for example, in specific cellular environments, such as the tumor microenvironment ("TME").

[0286] The proteases that cleave the protease cleavage sites of chimeric proteins may be expressed in the same cells that express the chimeric protein. The proteases that cleave the protease cleavage sites of chimeric proteins may be endogenous to the cells expressing the chimeric protein. In other words, cells engineered to express chimeric proteins can endogenously express proteases specific to the protease cleavage sites present in the chimeric protein. Endogenous protease expression generally refers to both expression under homeostatic conditions (e.g., in cells generally considered healthy) and differential expression under non-homeostatic conditions (e.g., upregulated expression in tumor cells). Protease cleavage sites can be selected based on known proteases endogenously expressed by the desired cell population. In such cases, the cleavage of the protease cleavage site (and thus, the release / secretion of the payload) can generally be restricted to only the cells of interest due to a cell-restricting protease that needs to come into contact with the protease cleavage site of the chimeric protein expressed in the same cell. For example, although we do not wish to be constrained by theory, ADAM17 is thought to have restricted endogenous expression in NK cells and T cells. Thus, the selection of an ADAM17-specific protease cleavage site can restrict the cleavage of the protease cleavage site to NK cells and T cells co-expressing the chimeric protein. In another example, the protease cleavage site can be selected for a specific tumor-associated protease that is known to be expressed in a particular tumor population of interest (e.g., in specific tumor cells engineered to express the chimeric protein).Using protease and / or expression databases, suitable protease cleavage sites are selected, and each of these is incorporated by reference for all purposes, such as selecting protease cleavage sites to be cleaved by tumor-associated proteases through consultation with, for example, Oncomine (www.oncomine.org), the European Institute for Bioinformatics (www.ebi.ac.uk), in particular (www.ebi.ac.uk / gxa), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org / tools / peptide cutter), and PMAP.Cut DB (cutdb.burnham.org).

[0287] The proteases that cleave the protease cleavage sites of chimeric proteins may be heterologous to the cells expressing the chimeric proteins. For example, cells engineered to express chimeric proteins may be engineered to express proteases that are not commonly expressed by cells and are specific to the protease cleavage sites present in the chimeric proteins. Cells engineered to express both chimeric proteins and proteases may be engineered to express each from separate engineered nucleic acids or from multi-cistron systems (multi-cistron and multi-promoter systems are described in more detail in a section of this specification titled "Multi-Cistron and Multi-Promoter Systems"). Heterologous proteases and their corresponding protease cleavage sites may be selected with reference to endogenous proteases as described above.

[0288] The proteases that cleave the protease cleavage sites of chimeric proteins may be expressed on different cells rather than on the cells expressing the chimeric protein. For example, proteases may generally be expressed in specific cellular environments, such as the tumor microenvironment. In such cases, cleavage of the protease cleavage site may generally be restricted to the cellular environment of interest (e.g., the tumor microenvironment) due to an environment-restricting protease that needs to come into contact with the protease cleavage site. In embodiments having membrane-cleavable chimeric proteins, the secretion of effector molecules may generally be restricted to the cellular environment of interest (e.g., the tumor microenvironment) due to an environment-restricting protease that needs to come into contact with the protease cleavage site. The proteases that cleave the protease cleavage sites of chimeric proteins may be endogenous to different cells. The proteases that cleave the protease cleavage sites of chimeric proteins may be heterogeneous to different cells. For example, different cells can be manipulated to express proteases that are not generally expressed by different cells.

[0289] Proteases include, but are not limited to, type 1 transmembrane proteases, type 2 transmembrane proteases, GPI-anchored proteases, ADAM8 proteases, ADAM9 proteases, ADAM10 proteases, ADAM12 proteases, ADAM15 proteases, ADAM17 proteases, ADAM19 proteases, ADAM20 proteases, ADAM21 proteases, ADAM28 proteases, ADAM30 proteases, ADAM33 proteases, BACE1 proteases, BACE2 proteases, SIP proteases, MT1-MMP proteases, MT3-MMP proteases, MT5-MMP proteases, furin proteases, PCSK7 proteases, matryptase proteases, matryptase 2 proteases, and MMP9 proteases. The protease may be an NS3 protease. The protease may be an ADAM17 protease. The protease may be a tumor-associated protease, such as a cathepsin, cysteine ​​protease, aspartyl protease, serine protease, or metalloprotease. Specific examples of tumor-associated proteases include cathepsin B, cathepsin L, cathepsin S, cathepsin D, cathepsin E, cathepsin A, cathepsin G, thrombin, plasmin, urokinase, tissue plasminogen activator, metalloproteinase 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, ADAMTS, CD10 (CALLA), or prostate-specific antigen. Proteases also include, but are not limited to, those listed in Table 13 below. Exemplary homologous protease cleavage sites for specific proteases are also listed in Table 13.

[0290] (Table 13) Exemplary proteases with homologous cleavage sites and inhibitors TIFF2026514757000028.tif253170TIFF2026514757000029.tif255165TIFF2026514757000030.tif255165TIFF2026514757000031.tif255165TIFF2026514757000032.tif255165TIFF2026514757000033.tif255165TIFF2026514757000034.tif255165TIFF2026514757000035.tif255164TIFF2026514757000036.tif114166

