Antigen-binding domains and methods of use thereof

Abstract

Provided herein are antibodies and antigen binding fragments thereof specific for V-set Immunoglobulin domain containing 2 (VSIG2). Also provided herein are cells, nucleic acids, vectors, compositions, and methods directed to antibodies or antigen-binding domains thereof specific for VSIG2.

Description

ANTIGEN-BINDING DOMAINS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 496,640, filed April 17, 2023, and to U.S. Provisional Application No. 63 / 590,361, filed October 13, 2023, each of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted via EFS- Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Month XX, 20XX, is named XXXXXUS_sequencelisting.xml, and is X, XXX, XXX bytes in size.BACKGROUND

[0003] Chimeric antigen receptor (CAR) based adoptive cell therapies used to redirect the specificity and function of immunoresponsive cells, such as T cells, have 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 TranslMed. (5): 177ra38 (2013)). CAR T cells have been shown to induce complete remission in patients with CD19-expressing malignancies for whom chemotherapies have led to drug resistance and tumor progression. The success of CD 19 CAR therapy provides optimism for treating other malignancies, such as solid tumors.

[0004] One challenge to developing CAR therapy for solid tumors is the lack of suitable targets. The ability to identify appropriate CAR targets is important to effectively targeting and treating the tumor without damaging normal cells that express the same target antigen. Thus, there remains a need for CAR-based solid tumor therapies that target tumor cells without targeting normal cells or tissues, such as therapies for the treatment of colorectal cancer (CRC). SUMMARY

[0005] Provided herein is an isolated antibody or antigen binding fragment thereof that specifically binds to human V-set Immunoglobulin domain containing 2 (VSIG2) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, and a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3; wherein the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, and a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7; and wherein: (i) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and theVL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of any one of SEQ ID NO: 67-87, and the VL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0006] Also provided herein is an isolated antibody or antigen binding fragment thereof that specifically binds to human V-set Immunoglobulin domain containing 2 (VSIG2) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having an amino acid sequence of SEQ ID NO: 2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7; wherein: (i) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and the VL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of any one of SEQ ID NO: 67-87, and the VL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0007] In some aspects, the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 88-107. In some aspects, the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some aspects, 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 theamino 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) the CDRH3 has the amino acid sequence of SEQ ID NO: 79, and the CDRL3 has 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 CDRH3 has the amino acid sequence of SEQ ID NO: 87, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8. In some aspects, 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.

[0008] Also provided herein is an isolated antibody or antigen binding fragment thereof that specifically binds to human V-set Immunoglobulin domain containing 2 (VSIG2) comprising 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 the amino acid sequence selected 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] Also provided herein is an isolated antibody or antigen binding fragment thereof that specifically binds to human V-set Immunoglobulin domain containing 2 (VSIG2) comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VL has an amino acid sequence of SEQ ID NO: 15. In some aspects, VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107.

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

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

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

[0013] Also provided herein is a chimeric protein comprising an antibody or antigen binding fragment thereof of any of the isolated antibodies or antigen binding fragments provided herein, and a heterologous molecule or moiety. In some aspects, the chimeric protein is an antibodydrug conjugate, and wherein the heterologous molecule or moiety comprises a therapeutic agent. In some aspects, the chimeric protein is a chimeric antigen receptor (CAR), and wherein 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 aspects, the CAR comprises a transmembrane domain. In some aspects, the CAR comprises one or more intracellular signaling domains. In some aspects, the CAR is an activating In some aspects, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some aspects, the one or more intracellular inhibitory domains comprise an ICD derived from the PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A , LAG3, LAIR1, SIRPa, 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 aspects, the intracellular inhibitory domain comprises the amino acid sequenceVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139) or an amino acid sequence with 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 to one or more of the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139).

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

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

[0017] Also provided herein is an expression vector comprising any one of the engineered nucleic acids provided herein or any one of the engineered expression systems provided herein.

[0018] Also provided herein is a composition comprising any one of the engineered nucleic acids or expression vectors provided herein or any one of the engineered expression systems provided herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

[0019] Also provided herein is a method of making an engineered cell, comprising transducing an isolated cells with any one of the engineered nucleic acids or expression vectors provided herein or any one of the engineered expression systems provided herein.

[0020] Also provided herein is an isolated cell comprising any one of the engineered nucleic acids or expression vectors provided herein.

[0021] Also provided herein is a population of engineered cells expressing any one of the engineered nucleic acids or expression vectors provided herein or any one of the engineered expression systems provided herein.

[0022] An isolated cell comprising any one of the antibodies or antigen binding fragments thereof provided herein or any one of the chimeric proteins provided herein or any one of the engineered expression systems provided herein.

[0023] Also provided herein is a population of engineered cells expressing any one of the antibodies or antigen binding fragments thereof provided herein or any one of the chimeric proteins provided herein.

[0024] In some aspects, the chimeric protein is recombinantly expressed. In some aspects, the chimeric protein is expressed from a vector or a selected locus from the genome of the cell. In some aspects, the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface. In some aspects, each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0025] In some aspects, the cell or population of cells is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0026] In some aspects, the cell is autologous. In some aspects, the cell is allogeneic.

[0027] Also provided herein is a pharmaceutical composition comprising an effective amount of any one of the cells or population of engineered cells provided herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

[0028] Also provided herein is a pharmaceutical composition comprising an effective amount of genetically modified cells expressing any one of the antibodies or antigen binding fragments thereof provided herein or any one of the chimeric proteins provided herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof. In some aspects, the pharmaceutical composition is for treating and / or preventing a tumor.

[0029] Also provided herein is a method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any one of the compositions, the cells, or the pharmaceutical compositions provided herein.

[0030] Also provided herein is a method of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any one of the compositions, the cells, or the pharmaceutical compositions provided herein.

[0031] In some aspects, the method comprises administering to the subject any one of the cells provided herein, wherein the isolated cell or population of cells express the chimeric protein comprising any one of the activating CARs provided herein.

[0032] Also provided herein is a method of inhibiting a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any one of the compositions, the cells, or the pharmaceutical compositions provided herein.

[0033] In some aspects, the method comprises administering to the subject any one of the cells provided herein, wherein the isolated cell or population of cells express the chimeric protein comprising any one of the inhibitory CARs provided herein.

[0034] Also provided herein is a method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of any one of the compositions, the cells, or the pharmaceutical compositions provided herein.

[0035] Also provided herein is a kit for treating and / or preventing a tumor, comprising any one of the chimeric proteins provided herein. In some aspects, the kit further comprises written instructions for using the chimeric protein for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0036] Also provided herein is a kit for treating and / or preventing a tumor, comprising any one of the cells or population of cells provided herein. In some aspects, the kit further comprises written instructions for using the cell for treating and / or preventing a tumor in a subject.

[0037] Also provided herein is a kit for treating and / or preventing a tumor, comprising any one of the engineered nucleic acids provided herein. In some aspects, the kit further comprises written instructions for using the nucleic acid for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0038] Also provided herein is a kit for treating and / or preventing a tumor, comprising any one of the vectors provided herein. In some aspects, the kit further comprises written instructions for using the vector for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0039] Also provided herein is a kit for treating and / or preventing a tumor, comprising any one of the compositions provided herein. In some aspects, the kit further comprises written instructions for using the composition for treating and / or preventing a tumor in a subject.

[0040] Also provided herein, is an engineered expression system comprising: a first nucleic acid sequence encoding a first CAR, wherein the first CAR comprises: a first extracellular antigenbinding domain that binds an antigen selected from the group consisting of CEACAM5, CEA, CEACAM1, and CEACAM6; a first transmembrane domain; and one or more intracellularsignaling domains; and a second nucleic acid sequence encoding a second CAR, wherein the second CAR comprises any one of the antibody or antigen binding fragments described herein, any one of the chimeric proteins described herein.