[0291] The protease may be one of the following human proteases (MEROPS peptidase database numbers provided in parentheses; Rawlings ND, Morton FR, Kok, CY, Kong, J. & Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320-325; incorporated herein by reference for all purposes): pepsin A (MER000885), gastricin (MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005 534), Napsin A (MER004981), Mername-AA034 peptidase (MER014038), Pepsin A4 (MER037290), Pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type presumptive peptidase (MER107386), Napsin B pseudogene (MER004982), CYMP gp(Homo sapiens)(MER002929), subfamily A1A unassigned peptidase(MER181559), mouse mammary tumor virus retropepsin(MER048030), rabbit endogenous retrovirus endopeptidase(MER043650), S71-related human endogenous retropepsin(MER001812), RTVL-H type presumptive peptidase(MER047117), RTVL-H type presumptive peptidase(MER047133), RTVL-H type presumptive peptidase(MER047160), RTVL-H RTVL-H type presumptive peptidase (MER047206), RTVL-H type presumptive peptidase (MER047253), RTVL-H type presumptive peptidase (MER047260), RTVL-H type presumptive peptidase (MER047291), RTVL-H type presumptive peptidase (MER047418), RTVL-H type presumptive peptidase (MER047440), RTVL-H type presumptive peptidase (MER047479), RTVL-H type presumptive peptidase (MER047559), RTVL-H type presumptive peptidase (MER047583),RTVL-H type presumptive peptidase (MER015446), human endogenous retrovirus retropepsin homolog 1 (MER015479), human endogenous retrovirus retropepsin homolog 2 (MER015481), endogenous retrovirus retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retrovirus retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous Sex retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retrovirus retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER002094), endogenous retrovirus retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retro Viral retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), Endogenous retrovirus retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), Endogenous retrovirus retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), Endogenous retrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), Endogenous retrovirus retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), Endogenous retro Viral retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), Endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), Endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), Endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), Endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182),Endogenous retrovirus retropepsin pseudogene 16 (MER047165), Endogenous retrovirus retropepsin pseudogene 16 (MER047178), Endogenous retrovirus retropepsin pseudogene 16 (MER047200), Endogenous retrovirus retropepsin pseudogene 16 (MER047315), Endogenous retrovirus retropepsin pseudogene 16 (MER047405), Endogenous retrovirus retropepsin pseudogene 16 (MER030292), Endogenous retrovirus retropepsin pseudogene 17 (Homo sapiens chromosome 8) (M ER005305), Endogenous retrovirus retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), Endogenous retrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047222), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047454), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047477), Endogenous retrovirus Retropepsin pseudogene 21 (Homo sapiens) (MER004403), Endogenous retrovirus retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), Subfamily A2A nonpeptidase homolog (MER047046), Subfamily A2A nonpeptidase homolog (MER047052), Subfamily A2A nonpeptidase homolog (MER047076), Subfamily A2A nonpeptidase homolog (MER047080), Subfamily A2A nonpeptidase homolog (MER047088), Subfamily - A2A nonpeptidase homolog (MER047089), subfamily A2A nonpeptidase homolog (MER047091), subfamily A2A nonpeptidase homolog (MER047092), subfamily A2A nonpeptidase homolog (MER047093), subfamily A2A nonpeptidase homolog (MER047094), subfamily A2A nonpeptidase homolog (MER047097), subfamily A2A nonpeptidase homolog (MER047099), subfamily A2A nonpeptidase homolog (MER047101),Subfamily A2A nonpeptidase homolog (MER047102), subfamily A2A nonpeptidase homolog (MER047107), subfamily A2A nonpeptidase homolog (MER047108), subfamily A2A nonpeptidase homolog (MER047109), subfamily A2A nonpeptidase homolog (MER047110), subfamily A2A nonpeptidase homolog (MER047111), subfamily A2A nonpeptidase homolog (MER047114), subfamily A2A nonpeptidase homolog (MER 047118), subfamily A2A nonpeptidase homolog (MER047121), subfamily A2A nonpeptidase homolog (MER047122), subfamily A2A nonpeptidase homolog (MER047126), subfamily A2A nonpeptidase homolog (MER047129), subfamily A2A nonpeptidase homolog (MER047130), subfamily A2A nonpeptidase homolog (MER047134), subfamily A2A nonpeptidase homolog (MER047135), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047137), subfamily A2A nonpeptidase homolog (MER047140), subfamily A2A nonpeptidase homolog (MER047141), subfamily A2A nonpeptidase homolog (MER047142), subfamily A2A nonpeptidase homolog (MER047148), subfamily A2A nonpeptidase homolog (MER047149), subfamily A2A nonpeptidase homolog (MER047151), subfamily A2A nonpeptidase homolog (MER047154), subfamily A 2A nonpeptidase homolog (MER047155), subfamily A2A nonpeptidase homolog (MER047156), subfamily A2A nonpeptidase homolog (MER047157), subfamily A2A nonpeptidase homolog (MER047159), subfamily A2A nonpeptidase homolog (MER047161), subfamily A2A nonpeptidase homolog (MER047163), subfamily A2A nonpeptidase homolog (MER047166), subfamily A2A nonpeptidase homolog (MER047171),Subfamily A2A nonpeptidase homolog (MER047173), subfamily A2A nonpeptidase homolog (MER047174), subfamily A2A nonpeptidase homolog (MER047179), subfamily A2A nonpeptidase homolog (MER047183), subfamily A2A nonpeptidase homolog (MER047186), subfamily A2A nonpeptidase homolog (MER047190), subfamily A2A nonpeptidase homolog (MER047191), subfamily A2A nonpeptidase homolog (MER 047196), subfamily A2A nonpeptidase homolog (MER047198), subfamily A2A nonpeptidase homolog (MER047199), subfamily A2A nonpeptidase homolog (MER047201), subfamily A2A nonpeptidase homolog (MER047202), subfamily A2A nonpeptidase homolog (MER047203), subfamily A2A nonpeptidase homolog (MER047204), subfamily A2A nonpeptidase homolog (MER047205), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047207), subfamily A2A nonpeptidase homolog (MER047208), subfamily A2A nonpeptidase homolog (MER047210), subfamily A2A nonpeptidase homolog (MER047211), subfamily A2A nonpeptidase homolog (MER047212), subfamily A2A nonpeptidase homolog (MER047213), subfamily A2A nonpeptidase homolog (MER047215), subfamily A2A nonpeptidase homolog (MER047216), subfamily A 2A nonpeptidase homolog (MER047218), subfamily A2A nonpeptidase homolog (MER047219), subfamily A2A nonpeptidase homolog (MER047221), subfamily A2A nonpeptidase homolog (MER047224), subfamily A2A nonpeptidase homolog (MER047225), subfamily A2A nonpeptidase homolog (MER047226), subfamily A2A nonpeptidase homolog (MER047227), subfamily A2A nonpeptidase homolog (MER047230),Subfamily A2A nonpeptidase homolog (MER047232), subfamily A2A nonpeptidase homolog (MER047233), subfamily A2A nonpeptidase homolog (MER047234), subfamily A2A nonpeptidase homolog (MER047236), subfamily A2A nonpeptidase homolog (MER047238), subfamily A2A nonpeptidase homolog (MER047239), Subfamily A2A nonpeptidase homolog (MER047240), subfamily A2A nonpeptidase homolog (MER047242), subfamily A2A nonpeptidase homolog (MER047243), subfamily A2A nonpeptidase homolog (MER047249), subfamily A2A nonpeptidase homolog (MER047251), subfamily A2A nonpeptidase homolog (MER047252), subfamily A2A nonpeptidase homolog (MER047254), subfamily A2A nonpeptidase homolog (MER 047255), subfamily A2A nonpeptidase homolog (MER047263), subfamily A2A nonpeptidase homolog (MER047265), subfamily A2A nonpeptidase homolog (MER047266), subfamily A2A nonpeptidase homolog (MER047267), subfamily A2A nonpeptidase homolog (MER047268), subfamily A2A nonpeptidase homolog (MER047269), subfamily A2A nonpeptidase homolog (MER047272), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047273), subfamily A2A nonpeptidase homolog (MER047274), subfamily A2A nonpeptidase homolog (MER047275), subfamily A2A nonpeptidase homolog (MER047276), subfamily A2A nonpeptidase homolog (MER047279), subfamily A2A nonpeptidase homolog (MER047280), subfamily A2A nonpeptidase homolog (MER047281), subfamily A2A nonpeptidase homolog (MER047282), subfamily A 2A nonpeptidase homolog (MER047284), subfamily A2A nonpeptidase homolog (MER047285), subfamily A2A nonpeptidase homolog (MER047289), subfamily A2A nonpeptidase homolog (MER047290), subfamily A2A nonpeptidase homolog (MER047294), subfamily A2A nonpeptidase homolog (MER047295), subfamily A2A nonpeptidase homolog (MER047298), subfamily A2A nonpeptidase homolog (MER047300),Subfamily A2A nonpeptidase homolog (MER047302), subfamily A2A nonpeptidase homolog (MER047304), subfamily A2A nonpeptidase homolog (MER047305), subfamily A2A nonpeptidase homolog (MER047306), subfamily A2A nonpeptidase homolog (MER047307), subfamily A2A nonpeptidase homolog (MER047310), subfamily A2A nonpeptidase homolog (MER047311), subfamily A2A nonpeptidase homolog (MER 047314), subfamily A2A nonpeptidase homolog (MER047318), subfamily A2A nonpeptidase homolog (MER047320), subfamily A2A nonpeptidase homolog (MER047321), subfamily A2A nonpeptidase homolog (MER047322), subfamily A2A nonpeptidase homolog (MER047326), subfamily A2A nonpeptidase homolog (MER047327), subfamily A2A nonpeptidase homolog (MER047330), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047333), subfamily A2A nonpeptidase homolog (MER047362), subfamily A2A nonpeptidase homolog (MER047366), subfamily A2A nonpeptidase homolog (MER047369), subfamily A2A nonpeptidase homolog (MER047370), subfamily A2A nonpeptidase homolog (MER047371), subfamily A2A nonpeptidase homolog (MER047375), subfamily A2A nonpeptidase homolog (MER047376), subfamily A 2A nonpeptidase homolog (MER047381), subfamily A2A nonpeptidase homolog (MER047383), subfamily A2A nonpeptidase homolog (MER047384), subfamily A2A nonpeptidase homolog (MER047385), subfamily A2A nonpeptidase homolog (MER047388), subfamily A2A nonpeptidase homolog (MER047389), subfamily A2A nonpeptidase homolog (MER047391), subfamily A2A nonpeptidase homolog (MER047394),Subfamily A2A nonpeptidase homolog (MER047396), subfamily A2A nonpeptidase homolog (MER047400), subfamily A2A nonpeptidase homolog (MER047401), subfamily A2A nonpeptidase homolog (MER047403), subfamily A2A nonpeptidase homolog (MER047406), subfamily A2A nonpeptidase homolog (MER047407), subfamily A2A nonpeptidase homolog (MER047410), subfamily A2A nonpeptidase homolog (MER 047411), subfamily A2A nonpeptidase homolog (MER047413), subfamily A2A nonpeptidase homolog (MER047414), subfamily A2A nonpeptidase homolog (MER047416), subfamily A2A nonpeptidase homolog (MER047417), subfamily A2A nonpeptidase homolog (MER047420), subfamily A2A nonpeptidase homolog (MER047423), subfamily A2A nonpeptidase homolog (MER047424), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047428), subfamily A2A nonpeptidase homolog (MER047429), subfamily A2A nonpeptidase homolog (MER047431), subfamily A2A nonpeptidase homolog (MER047434), subfamily A2A nonpeptidase homolog (MER047439), subfamily A2A nonpeptidase homolog (MER047442), subfamily A2A nonpeptidase homolog (MER047445), subfamily A2A nonpeptidase homolog (MER047449), subfamily A 2A nonpeptidase homolog (MER047450), subfamily A2A nonpeptidase homolog (MER047452), subfamily A2A nonpeptidase homolog (MER047455), subfamily A2A nonpeptidase homolog (MER047457), subfamily A2A nonpeptidase homolog (MER047458), subfamily A2A nonpeptidase homolog (MER047459), subfamily A2A nonpeptidase homolog (MER047463), subfamily A2A nonpeptidase homolog (MER047468),Subfamily A2A nonpeptidase homolog (MER047469), subfamily A2A nonpeptidase homolog (MER047470), subfamily A2A nonpeptidase homolog (MER047476), subfamily A2A nonpeptidase homolog (MER047478), subfamily A2A nonpeptidase homolog (MER047483), subfamily A2A nonpeptidase homolog (MER047488), subfamily A2A nonpeptidase homolog (MER047489), subfamily A2A nonpeptidase homolog (MER 047490), subfamily A2A nonpeptidase homolog (MER047493), subfamily A2A nonpeptidase homolog (MER047494), subfamily A2A nonpeptidase homolog (MER047495), subfamily A2A nonpeptidase homolog (MER047496), subfamily A2A nonpeptidase homolog (MER047497), subfamily A2A nonpeptidase homolog (MER047499), subfamily A2A nonpeptidase homolog (MER047502), subfamily A2A nonpeptidase A2A nonpeptidase homolog (MER047504), subfamily A2A nonpeptidase homolog (MER047511), subfamily A2A nonpeptidase homolog (MER047513), subfamily A2A nonpeptidase homolog (MER047514), subfamily A2A nonpeptidase homolog (MER047515), subfamily A2A nonpeptidase homolog (MER047516), subfamily A2A nonpeptidase homolog (MER047520), subfamily A2A nonpeptidase homolog (MER047533), subfamily A 2A nonpeptidase homolog (MER047537), subfamily A2A nonpeptidase homolog (MER047569), subfamily A2A nonpeptidase homolog (MER047570), subfamily A2A nonpeptidase homolog (MER047584), subfamily A2A nonpeptidase homolog (MER047603), subfamily A2A nonpeptidase homolog (MER047604), subfamily A2A nonpeptidase homolog (MER047606), subfamily A2A nonpeptidase homolog (MER047609),Subfamily A2A nonpeptidase homolog (MER047616), subfamily A2A nonpeptidase homolog (MER047619), subfamily A2A nonpeptidase homolog (MER047648), subfamily A2A nonpeptidase homolog (MER047649), subfamily A2A nonpeptidase homolog (MER047662), subfamily A2A nonpeptidase homolog (MER048004), subfamily A2A nonpeptidase homolog (MER048018), subfamily A2A nonpeptidase homolog (MER048019), Subfamily A2A nonpeptidase homolog (MER048023), Subfamily A2A nonpeptidase homolog (MER048037), Subfamily A2A unassigned peptidase (MER047164), Subfamily A2A unassigned peptidase (MER047231), Subfamily A2A unassigned peptidase (MER047386), Cutaneous aspartate protease (MER057097), Presenilin 1 (MER005221), Presenilin 2 (MER005223), impas 1 peptidase (MER019701), impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsin F (MER004980), cathepsin L (MER000622), cathe Cathepsin S (MER000633), Cathepsin O (MER001690), Cathepsin K (MER000644), Cathepsin W (MER003756), Cathepsin H (MER000629), Cathepsin B (MER000686), Dipeptidyl-peptidase I (MER001937), Bleomycin hydrolase (animal) (MER002481), Tubulointerstitial nephritis antigen (MER016137),Tubulointerstitial nephritis antigen-related protein (MER021799), cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 gp (Homo sapiens) (MER005210), CTSLL3 g, .p.