[0041] In some aspects of the engineered expression system described herein, the first CAR comprises a first spacer between the first extracellular antigen-binding domain and the first transmembrane domain. In some aspects of the engineered expression system described herein, the first spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52. In some aspects of the engineered expression system described herein, the first spacer comprises the amino acid sequence of SEQ ID NO: 50. In some aspects of the engineered expression system described herein, the second CAR comprises a second spacer between the second extracellular antigen-binding domain and the second transmembrane domain. In some aspects of the engineered expression system described herein, the second spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52. In some aspects of the engineered expression system described herein, the second spacer comprises the amino acid sequence of SEQ ID NO: 50. In some aspects of the engineered expression system described herein, the one or more intracellular signaling domains of the first CAR are selected from the group consisting of: a CD3zeta-chain intracellular signaling domain, a CD3epsilon- chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD1 la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, an NKp46 intracellular signaling domain, an NKp30 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKG2D intracellular signaling domain, a CD226 intracellular signaling domain, and a CD 160 intracellular signaling domain. In some aspects of the engineered expression system described herein, the first CAR comprises a CD28 intracellular signaling domain and a CD3zeta-chain intracellular signaling domain. In some aspects of the engineered expression system described herein, the first transmembrane domain are selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- IBB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4transmembrane 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 aspects of the engineered expression system described herein, the first CAR comprises a CD28 transmembrane domain. In some aspects of the engineered expression system described herein, the first and second nucleic acid sequences are comprised within a single expression vector. In some aspects of the engineered expression system described herein, the first nucleic acid sequence is comprised within a first expression vector and the second nucleic acid sequence is comprised within a second expression vector. In some aspects of the engineered expression system described herein, the first antigen-binding domain binds CEACAM5. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1) , a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an hMN14 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an hMN14 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of an hMN14 VH, and 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 to the amino acid sequence of an hMN 14 VL. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence of an hMN14 VH, and the VL comprises the amino acid sequence of an hMN14 VL. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a BW431 / 26 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a BW431 / 26 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some embodiments, the VH comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of a BW431 / 26 VH, and 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 to the amino acid sequence of a BW431 / 26 VL. In some aspects of the CAR or engineered expression system described herein, the VH comprises the amino acid sequence of a BW431 / 26 VH, and the VL comprises the amino acid sequence of a BW431 / 26 VL. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an A5B7 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an A5B7 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of an A5B7 VH, and 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 to the amino acid sequence of an A5B7 VL. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence of an A5B7 VH, and the VL comprises the amino acid sequence of an A5B7 VL. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an MFE23 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an MFE23 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the CAR or engineered expression system described herein, 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 to the amino acid sequence of an MFE23 VH, and 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 to the amino acid sequence of an MFE23 VL. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence ofan MFE23 VH, and the VL comprises the amino acid sequence of an MFE23 VH. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an hMFE23 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an hMFE23 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of an hMFE23 VH,and 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 to the amino acid sequence of an hMFE23 VL,. In some embodiments, the VH comprises the amino acid sequence of an hMFE23 VH, and the VL comprises the amino acid sequence of an hMFE23 VL. In some aspects of the engineered expression system described herein, the first antigenbinding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an FM4 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an FM4 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of an FM4 VH, and 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 to the amino acid sequence of an FM4 VL. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence of an FM4 VH, and the VL comprises the amino acid sequence of an FM4 VL. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a cibisatamab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2),and a VL complementarity region 3 (CDRL3) of a cibisatamab LC, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of the VH of a cibisatamab HC, and 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 to the amino acid sequence of a cibisatamab LC. In some aspects of the engineered expression system described herein, the HC comprises the amino acid sequence of a cibisatamab HC and the LC comprises the amino acid sequence of the VL of a cibisatamab LC. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a tusamitamab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a tusamitamab LC, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, the 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 to the amino acid sequence of a tusamitamab HC, and the 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 to the amino acid sequence a tusamitamab LC. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence of the VH of a tusamitamab HC, and the VL comprises the amino acid sequence the VL of a tusamitamab LC. In some aspects of the engineered expression system described herein, the first antigen-binding domain binds CECAM1. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an MRG1 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an MRG1 VL, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, the VH comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of an MRG1 VH, and 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 to the amino acid sequence of an MRG1 VL. In some aspects of the engineered expression system described herein, the VH comprises the amino acid sequence of an MRG1 VH, and the VL comprises the amino acid sequence of an MRG1 VL. In some aspects of the engineered expression system described herein, the first antigen-binding domain binds CEACAM6. In some aspects 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), wherein the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a tinurilimab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a tinurilimab LC, and wherein the antibody or antigen binding fragment thereof is humanized. In some aspects of the engineered expression system described herein, 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 to the amino acid sequence of the VH of a tinurilimab HC and 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 to the amino acid sequence of the VL of a tinurilimab LC. In some aspects of the engineered expression system described herein, the VL comprises the amino acid sequence of the VH of a tinurilimab HC and the VL comprises the amino acid sequence of the VL of a tinurilimab LC. In some aspects, the engineered expression system further comprises: a fourth nucleotide sequence encoding a first cytokine; and a fifth nucleotide sequence encoding a second cytokine. In some aspects of the engineered expression system described herein, at least one of the first and the second cytokines is a controlled release cytokine. In some aspects of the engineered expression system described herein, the controlled release cytokine has the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule; C comprises a protease cleavage site; and MT comprises a cell membrane tethering domain, optionally wherein the protease cleavage site is cleaved by ADAM10 and / or ADAM17, optionally wherein the protease cleavage site comprises the amino acid sequence of PRAEALKGG or VTPEPIFSLI, optionally wherein the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA, optionallywherein the cell membrane tethering domain comprises a B7-1 transmembrane domain comprising the amino acid sequence set forth in Table 14. In some aspects of the engineered expression system described herein, the first cytokine is IL 15, optionally wherein the IL 15 comprises the amino acid sequence of IL15 set forth in Table 10. In some aspects of the engineered expression system described herein, the IL15 is controlled-release IL15 (crIL15). In some aspects of the engineered expression system described herein, the second cytokine is IL21, optionally wherein the IL21 comprises the amino acid sequence set forth in Table 10, optionally wherein the IL21 is controlled-release IL21 (crIL21). In some aspects of the engineered expression system described herein, the first or second cytokine comprises an amino acid sequence set forth in Table 10. In some aspects of the engineered expression system described herein, the first or second cytokine is encoded by a nucleic acid sequence set forth in any one of the nucleic acid sequences set forth in Table 10.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0043] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.

[0044] FIG. 1 shows killing of CEA+ / VSIG2+ target cells (left column) in comparison to killing of CEA+ target cells that did not express VSIG2 (right column), and anti-VISG2 mediated protection, as assessed by percent suppression of killing.

[0045] FIG. 2 shows a summary of results for the VSIG2 iCAR protection assay of FIG. 1.

[0046] FIG. 3 shows expression of the iCARs with various inhibitory ICDs.

[0047] FIG. 4 shows anti-VSIG2 iCAR-mediated protection with various inhibitory ICDs.

[0048] FIG. 5A and 5B shows expression of aCAR, iCAR and membrane-associated IL- 15 in a quadcistronic payload in different orientations.

[0049] FIGs. 6A-6D show expression of both CARs (aCAR and iCAR) and IL15 and IL21 on engineered NK cells and iCAR NOT gate function

[0050] FIG. 6A shows expression of both aCAR, iCAR, and membrane-associated 11.1 .

[0051] FIG. 6B shows expression of soluble IL 1 in transduced NK cells.

[0052] FIG. 6C shows expression of soluble IL21 in transduced NK cells.

[0053] FIG. 6D shows results of an m vitro cytotoxicity assay to demonstrate protection ofVSIG2+ cells.

[0054] FIGs. 7A-7F show results from 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.

[0055] FIG. 7 A shows an exemplary schematic of a NOT-gate gene circuit introduced into the NK celi .

[0056] FIG. 7B shows the expression of a VSIG2 inhibitory GAR on engineered NK cells.

[0057] FIG. 7C shows the expression of the membrane-associated mIL-15 on engineered NK ceils.

[0058] FIG. 7D shows the experimental scheme of the in vivo subcutaneous solid tumor model.

[0059] FIG. 7E shows an exemplary flow cytometry result of a no treatment and CAR NK treatment.

[0060] FIG. 7F shows the results of the in vivo co-culture assay shown in FIG. 7D.DETAILED DESCRIPTION

[0061] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan ed., Taylor & Francis, 2006); Fundamental Virology, 3rdEdition, vol. I & II (B.N. Fields and D.M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rdEdition, 2001); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).Definitions

[0062] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0063] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0064] As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise.

[0065] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).