(Homo sapiens)(MER005209), Calpain-1(MER000770), Calpain-2(MER000964), Calpain-3(MER001446), Calpain-9(MER004042), Calpain-8(MER021474), Calpain-15(MER004745), Calpain-5(MER002939), Calpain-11(MER005844), Calpain-12(MER029889), Calpain-10(MER013510), Calpain-13(MER020139), Calpain-14(ME R029744), Mername-AA253 peptidase (MER005537), carpamodulin (MER000718), virtual protein 940251 (MER003201), ubiquitinyl hydrolase-L1 (MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinyl hydrolase-BAP1 (MER003989), ubiquitinyl hydrolase-UCH37 (MER005539), ubiquitin-specific peptidase 5 (MER002066), ubiquitin-specific peptidase 6 (MER000863) ), ubiquitin-specific peptidase 4 (MER001795), ubiquitin-specific peptidase 8 (MER001884), ubiquitin-specific peptidase 13 (MER002627), ubiquitin-specific peptidase 2 (MER004834), ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), Ubiquitin-specific peptidase 1 (MER004978), Ubiquitin-specific peptidase 12 (MER005454), Ubiquitin-specific peptidase 16 (MER005493), Ubiquitin-specific peptidase 15 (MER005427), Ubiquitin-specific peptidase 17 (MER002900), Ubiquitin-specific peptidase 19 (MER005428), Ubiquitin-specific peptidase 20 (MER005494), Ubiquitin-specific peptidase 3 (MER005513), Ubiquitin-specific peptidase 9Y (MER004314),Ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specific peptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specific peptidase 26 ( Homo sapiens type) (MER014292), ubiquitin-specific peptidase 24 (MER005706), ubiquitin-specific peptidase 42 (MER011852), ubiquitin-specific peptidase 46 (MER014629), ubiquitin-specific peptidase 37 (MER014633), ubiquitin-specific peptidase 28 (MER014634), ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specific peptidase 38 (MER014637), ubiquitin-specific peptidase 44 (MER01463 8) Ubiquitin-specific peptidase 50 (MER030315), Ubiquitin-specific peptidase 35 (MER014646), Ubiquitin-specific peptidase 30 (MER014649), Mername-AA091 peptidase (MER014743), Ubiquitin-specific peptidase 45 (MER030314), Ubiquitin-specific peptidase 51 (MER014769), Ubiquitin-specific peptidase 34 (MER014780), Ubiquitin-specific peptidase 48 (MER064620), Ubiquitin-specific peptidase -ze 40 (MER015483), ubiquitin-specific peptidase 41 (MER045268), ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), ubiquitin-specific peptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER027329),Ubiquitin-specific endopeptidase 39 [misleading] (MER064621), Mername-AA090 non-peptidase homolog (MER014739), ubiquitin-specific peptidase 43 [misleading] (MER030140), ubiquitin-specific peptidase 52 [misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudo Gene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750), autofadin-2 (MER013564), autofadin-1 (MER013561), autofadin-3 (MER014316), autofadin-4 (MER064622), Cezanne deubiquitination peptidase (MER029 042), Cezanne-2 peptidase (MER029044), Tumor necrosis factor alpha-inducing protein 3 (MER029050), Trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), Otubain-1 (MER029056), Otubain-2 (MER029061), CylD protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), Glycosyltransferase 28-domain-containing 1, Isoform CRA_c (Homo sapiens) (MER123606), hin1L gp (Homo sapiens) (MER139816), Ataxin-3 (MER099998), ATXN3L presumptive peptidase (MER115261), Josephine domain-containing 1 (Homo sapiens) (MER125334), Josephine domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), Regmine (plant alpha type) (MER044591), Regmine (MER001800), Glycosylphosphatidylinositol:protein transamidase (MER002479), Regmine pseudogene (Homo sapiens) (MER029741),Family C13 unassigned peptidase (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), ca caspase-14 (MER012083), paracaspase (MER019325), Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 Caspase pseudogene (MER014766), subfamily C14A nonpeptidase homolog (MER185329), subfamily C14A nonpeptidase homolog (MER179956), separin (Homo sapiens type) (MER011775), separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl-peptidase I (chordate) (MER011032), Mername-AA073 peptidase (MER029978), sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Mername-AA164 protein (MER020410),LOC138971 gp (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl peptidase II (MER012221), cytozolaranyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (MER001494) ), Aminopeptidase PILS (MER005331), Arginyl Aminopeptidase-like 1 (MER012271), Leukocyte-derived Arginine Aminopeptidase (MER002968), Aminopeptidase Q (MER052595), Aminopeptidase O (MER019730), Tata-binding protein-related factor (MER026493), Angiotensin-converting enzyme peptidase unit 1 (MER004967), Angiotensin-converting enzyme peptidase unit 2 (MER001019), Angiotensin-converting enzyme-2 (ME R011061), Mername-AA153 protein (MER020514), thymet oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein (MER021317), reishi manorisin-2 (MER014492), reishi manorisin-3 (MER180031), matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8 (MER001084), mat Rix metallopeptidase-2 (MER001080), Matrix metallopeptidase-9 (MER001085), Matrix metallopeptidase-3 (MER001068), Matrix metallopeptidase-10 (Homo sapiens type) (MER001072), Matrix metallopeptidase-11 (MER001075), Matrix metallopeptidase-7 (MER001092), Matrix metallopeptidase-12 (MER001089), Matrix metallopeptidase-13 (MER001411),Membrane-type matrix metallopeptidase-1 (MER001077), Membrane-type matrix metallopeptidase-2 (MER002383), Membrane-type matrix metallopeptidase-3 (MER002384), Membrane-type matrix metallopeptidase-4 (MER002595), Matrix metallopeptidase-20 (MER003021), Matrix metallopeptidase-19 (MER002076), Matrix metallopeptidase-23B (MER, 004766), Membrane-type matrix metallopeptidase-5 (MER005638), Membrane-type matrix metallopeptidase-6 (MER012071), Matrix metallopeptidase-21 (MER006101), Matrix metallopeptidase-22 (MER014098), Matrix metallopeptidase-26 (MER012072), Matrix metallopeptidase-28 (MER013587), Matrix metallopeptidase-23A (MER037217), Macrophage Elastomer -ze homolog (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), Matrix metallopeptidase-like 1 (MER045280), Subfamily M10A nonpeptidase homolog (MER175912), Subfamily M10A nonpeptidase homolog (MER187997), Subfamily M10A nonpeptidase homolog (MER187998), Subfamily M10A nonpeptidase homolog (MER180000), Meprin alpha Subunit (MER001111), Meprin beta subunit (MER005213), Procollagen C-peptidase (MER001113), Mammalian toroid-like 1 protein (MER005124), Mammalian toroid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS1 8 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMMDEC1 peptidase (MER000743),ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 Peptidase (MER006268), ADAM21 peptidase (Homo sapiens type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), Ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens type) (MER026906), Procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homolog (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homolog (Homo sapiens chromosome 15) (MER047250), AD AM3B protein (Homo sapiens type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), a protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Mername-AA225 peptidase homolog (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A gp (Homo sapiens) (MER005200),ADAM1 gp (Homo sapiens) (MER003912), subfamily M12B non-peptidase homolog (MER188210), subfamily M12B non-peptidase homolog (MER188211), subfamily M12B non-peptidase homolog (MER188212), subfamily M12B non-peptidase homolog (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER0051) 97), Neprilysin-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), Parapregin (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), Paparin-1 (MER002217), Paparin-2 (MER014521), Farnesylated protein-converting enzyme 1 (MER00 2646), Metalloproteinase-related protein-1 (MER030873), Aminopeptidase AMZ2 (MER011907), Aminopeptidase AMZ1 (MER058242), Carboxypeptidase A1 (MER001190), Carboxypeptidase A2 (MER001608), Carboxypeptidase B (MER001194), Carboxypeptidase N (MER001198), Carboxypeptidase E (MER001199), Carboxypeptidase M (MER001205), Carboxypeptidase U (MER001193), Carboxypeptidase A3 (MER001187), Metallocarboxypeptidase D peptidase unit 1 (MER003781), Metallocarboxypeptidase Z (MER003428), Metallocarboxypeptidase D peptidase unit 2 (MER004963), Carboxypeptidase A4 (MER013421), Carboxypeptidase A6 (MER013456), Carboxypeptidase A5 (MER017121), Metallocarboxypeptidase O (MER016044),Cytosolic carboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte enhancer-binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homolog (MER199530), insulin (MER001214), mitochondria Processing peptidase beta subunit (MER004497), Nardilysin (MER003883), Eupitrilidine (MER004877), Mitochondria Processing peptidase non-peptidase alpha subunit (MER001413), Ubiquinol-cytochrome c reductase core protein I (MER003543), Ubiquinol-cytochrome c reductase core protein II (MER003544), Ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), Insulin unit 2 (MER046821), Nardilysin unit 2 (MER046874), Insulin unit 3 (MER078753), Mitochondria Processing peptidase subunit alpha unit 2 (MER124489), Nardilyzin unit 3 (MER142856), LOC133083 gp (Homo sapiens) (MER021876), subfamily M16B non-peptidase homolog (MER188757), leucylaminopeptidase (animal) (MER003100), Mername-AA040 peptidase (MER003919), leucylaminopeptidase-1 (senorhabditis type) (MER013416), methionylaminopeptidase 1 (MER001342), methionylaminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498),Xaa-Pro dipeptidase (eukaryotes) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate cleavage peptidase 55kDa (MER013463), mitochondrial methionylaminopeptidase (MER014055), Mername-AA020 peptidase homolog (MER010972), proliferation-related protein 1 (MER005497), chromatin-specific transcription elongation factor 140kDa subunit (MER026495), proliferation-related protein 1-like (human chromosome X) (MER029983), Mername-AA226 peptidase homolog (Homo sapiens) (MER 056262), Mername-AA227 peptidase homolog (Homo sapiens) (MER047299), subfamily M24A non-peptidase homolog (MER179893), aspartylaminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidase I (MER015142), Mername-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L-peptidase (MER005239), glutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclise (MER015095) ), glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), nicarin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase 2 (MER013499), membrane-bound dipeptidase 3 (MER013496), dihydroorotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase-related protein-1 (MER030143),Virtual proteins such as dihydropyrimidinase-related protein-2 (MER030155), dihydropyrimidinase-related protein-3 (MER030151), dihydropyrimidinase-related protein-4 (MER030149), dihydropyrimidinase-related protein-5 (MER030136), 5730457F11RIK (MER033184), 1300019j08r, IK protein (MER033186), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homolog (MER199845), subfamily M23B non-peptidase homolog (MER199846), subfamily M23B non-peptidase homolog (MER199847), subfamily M23B non-peptidase homolog (MER13 7320), subfamily M23B nonpeptidase homolog (MER201557), subfamily M23B nonpeptidase homolog (MER199417), subfamily M23B nonpeptidase homolog (MER199418), subfamily M23B nonpeptidase homolog (MER199419), subfamily M23B nonpeptidase homolog (MER199420), subfamily M23B nonpeptidase homolog (MER175932), subfamily M23B nonpeptidase homolog (MER199665), Po h1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinating enzyme MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021886), COP9 signalosome subunit Subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MER030133), IFP38 peptidase homolog (MER030132), subfamily M67A non-peptidase homolog (MER191181), subfamily M67A unassigned peptidase (MER191144), granzyme B (Homo sapiens type) (MER000168), testisin (MER005212), tryptase beta (MER000136),Kallikrein-related peptidase 5 (MER005544), choline (MER005881), kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronic acid-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), enteric serine peptidase (rodents) (MER016130), adrenal-secreted serine peptidase (MER00 3734), Tryptase Delta 1 (Homo sapiens) (MER005948), Matryptase-3 (MER029902), Malapsin (MER006119), Tryptase-6 (MER006118), Ovochymase-1 Domain 1 (MER099182), Transmembrane Peptidase, Serine 3 (MER005926), Kallikrein-related Peptidase 15 (MER000064), Mername-AA031 Peptidase (MER014054), TMPRSS13 Peptidase (MER014226), Mername-AA038 Peptidase Peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-related serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase (Homo sapiens type) (MER000123), tryptase Rufa (MER000135), Granzyme K (MER001936), Granzyme H (MER000166), Chymotrypsin B (MER000001), Elastase-1 (MER003733), Pancreatic Endopeptidase E (MER000149), Pancreatic Elastase II (MER000146), Enteropeptidase (MER002068), Chymotrypsin C (MER000761), Prostasin (MER002460), Kallikrein 1 (MER000093), Kallikrein-related Peptidase 2 (MER000094),Kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022), complement component C1r-like peptidase (MER016352), complement factor D (MER000130), complement component activated C1r (MER000238), complement component activated C1s (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-related serine peptidase 1 (MER000244), complement factor I (MER000228), pancreatic endopeptidase E form B (MER000 150), pancreatic elastase IIB (MER000147), coagulation factor XIIa (MER000187), plasma kallikrein (MER000203), coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MER000188), protein C (active form) (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), manna Kallikrein-binding lectin-related serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epiceriacin (MER003736), kallikrein-related peptidase 4 (M ER005266), Procemin (MER004214), Kymopathin (MER001503), Kallikrein-related peptidase 11 (MER004861), Kallikrein-related peptidase 11 (MER216142), Trypsin type 2A (MER000021), HtrA1 peptidase (Homo sapiens type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351),Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like presumptive peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), matryptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matryptase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase Dase (MER001900), spinesin (MER014385), malapsin-2 (MER021929), complement factor D-like presumptive peptidase (MER056164), ovochimase-2 (MER022410), HAT-like 4-peptidase (MER044589), ovochimase 1 domain 1 (MER022412), epidermal-specific SP-like presumptive peptidase (MER029900), testicular serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), polycellase IA unit 1 (MER 030879), Polycellase-IA Unit 2 (MER030880), Testicular Serine Peptidase 2 (Human Type) (MER033187), Virtual Acrosin-like Peptidase (Homo Sapiens) (MER033253), HAT-like 5 Peptidase (MER028215), Polycellase-3 Unit 1 (MER061763), Polycellase-3 Unit 2 (MER061748), Tryptophan / Serine Protease-like Peptidase (MER056263), Polycellase-2 Unit 1 (MER061777), Mername-A A123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), Brain rescue factor-1 (Human) (MER098873), hCG2041108 (Homo sapiens) (MER099173), Polycellase-2 unit 2 (MER061760), Polycellase-2 unit 3 (MER065694), Mername-AA201 (Peptidase homolog) MER099175,Secretory trypsin-like serine peptidase homolog (MER030000), polycellase-1A unit 3 (MER029880), azulocidine (MER000119), haptoglobin-1 (MER000233), haptoglobin-related protein (MER000235), macrophage-stimulating protein (MER001546), hepatocyte growth factor (MER000185), protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), plasma kallikrein-like protein Protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unassigned peptidase (MER216982), subfamily S1A unassigned peptidase (MER216148), amide phosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidetransferase (MER012158), Mername-AA144 protein (MER021319), asparagine synthase (MER033254), Family C44 non-peptidase homolog (MER159286), Family C44 unassigned peptidase (MER185625), Family C4 4 Unassigned peptidase (MER185626), cesernin 1 (MER045376), cesernin 2 (MER064573), cesernin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamino acid amidase precursor (MER141667), proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit 1i (MER000552),Proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit alpha 6 (MER000557), proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4 (MER000554), proteasome sub, Proteasome subunit Alpha 7 (MER033250), Proteasome subunit Alpha 5 (MER000558), Proteasome subunit Alpha 1 (MER000549), Proteasome subunit Alpha 3 (MER000553), Proteasome subunit XAPC7 (MER004372), Proteasome subunit Beta 3 (MER001710), Proteasome subunit Beta 2 (MER002676), Proteasome subunit Beta 1 (MER000551), Proteasome subunit Beta 4 (MER001711), Mername-AA230 peptidase homolog (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosyl asparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), tapase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (ME Similar to gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), gamma-glutamyltransferase-like protein 3 (MER016970), gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER02 Similar to 6205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyltranspeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamylhydrolase (MER002963), guanine 5''-monophosphate synthase (MER043387),Carbamoyl phosphate synthase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (M EGF-like modules including ER160094), Family C56 non-peptidase homolog (MER177016), Family C56 non-peptidase homolog (MER176613), Family C56 non-peptidase homolog (MER176918), Mucinoid hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), Mucinoid hormone receptor-like 3 (MER037288), Mucinoid hormone receptor-like 1 (MER037278), Mucinoid hormone receptor-like 4 (MER037294), Cadherin EGF LAG7 path G receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), Latrophyllin 2 (MER122199), Latrophyllin-1 (MER126380), Latrophyllin 3 (MER124612), Protocadherin flamingo 2 (MER124239), ETL protein (MER126267), G protein GPR112-type G protein-coupled receptor (MER126114), 7-transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular-induced G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280), GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015),GPR133 (Homo sapiens) type protein (MER159334), GPR110G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER1597 46) PIDD Automated Protein Unit 1 (MER020001), PIDD Automated Protein Unit 2 (MER063690), MUC1 Self-Cutting Mucin (MER074260), Dystroglycan (MER054741), Proprotein Convertase 9 (MER022416), Site 1 Peptidase (MER001948), Furin (MER000375), Proprotein Convertase 1 (MER000376), Proprotein Convertase 2 (MER000377), P Proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355), subfamily S8A nonpeptidase homolog (MER201339), subfamily S8A nonpeptidase homolog (MER191613), subfamily S8A unassigned peptidase (MER191 611), Subfamily S8A unassigned peptidase (MER191612), Subfamily S8A unassigned peptidase (MER191614), Tripeptidyl peptidase I (MER003575), Prolyl oligopeptidase (MER000393), Dipeptidyl peptidase IV (Eukaryotes) (MER000401), Acylaminoacyl peptidase (MER000408), Fibroblast-activating protein alpha subunit (MER000399), PREPL A protein (MER004227), Dipeptidyl peptidase 8 (MER013484), Dipeptidyl peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240),Mername-AA194 presumptive peptidase (MER017353), Mername-AA195 presumptive peptidase (MER017367), Mername-AA196 presumptive peptidase (MER017368), Mername-AA197 presumptive peptidase (MER017371), C14orf29 protein (MER033244), virtual protein (MER033245), virtual esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), virtual protein flj40 219 (MER033212), virtual protein flj37464 (MER033240), virtual protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to mouse chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), Cholinesterase (MER033198), Carboxylesterase D1 (MER033213), Liver Carboxylesterase (MER033220), Carboxylesterase 3 (MER033224), Carboxylesterase 2 (MER033226), Bile Salt-Dependent Lipase (MER033227), Carboxylesterase-Related Protein (MER033231), Neurolysin 3 (MER033232), Neurolysin 4, X-linked (MER033235), Neurolysin 4, Y Binding (MER033236), Esterase D (MER043126), Arylacetamide Deacetylase (MER033237), KIAA1363-like protein (MER033242), Hormone-sensitive lipase (MER033274), Neurolysin 1 (MER033280), Neurolysin 2 (MER033283), Family S9 non-peptidase homolog (MER212939), Family S9 non-peptidase homolog (MER211490), Subfamily S9C unassigned peptidase (MER192341),Family S9 unassigned peptidase (MER209181), Family S9 unassigned peptidase (MER200434), Family S9 unassigned peptidase (MER209507), Family S9 unassigned peptidase (MER209142), Serine carboxypeptidase A (MER000430), Yolk-forming carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), Family S15 unassigned peptidase (MER1994 42) Family S15 unassigned peptidase (MER200437), Family S15 unassigned peptidase (MER212825), Lysosomal Pro-Xaa carboxypeptidase (MER000446), Dipeptidyl peptidase II (MER004952), Thymus-specific serine peptidase (MER005538), Epoxyd hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), Abhydrolase domain-containing protein Protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866) similar to the virtual protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Buren syndrome critical region protein 21 epoxide hydrolase Hydrolytic enzyme (MER031610), epoxide hydrolase (MER031612), virtual protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), virtual protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interaction factor b (MER210738), glycosyl asparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622). Tassase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977),Gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721). Gamma-glutamyltransferase-like protein 3 (MER016970). Similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204). Gamma-glutamyltransferase 1 precursor (Homo sapiens) (M, Similar to ER026205). Mername-AA211 presumptive peptidase (MER026207). Gamma-glutamyltransferase 6 (MER159283). Gamma-glutamyltranspeptidase homolog (chromosome 2, Homo sapiens) (MER037241). Polycystin-1 (MER126824), KIAA1879 protein (MER159329). Polycystic kidney disease 1-like 3 (MER172554). Gamma-glutamylhydrolase (MER002963). Guanine 5″ monophosphate synthetase (MER043387). Carbamoyl phosphate synthase (Homo sapiens type) (MER078640). Dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647). DJ-1 putative peptidase (MER003390). Mername-AA100 putative peptidase (MER014802). Mername-AA101 non-peptidase homolog (MER014803). KIAA0361 protein (Homo sapiens type) (MER042827). F1134283 protein (Homo sapiens) (MER044553). Non-peptidase homolog chromosome 21 open reading frame Mu33 (Homo sapiens) (MER160094). Family C56 nonpeptidase homolog (MER177016), Family C56 nonpeptidase homolog (MER176613). Family C56 nonpeptidase homolog (MER176918). EGF-like module containing mucin-like hormone receptor 2 (MER037230). CD97 antigen (human type) (MER037286). EGF-like module containing mucin-like hormone receptor 3 (MER037288). EGF-like module containing mucin-like hormone receptor 1 (MER037278). EGF-like module containing mucin-like hormone receptor 4 (MER037294). Cadherin EGF LAG 7-transmembrane G receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophyllin 2 (MER122199), latrophyllin-1 (MER126380), latrophyllin 3 (MER124612),Protocadherin Flamingo 2 (MER124239). ETL protein (MER126267). G protein-coupled receptor 112 (MER126114). 7-transmembrane helix receptor (MER125448). Gpr114 protein (MER159320). GPR126 vascular-induced G protein-coupled receptor (MER140015). GPR125 (Homo sapiens) type protein (MER159279). GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280). GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015). GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 autocleaving mucin (MER074260), dystroglycan (MER054741), proprotein converter 9 (MER022416), site 1 peptidase (MER001 948), Furin (MER000375), Proprotein Combatase 1 (MER000376), Proprotein Combatase 2 (MER000377), Proprotein Combatase 4 (MER028255), PACE4 Proprotein Combatase (MER000383), Proprotein Combatase 5 (MER002578), Proprotein Combatase 7 (MER002984), Tripeptidyl Peptidase II (MER000355), Subfamily S8A Non-Peptidase Homolog (MER201339), Subfamily S8A Non-Peptidase Homolog (MER191613), Subfamily S8A Unassigned Peptidase (MER191611), Subfamily S8A Unassigned Peptidase (MER191612),Subfamily S8A unassigned peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotes) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast-activating protein alpha subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 presumptive peptidase (MER017240), Mername-AA194 presumptive peptidase (MER017353), Mername-AA195 presumptive peptidase (MER017367), Mername-AA196 presumptive peptidase (MER017368), Mername-AA197 presumptive peptidase -ase (MER017371), C14orf29 protein (MER033244), virtual protein (MER033245), virtual esterase / lipase / thioesterase (MER047309), protein bat 5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), a protein similar to mouse chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neurolysin 3 (MER033232), neurolysin 4, X-linked (MER033235),Neurolysin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neurolysin 1 (MER033280), neurolysin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211 490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), yolk-forming carboxypeptidase-like protein (MER00549 2) RISC peptidase (MER010960), Family S15 unassigned peptidase (MER199442), Family S15 unassigned peptidase (MER200437), Family S15 unassigned peptidase (MER212825), Lysosomal Pro-Xaa carboxypeptidase (MER000446), Dipeptidyl peptidase II (MER004952), Thymus-specific serine peptidase (MER005538), Epoxydohydrolase Presumptive peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to virtual protein FLJ22408 (MER031608), CGI-58 presumptive peptidase (MER030163), Williams-Buren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612),Hypothetical proteins: flj22408 (epoxydohydrolase) (MER031617), monoglyceride lipase (MER033247), virtual protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0292] Protease enzyme activity can be regulated. For example, certain proteases can be inactivated by the presence or absence of certain drugs (e.g., those that bind to proteases, such as certain small molecule inhibitors). Such proteases can be called “inhibitory proteases.” Exemplary inhibitors for certain proteases are listed in Table 13. For example, NS3 protease can be inhibited by protease inhibitors, including, but not limited to, simeprevir, danoprevir, asunaprevir, silprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In another embodiment, protease activity can be regulated by modulating the expression of the protease itself, for example, by manipulating cells to express the protease using an inducible promoter system (e.g., a Tet On / Off system) or a cell-specific promoter (promoters that can be used to express heterologous proteases are described in more detail in the section titled “Promoters” herein). The protease may also include degron, for example, any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0293] Protease enzyme activity can also be regulated through the selection of specific protease cleavage sites. For example, protease cleavage sites may be selected and / or manipulated so that the sequence exhibits a desired cleavage rate by the desired protease, e.g., a reduced cleavage rate relative to the endogenous sequence of a substrate spontaneously cleaved by the desired protease. Another example is selecting and / or manipulating protease cleavage sites so that the sequence exhibits a desired cleavage rate in a cell-state-specific manner. For example, various cell states (e.g., after cell signaling, e.g., immune cell activation) can affect the expression and / or localization of specific proteases. As an illustrative example, ADAM17 protein levels and localization are known to be affected by signaling, e.g., through the protein kinase C (PKC) signaling pathway (e.g., activation by the PKC activator phorbol-12-myristate-13-acetat [PMA]). Therefore, protease cleavage sites can be selected and / or manipulated so that the cleavage of the protease cleavage site and the subsequent release of effector molecules are increased or decreased, as desired, depending on the protease properties (e.g., expression and / or localization) of a particular cellular state. As another example, protease cleavage sites (particularly in combination with specific membrane anchoring domains) can be selected and / or manipulated for optimal protein expression of chimeric proteins.

[0294] Cell membrane anchoring domain The membrane-cleavable chimeric proteins provided herein contain a cell membrane anchoring domain (referred to as "MT" in formulas SC-MT or MT-CS). Generally, the cell membrane anchoring domain may be any amino acid sequence motif that is localized to the cell membrane of a cell expressing the chimeric protein (e.g., inserted into it) or otherwise capable of orienting the chimeric protein to associate with it. The cell membrane anchoring domain may be a transmembrane-intracellular domain. The cell membrane anchoring domain may be a transmembrane domain. The cell membrane anchoring domain may be an endogenous membrane protein domain (e.g., a transmembrane domain). The cell membrane anchoring domain may be derived from type I, type II, or type III transmembrane proteins. The cell membrane anchoring domain may contain a post-translational modification tag, or a post-translationally modifiable motif for modifying the chimeric protein to include the post-translational modification tag, where the post-translational modification tag enables association with the cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchoring domains (e.g., GPI lipid anchors, myristoylation tags, or palmitoylation tags). Examples of cell membrane anchoring domains include, but are not limited to, transmembrane-intracellular domains and / or transmembrane domains derived from PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. Cell membrane anchoring domains may be cell surface receptors or their cell membrane binding portions. Sequences of exemplary cell membrane anchoring domains are provided in Table 14.