[0066] As used herein, the term "activating an immuneresponsive cell" refers to the induction of signal transduction or changes in protein expression in the cell that results in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibition motifs (ITAMs) a signal transduction cascade is produced. In certain embodiments, when an endogenous TCR or an exogenous CAR binds antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g. CD4 or CD8, CDSy / b / e / ^, etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation in turn initiates a T cell activation pathway ultimately activating transcription factors, such as NF-KB and AP-1. These transcription factors induce global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response.

[0067] As used herein, the term "stimulating a cell -mediated immune response " or “stimulating an immune response" refers to generating a signal that results in a immune response by one or more cell types or cell populations. Immunostimulatory activity may include pro-inflammatory activity. In various embodiments, the immune response occurs after immune cell (e.g., T-cell or NK cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), 0X40, 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 can activate immune responses to neoplastic cells. In various embodiments, promoting, stimulating, or otherwise agonizing pro-inflammatory polypeptides and / or their ligands may enhance the immune response of an immunoresponsive cell. Withoutbeing bound to a particular theory, receiving multiple stimulatory signals (e.g., co-stimulation) is important to mount a robust and long-term cell-mediated immune response, such as a T cell mediated immune response where T cells can become inhibited and unresponsive to antigen (also referred to as “T cell anergy”) in the absence of co-stimulatory signals. Without receiving these stimulatory signals, T cells quickly become inhibited and unresponsive to antigen. While the effects of the variety of co-stimulatory signals, particularly in combination with one another, can vary and remain only partially understood, co-stimulation generally results in increasing gene expression in order to generate long-lived, proliferative, and anti-apoptotic resistant cells, such as T cells or NK cells, that robustly respond to antigen, for example in meditating complete and / or sustained eradication of targets cells expressing a cognate antigen.

[0068] As used herein, the term "chimeric antigen receptor" or alternatively a "CAR" refers 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 "an intracellular signaling domain") comprising a functional signaling domain.

[0069] As used herein, the term "activating CAR" or “aCAR” refers to CAR constructs / architectures capable of inducing signal transduction or changes in protein expression in the activating CAR-expressing cell that initiate, activate, stimulate, or increase an immune response upon binding to a cognate aCAR ligand.

[0070] As used herein, the term "inhibitory CAR" or “iCAR” refers to CAR constructs / architectures capable of inducing signal transduction or changes in protein expression in the inhibitory CAR-expressing cell that prevent, attenuate, inhibit, reduce, decrease, inhibit, or suppress an immune response upon binding to a cognate iCAR ligand, such as reduced activation of immunoresponsive cells receiving or having received one or more stimulatory signals, including co-stimulatory signals.

[0071] As used herein, the term “enzymatic inhibitory domain” refers to a protein domain that inhibits an intracellular signal transduction cascade, for example a native T cell activation cascade. In some embodiments, the enzymatic inhibitory domain of a chimeric inhibitory receptor of the present disclosure comprises at least a portion of an extracellular domain, a transmembrane domain, and / or an intracellular domain. In some embodiments, the enzymatic inhibitory domain comprises 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 e.g., Stanford el 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 comprises an enzyme domain(s), an enzyme fragment(s), or a mutant(s) thereof. In some embodiments, theportion of the enzyme is a catalytic domain of the enzyme. In some embodiments, the enzyme domain(s), enzyme fragment(s), or mutants(s) thereof are selected to maximize efficacy and minimize basal inhibition.

[0072] As used herein, the term "intracellular signaling domain" refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0073] As used herein, the term “extracellular antigen-binding domain” or “antigen-binding domain” (ABD) refers to a polypeptide sequence or polypeptide complex that specifically recognizes or binds to a given antigen or epitope, such as the polypeptide sequence or polypeptide complex portion of the chimeric proteins described herein that provide, for example, the VSIG2-specific binding. An ABD (or antibody, antigen-binding fragment, and / or the chimeric protein including the same) is said to “recognize” the epitope (or more generally, the antigen) to which the ABD specifically binds, and the epitope is said to be the “recognition specificity” or “binding specificity” of the ABD. The ABD is said to bind to its specific antigen or epitope with a particular affinity. As described herein, “affinity” refers to the strength of interaction of non-covalent intermolecular forces between one molecule and another. The affinity, i. e. , the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), wherein a lower KD value refers to a stronger interaction between molecules. KD values of antibody constructs are measured by methods well known in the art including, but not limited to, bio-layer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays (e.g., Flow-cytometry). Specific binding, as assessed by affinity, can refer to a binding molecule with an affinity between an ABD and its cognate antigen or epitope in which the KD value is below 106M. 107M. 10SM.109M. or 10l0M. Specific binding can also include recognition and binding of a biological molecule of interest (e.g., a polypeptide) while not specifically recognizing and binding other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the present disclosure. In certain embodiments, specific binding refers to binding between an ABD, antibody, or antigen-binding fragment to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.

[0074] An ABD can be an antibody. The term "antibody," as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intactimmunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0075] 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 recombinant variants thereof, that is sufficient to confer recognition and specific binding of the antigenbinding fragment to a target, such as an antigen or epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antigen-binding fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen-binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1 136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Patent No. : 6,703,199, which describes fibronectin polypeptide minibodies).

[0076] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the “valency” of the binding molecule. A binding molecule having a single ABD is “monovalent”. A binding molecule having a plurality of ABDs is said to be “multivalent”. A multivalent binding molecule having two ABDs is “bivalent.” 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 plurality of ABDs have the same recognition specificity and can be referred to as a “monospecific multivalent” binding molecule. In other multivalent embodiments, at least two of the plurality of ABDs have different recognition specificities. Such binding molecules are multivalent and “multispecific.” In multivalent embodiments in which the ABDs collectively have two recognition specificities, the binding molecule is “bispecific.” In multivalent embodiments in which the ABDs collectively have three recognition specificities, the binding molecule is “trispecific.” In multivalent embodiments in which the ABDs collectively have a plurality of recognition specificities for different epitopes present on the same antigen, the binding molecule is “multiparatopic.” Multivalent embodiments in which the ABDs collectively recognize two epitopes on the same antigen are “biparatopic.”

[0077] In various multivalent embodiments, multivalency of the binding molecule improves the avidity of the binding molecule for a specific target. As described herein, “avidity” refers to the overall strength of interaction between two or more molecules, e.g., a multivalent bindingmolecule for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 106M. 107M. 10SM.109M. or 10l0M. In certain embodiments, the avidity of a binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABDs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate epitopes on a shared individual antigen.

[0078] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising at least one antigen-binding fragment comprising a variable region of a light chain and at least one antigen-binding fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0079] As used herein, “variable region” refers to a variable sequence that arises from a recombination event, for example, following V, J, and / or D segment recombination in an immunoglobulin gene in a B cell or T cell receptor (TCR) gene in a T cell. In immunoglobulin genes, variable regions are typically defined from the antibody chain from which they are derived, e.g. , VH refers to the variable region of an antibody heavy chain and VL refers to the variable region of an antibody light chain. A select VH and select VL can associate together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0080] The term "complementarity determining region" or "CDR," as used herein, refers to the sequences within antibody variable regions VH and VL which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described byKabat 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 in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in 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 in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VE are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL. In a variety of embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences.

[0081] The term "framework region" or "FR," as used herein, refers to the generally conserved sequences within antibody variable regions VH and VL that act as a scaffold for interspersed CDRs, typically in a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus). In a variety of embodiments, the FRs are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In specific embodiments, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthesized sequences including, but not limited, rationally designed sequences. In some embodiments, human FR sequences are naturally occurring sequences (e.g., human germline antibody sequences, such as IGHV3-21 for the heavy chain and IGKV1-5 for the light chain). In some embodiments, human FR sequences are naturally occurring sequences that include one or more mutations, such as “back” mutations to match the species of origin of the CDRs grafted onto the human framework.

[0082] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0083] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (X) light chains refer to the two major antibody light chain isotypes.

[0084] As used herein, the term "recombinant antibody" refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0085] As used herein, the term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0086] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place, such as in a prophylactic therapy or treatment.

[0087] As used herein, the term "autologous" refers to any material derived from the same subject to whom it is later to be re-introduced into the subject.

[0088] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the subject to whom the material is introduced. Two or more subjects are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically distinct, e.g., at particular genes such as MHC alleles, to interact antigenically. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically similar, e.g., at particular genes such as MHC alleles, to not interact antigenically.