[0295] (Table 14) TIFF2026514757000037.tif53170

[0296] Generally, for all membrane-cleavable chimeric proteins described herein, the cell membrane anchoring domain is either (1) the C-terminus of the protease cleavage site and the N-terminus of any intracellular domain, if present (in other words, the cell membrane anchoring domain is between the protease cleavage site and, if present, the intracellular domain), or (2) the N-terminus of the protease cleavage site and the C-terminus of any intracellular domain, if present (also between the protease cleavage site and, if present, the intracellular domain with reversed domain orientation). In embodiments characterized by a degron associated with a chimeric protein, the degron domain is specifically a terminal cytoplasm-directed domain with respect to cell membrane anchoring (in other words, the cell membrane anchoring domain is between the protease cleavage site and the degron). The cell membrane anchoring domain can be linked to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the protease cleavage site. The cell membrane anchoring domain, if present, can be linked to the intracellular domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring domain or the intracellular domain. The cell membrane anchoring domain, if present, can be linked to the degron by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring domain or the degron. The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a mobile linker (e.g., a Gly-Ser-Gly sequence).Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG), A(EAAAK)3A, and Whitlow linkers (e.g., "KEGS" linkers, e.g., amino acid sequence KESGSVSSEQLAQFRSLD, eGK linkers, e.g., amino acid sequence EGKSSGSGSESKST, LR1 linkers, e.g., amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ, and linkers described in detail in published U.S. Patent No. 5,990,275 incorporated herein by reference). Additional polypeptide linkers include SGGGGSGGGGSG, TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, and GGGSGGGGSGGGSLQ. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art.

[0297] Generally, the cell membrane anchoring domain is oriented so that the secreted effector molecule and protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane, and the protease cleavage site is cleaved by its respective protease, allowing the effector molecule to be released (secreted) into the extracellular space.

[0298] Deglon system and domain In some embodiments, any of the proteins described herein may include a degron domain, which may include, but is not limited to, a cytokine, CAR, protease, transcription factor, promoter or component of a promoter system (e.g., ACP), and / or a membrane-cleavable chimeric protein described herein. Generally, a degron domain may be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation via a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs the degradation of a ubiquitin-mediated degron fusion protein.

[0299] The degron domain may be a cerebron (CRBN) polypeptide substrate domain capable of binding to CRBN in response to an immunomodulatory drug (IMiD), including, but not limited to, IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof capable of drug-inducible binding to CRBN. The CRBN polypeptide substrate domain may be a chimeric fusion product of a natural CRBN polypeptide sequence, such as an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL. The degron domain, and in particular the CRBN degron system, are described in more detail in International Publication No. 2019 / 089592Al, which is incorporated herein by reference for all purposes. Other examples of degron domains include, but are not limited to, HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα; LQMLPESEDEESYDTESEFTEFTEDELPYDDGSLQMLPESEDEESYDTESEFTEFTEDELPYDD), GRR (residues 352-408 of human p105; EIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPH), and DRR (residues 210-295 of yeast Cdc34; IDDENGSVILQDDDYDDGNNHIPFEDDDV YNYNDNDDDDERIEFEDDDDDDDDSIDNDSVMDRKQPHKAEDESEDVEDVERVSKKD), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B; e.g., IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDNDSVMDRKQPHKAEDESEDVEDVERVSKKD), RPB (four copies of residues 1688-1702 of yeast RPB;IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDNDSVMDRKQPHKAEDESEDVEDVERVSKKD), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein; PESMREEYRKEGSSLLTEVETPGSPESMREEYRKEGSSLLTEVETPGSPESMREEYRKE), NS2 (three residues 79-93 of influenza A virus NS protein) Copy;LIEEVRHRLKTTENSGSLIEEVRHRLKTTENSGSLIEEVRHRLKTTENSGS), ODC (residues 106-142 of ornithine decarboxylase;FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV), Nek2A, mouse ODC (residues 422-461,SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), APC / C degron, COP1 Examples include E3 ligase-binding degron motifs, CRL4-Cdt2-binding PIP degrons, actinphylline-binding degrons, KEAP1-binding degrons, KLHL2 and KLHL3-binding degrons, MDM2-binding motifs, N-degrons, hydroxyproline modifications in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degrons, SCF ubiquitin ligase-binding phosphodegrons, plant hormone-dependent SCF-LRR-binding degrons, DSGxxS phosphate-dependent degrons, Siah-binding motifs, SPOP SBC docking motifs, or PCNA-binding PIP boxes.

[0300] Modulated degradation can be drug-inducible. Drugs capable of mediating / modulating degradation can be small molecule compounds. Drugs capable of mediating / modulating degradation may include “immunomodulatory drugs” (IMiDs). Generally, as used herein, IMiD refers to a class of small molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is a known target of IMiDs, and binding of an IMiD to CRBN or a CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, leading to the degradation of proteins having a CRBN polypeptide substrate domain (e.g., secreted effector molecules or other proteins of interest as described herein). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. IMiDs may be FDA-approved drugs.

[0301] The proteins described herein may contain a degron domain (e.g., referred to as "D" in formulas SC-MT-D or D-MT-CS for membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of chimeric proteins does not occur. Following the expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, causing the effector molecule to be released (secreted) into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs the ubiquitin-mediated degradation of the chimeric protein, causing the secretion of the effector molecule to be reduced or eliminated. Generally, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is a terminal cytoplasm-oriented domain, particularly relative to the cell membrane anchoring domain, e.g., the C-terminal domain in formula SC-MT-D or the N-terminal domain in formula D-MT-CS. The degron domain can be linked to the cell membrane anchoring domain by polypeptide linkers, i.e., polypeptide sequences that are not generally considered to be part of the cell membrane anchoring domain or the degron domain. The polypeptide linker may be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker may be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG), A(EAAAK)3A, and Whitlow linkers (e.g., "KEGS" linkers, e.g., amino acid sequence KESGSVSSEQLAQFRSLD, eGK linkers, e.g., amino acid sequence EGKSSGSGSESKST, LR1 linkers, e.g., amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ, and linkers described in detail in published U.S. Patent No. 5,990,275, incorporated herein by reference).Additional polypeptide linkers include SGGGGSGGGGSG, TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, and GGGSGGGGSGGGSLQ. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art. Generally, the degron is oriented in relation to the cell membrane anchoring domain, and after localization to the cell membrane, the degron is exposed to the cytosol, allowing the degron domain to mediate degradation (e.g., cytosol and exposure to the cytosol) and to mediate ubiquitin-mediated degradation.

[0302] For degron fusion proteins, the degron domain can be the N-terminus or C-terminus of the target protein, for example, an effector molecule. The degron domain can be linked to the target protein by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the target protein or the degron domain. The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS...

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to human V-set immunoglobulin domain 2 (VSIG2), which includes a heavy chain variable (VH) region and a light chain variable (VL) region, The VH comprises a VH complementary region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1 or 2 and a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3 or 4. The VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6 and a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and (i) The VH includes a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and the VL includes a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) The VH includes a VH complementary region 3 (CDRH3) having one amino acid sequence from SEQ ID NOs. 67 to 87, and the VL includes a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NOs. 8 or 9, and optionally The VH is an isolated antibody or antigen-binding fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 88-107, and optionally, the VL is an isolated antibody or antigen-binding fragment having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:

15.

2. A) The CDRH3 has the amino acid sequence of SEQ ID NO: 5, and the CDRL3 has the amino acid sequence of SEQ ID NO: 9, or B) The CDRH3 has the amino acid sequence of SEQ ID NO: 67, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or C) The CDRH3 has the amino acid sequence of SEQ ID NO: 68, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or D) The CDRH3 has the amino acid sequence of SEQ ID NO: 67, and the CDRL3 has the amino acid sequence of SEQ ID NO: 9, or E) The CDRH3 has the amino acid sequence of SEQ ID NO: 68, and the CDRL3 has the amino acid sequence of SEQ ID NO: 9, or F) The CDRH3 has the amino acid sequence of SEQ ID NO: 69, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or G) The CDRH3 has the amino acid sequence of SEQ ID NO: 69, and the CDRL3 has the amino acid sequence of SEQ ID NO: 9, or H) The CDRH3 has the amino acid sequence of SEQ ID NO: 70, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or I) The CDRH3 has the amino acid sequence of SEQ ID NO: 71, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or J) The CDRH3 has the amino acid sequence of SEQ ID NO: 72, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or K) The CDRH3 has the amino acid sequence of SEQ ID NO: 73, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or L) The CDRH3 has the amino acid sequence of SEQ ID NO: 74, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or M) The CDRH3 has the amino acid sequence of SEQ ID NO: 75, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or N) The CDRH3 has the amino acid sequence of SEQ ID NO: 76, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or O) The CDRH3 has the amino acid sequence of SEQ ID NO: 77, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or P) The CDRH3 has the amino acid sequence of SEQ ID NO: 78, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Q) Does CDRH3 have the amino acid sequence of SEQ ID NO: 79, and does CDRL3 have the amino acid sequence of SEQ ID NO: 8, or R) The CDRH3 has the amino acid sequence of SEQ ID NO: 80, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or S) The CDRH3 has the amino acid sequence of SEQ ID NO: 81, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or T) The CDRH3 has the amino acid sequence of SEQ ID NO: 82, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or U) The CDRH3 has the amino acid sequence of SEQ ID NO: 83, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or V) The CDRH3 has the amino acid sequence of SEQ ID NO: 84, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or W) The CDRH3 has the amino acid sequence of SEQ ID NO: 85, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or X) The CDRH3 has the amino acid sequence of SEQ ID NO: 86, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8, or Y) The antibody or antigen-binding fragment according to claim 1, wherein CDRH3 has the amino acid sequence of SEQ ID NO: 87 and CDRL3 has the amino acid sequence of SEQ ID NO:

8.

3. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the antibody or antigen-binding fragment is an antigen-binding fragment, optionally comprising an F(ab) fragment, an F(ab') fragment, or a single-strand variable fragment (scFv), optionally comprising a single-strand variable fragment (scFv), optionally comprising VH and VL separated by a peptide linker, optionally comprising the structure VH-L-VL or VL-L-VH, where VH is a heavy-chain variable domain, L is the peptide linker, and VL is a light-chain variable domain, optionally comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 37, and optionally comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 108 to 132.

4. A chimeric protein comprising an antibody or its antigen-binding fragment and a heterologous molecule or portion according to any one of claims 1 to 3, wherein the chimeric protein is an antibody-drug conjugate, the heterologous molecule or portion comprises a therapeutic agent, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or portion comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. Optionally, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit the immune response, and optionally, the one or more intracellular inhibitory domains are PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KI R3DL2, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, SIGLEC-10, PECAM-1, CD72, IRTA2, IRTA4, NKIR, T LT1, PCDHGC3, MPZL1, FCGR2B, SIGLEC-6, MPIG6B, SIGLEC-12, LIR8, IRTA1, KIR2DL4, KIR2DL5, SIGLEC-7 Alternatively, the ICD derived from FCRH3 may optionally include the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSSEYASVQVPRK (Sequence No. 139), or the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSSEYASVQVPRK (Sequence No. 139), or the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSSEYASVQVPRK (Sequence No. 139). A chimeric protein comprising an amino acid sequence identical to 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%, at least 99%, or 100% of one or more of the sequences in (No. 139), wherein the intracellular inhibitory domain further comprises an enzyme inhibitory domain and / or an additional intracellular inhibitory co-signaling domain, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, and optionally the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 52.

5. A manipulated expression system, a. A first nucleic acid sequence encoding a first CAR, wherein the first CAR is i. A first extracellular antigen-binding domain that binds to an antigen selected from the group consisting of CEACAM5, CEA, CEACAM1, and CEACAM6, ii. The first transmembrane domain, iii. A first nucleic acid sequence comprising one or more intracellular signaling domains, and b. An engineered expression system comprising a second nucleic acid sequence encoding a second CAR, wherein the second CAR comprises an antibody or antigen-binding fragment according to any one of claims 1 to 3, or a chimeric protein according to claim 4.

6. The manipulated expression system according to claim 5, wherein the first CAR includes a first spacer between the first extracellular antigen-binding domain and the first transmembrane domain, optionally comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52, optionally comprising the amino acid sequence of SEQ ID NO: 5, and / or the second CAR includes a second spacer between the second extracellular antigen-binding domain and the second transmembrane domain, optionally comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52, optionally comprising the amino acid sequence of SEQ ID NO:

50.

7. The one or more intracellular signaling domains of the first CAR are: CD3 zeta chain intracellular signaling domain, CD3 epsilon chain intracellular signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8 intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, NKp46 intracellular signaling domain, NKp30 intracellular signaling domain, NKp44 intracellular signaling domain, NKG2D intracellular signaling domain Selected from the group consisting of the CD226 intracellular signaling domain and the CD160 intracellular signaling domain, the first CAR optionally includes the CD28 intracellular signaling domain and the CD3 zeta chain intracellular signaling domain, and / or optionally the first transmembrane domain includes the CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain, CD3 zeta chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, and ICOS The manipulated expression system according to claim 5 or 6, wherein the first CAR is selected from the group consisting of transmembrane domains, CTLA-4 transmembrane domain, LAX transmembrane domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane domain, TIGIT transmembrane domain, 2B4 transmembrane domain, and BTLA transmembrane domain, and optionally comprises a CD28 transmembrane domain.

8. The manipulated expression system according to any one of claims 5 to 7, wherein the first and second nucleic acid sequences are contained in a single expression vector, or the first nucleic acid sequence is contained in a first expression vector and the second nucleic acid sequence is contained in a second expression vector.

9. The first antigen-binding domain is bound to one of CEACAM5, CEACAM6, or CEACAM1, and the first antigen-binding domain includes a heavy chain variable domain (VH) and a light chain variable domain (VL), optionally i) The VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of hMN14 VH, the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of hMN14 VL, the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of hMN14 VH, and the VL comprises hMN14 The first antigen-binding domain comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of hMN14 VH, and the VL comprises the amino acid sequence of hMN14 VL, and optionally, the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or ii) The VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of BW431 / 26 VH, and optionally, the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of BW431 / 26 VL, the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of BW431 / 26 VH, and the VL comprises BW431 / 26 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of BW431 / 26 VH, the VL comprises the amino acid sequence of BW431 / 26 VL, or iii) The first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of A5B7 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of A5B7 VL, and the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of A5B7 VH, and the VL comprises A5B7 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of A5B7 VH, and the VL comprises the amino acid sequence of A5B7 VL. iv) The VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of MFE23 VH, the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of MFE23 VL, the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of MFE23 VH, and the VL comprises MFE23 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of MFE23 VH, the VL comprises the amino acid sequence of MFE23 VH, or v) The VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of hMFE23 VH, the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of hMFE23 VL, the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of hMFE23 VH, and the VL comprises hMFE23 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of hMFE23 VH, the VL comprises the amino acid sequence of hMFE23 VL, or vi) The VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of FM4 VH, the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of FM4 VL, the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of FM4 VH, and the VL is FM4 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of FM4 VH, the VL comprises the amino acid sequence of FM4 VL, or vii) The VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of cibisatamab HC, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of cibisatamab LC, and the antibody or its antigen-binding fragment is humanized, and optionally, the VH has 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% identity with the amino acid sequence of the VH of cibisatamab HC. The amino acid sequence is included, and the VL includes an amino acid sequence having 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% identity with the amino acid sequence of the VL of sibisatamab LC, and optionally, the VH includes the amino acid sequence of the VH of sibisatamab HC, and the VL includes the amino acid sequence of the VL of sibisatamab LC, or viiii) The VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of tusamitamab HC, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of tusamitamab LC, and the antibody or its antigen-binding fragment is humanized, and optionally the VH comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, and at least the amino acid sequence of the VH of tusamitamab HC. The amino acid sequence comprises 97%, at least 98%, or at least 99% identity, and the LC comprises an amino acid sequence that comprises 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% identity with the amino acid sequence of the VL of tusamitamab LC, and optionally, the HC comprises the amino acid sequence of the VH of tusamitamab HC, and the VL comprises the amino acid sequence of the VL of tusamitamab LC, or ix) The first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of MRG1 VH, and the VL comprises VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of MRG1 VL, and the antibody or its antigen-binding fragment is humanized, and optionally, the VH comprises an amino acid sequence having 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% identity with the amino acid sequence of MRG1 VH, and the VL comprises MRG1 The amino acid sequence comprises an amino acid sequence having 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% identity with the amino acid sequence of VL, and optionally, the VH comprises the amino acid sequence of MRG1 VH, the VL comprises the amino acid sequence of MRG1 VL, or x) The first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the VH complementarity region 1 (CDRH1), VH complementarity region 2 (CDRH2), and VH complementarity region 3 (CDRH3) of tinuririmab HC, and the VL comprises the VL complementarity region 1 (CDRL1), VL complementarity region 2 (CDRL2), and VL complementarity region 3 (CDRL3) of tinuririmab LC, wherein the antibody or its antigen-binding fragment is humanized, and optionally, the VH has at least 90%, at least 91%, at least 92%, at least 93%, and at least 94% of the amino acid sequence of the VH of tinuririmab HC. The manipulated expression system according to any one of claims 5 to 8, comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the VL, wherein the VL comprises an amino acid sequence having 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% identity with the amino acid sequence of the VL of chinuririmab LC, and optionally, the VH comprises the amino acid sequence of the VH of chinuririmab HC, and the VL comprises the amino acid sequence of the VL of chinuririmab LC.

10. a. The fourth nucleotide sequence encoding the first cytokine, b. The manipulated expression system according to any one of claims 5 to 9, further comprising a fifth nucleotide sequence encoding a second cytokine.

11. At least one of the first and second cytokines is a controlled-release cytokine, and optionally, the controlled-release cytokine has the following formula: S-C-MT or MT-C-S During the ceremony, S contains secretory effector molecules, C includes a protease cleavage site, and MT includes a cell membrane anchoring domain, Optionally, the protease cleavage site is cleaved by ADAM10 and / or ADAM17, and optionally, the protease cleavage site comprises the amino acid sequence PRAEALKGG or VTPEPIFSLI. Optionally, the cell membrane anchoring domain includes a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA. The manipulated expression system according to any one of claims 5 to 10, wherein the cell membrane anchoring domain optionally comprises a B7-1 transmembrane domain containing the B7-1 transmembrane domain amino acid sequence described in Table 14.

12. The manipulated expression system according to any one of claims 5 to 11, wherein the first cytokine is IL15, optionally comprising the amino acid sequence of IL15 listed in Table 10, and optionally being controlled-release IL15 (crIL15).

13. The manipulated expression system according to any one of claims 5 to 12, wherein the second cytokine is IL21, optionally comprising an amino acid sequence listed in Table 10, and optionally being controlled-release IL21 (crIL21).

14. An antibody or antigen-binding fragment according to any one of claims 1 to 3, a chimeric protein according to claim 4, or an engineered nucleic acid encoding an engineered expression system according to any one of claims 5 to 13, or optionally a vector comprising the engineered nucleic acid.

15. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or a chimeric protein according to claim 4, an engineered nucleic acid according to claim 14, or an engineered expression system according to any one of claims 5 to 13, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

16. An isolated cell or population of manipulated cells comprising the manipulated nucleic acid according to claim 14, the composition according to claim 15, the manipulated expression system according to any one of claims 5 to 13, the antigen-binding fragment according to any one of claims 1 to 3, and the chimeric protein according to claim 4, wherein the cell or population of cells optionally comprises T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphocytes, mast cells, eosinophils, basophils, neutrophils, A population of isolated or engineered cells selected from the group consisting of bone marrow cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells, wherein, optionally, the cells are autologous or allogeneic, and optionally, the cells or population of cells further include one or more tumor-targeting chimeric receptors expressed on the cell surface, and optionally, at least one of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

17. A pharmaceutical composition, a. An effective amount of the cells or manipulated cell population described in claim 16, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, b. A gene-modified cell expressing the antigen-binding fragment described in any one of claims 1 to 3 or the chimeric protein described in claim 4, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. Optionally, the pharmaceutical composition is for treating and / or preventing tumors.

18. A method for inhibiting the cell-mediated immune response against normal cells in a target, a. The composition according to claim 15 for a therapeutically effective dose, b. Any of the cells described in claim 16, wherein the isolated cells or cell population expresses any of the cells that express the chimeric protein containing the inhibitory CAR described in claim 4. c. The composition according to claim 17 is administered to a subject, Optionally, the method further comprises stimulating a cell-mediated immune response against tumor cells in the subject, wherein the isolated cells or population of cells further comprises one or more tumor-targeted chimeric receptors expressed on the cell surface, optionally, at least one of the one or more tumor-targeted chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor, optionally, the normal cells comprises human VSIG2, optionally, the human VSIG2 is expressed on the surface of the normal cells, optionally, the normal cells are healthy (e.g., non-tumor) epithelial cells.

19. A method for treating a subject having a tumor, comprising administering either the composition according to claim 15, or the cells according to claim 16, or the composition according to claim 17, in a therapeutically effective dose.

20. A kit for treating and / or preventing tumors, a. The chimeric protein according to claim 4, and optionally the kit further comprising written instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject. b. The cells or population of cells according to claim 16, and optionally the kit further comprising written instructions for using the cells to treat and / or prevent tumors in a subject. c. The vector according to claim 14, and optionally the kit further comprising written instructions for using the vector to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject. d. A composition according to claim 15 or claim 17, and optionally the kit further comprising written instructions for using the composition to treat and / or prevent a tumor in a subject. e. A kit comprising the engineered nucleic acid described in claim 14, and optionally further comprising written instructions for using the nucleic acid to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

Images