[0089] Isolated nucleic acid molecules of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure, or fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical with an endogenous nucleic acidsequence, but will typically exhibit substantial identity. Nucleic acids having "substantial identity" or "substantial homology" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double -stranded nucleic acid molecule. As used herein, "hybridize" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. For example, stringent salt concentration may be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency may be accomplished by combining these various conditions as needed.

[0090] By "substantially identical" or "substantially homologous" is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University ofWisconsin 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, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0091] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either adefined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0092] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and / or interaction.

[0093] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. In some embodiments, an "effective amount" or a "therapeutically effective amount" is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis of a disease or disorder of interest, e.g., a myeloid disorder.

[0094] As used herein, the term "immunoresponsive cell" refers to a cell that functions in an immune response (e.g., an immune effector response) or a progenitor, or progeny thereof. Examples of immune effector cells include, without limitation, alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid- derived phagocytes.

[0095] As used herein, the term "immune effector response" or "immune effector function" refers to a function or response, e.g., of an immunoresponsive cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response may refer to a property of a T cell or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0096] As used herein, the term "flexible polypeptide linker" or "linker" refers to a peptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acidsequence (Gly-Gly-Gly-Gly-Ser)nor (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10. In some embodiments, the flexible polypeptide linkers include, but are not limited to, GlyrScr or (Gly4Ser)3. In other embodiments, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (GlysSer). In some embodiments, the flexible polypeptide linkers include a Whitlow linker (e.g., GSTSGSGKPGSGEGSTKG [SEQ ID NO: 36]). Also included within the scope of the present disclosure are linkers described, for example, in WO2012 / 138475.

[0097] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of a proliferative disorder (e.g., cancer), or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of the present disclosure). In some embodiments, reduction or amelioration refers to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In some embodiments, reduction or amelioration include reduction or stabilization of tumor size or cancerous cell count.

[0098] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[0099] Other aspects of the present disclosure are described in the following sections and are within the ambit of the claimed invention.

[0100] Other interpretational conventions

[0101] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from 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.

[0102] Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof.Solid tumor antigens

[0103] Certain aspects of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, that have been genetically modified to express one or more of such chimeric receptors that bind to an antigen of interest, and to methods of using such receptors andcells to treat and / or prevent solid malignancies, such as lung cancer, pancreatic cancer, gastrointestinal cancer, colon cancer, brain cancer, cancer of the neuronal tissue, endocrine tumors, bone cancer, cancer of the bone marrow, cancer of the immune system, muscle cancer, liver cancer, gallbladder cancer, kidney cancer, urinary bladder cancer, cancer of the male reproductive organs, cancer of the female reproductive organs, adipose cancer, soft tissue cancer, and skin cancer, and other pathologies where an antigen-specific immune response is desired. Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination. The present disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0104] Certain aspects of the present disclosure related to chimeric receptors that specifically bind one or more antigens expressed on a myeloid cell useful for treating solid tumor malignancies, and to immunoresponsive cells genetically modified to express such chimeric receptors. Solid cancers are clonal diseases caused by genetic and epigenetic alterations that disrupt key processes such as cell proliferation and differentiation. Solid tumor malignancies can be chronic or acute.

[0105] Certain aspects of the present disclosure relate, in general, to chimeric receptors, engineered expression systems, cells, and methods of treatment for the combined targeting of a first solid tumor antigen that is a CEA family member (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6) and a second antigen that is-VSIG2. In various embodiments, the present disclosure relates to aNOT-logic gate to control, modulate, or otherwise inhibit one or more activities of the one or more activating chimeric receptors on healthy cells (e.g., cells expressing VSIG2).

[0106] In some embodiments, the present disclosure relates to engineered expression systems, cells, and methods of treatment comprising a bivalent chimeric receptor comprising a first antigen binding domain that binds a CEA family member (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6) and a second antigen binding domain that binds VSIG2. In some embodiments, the present disclosure relates to engineered expression systems, cells, and methods of treatment comprising a first chimeric receptor comprising an antigen binding domain that binds a CEA family member (e.g., CEACAM5, CEA, CEACAM1, and CEACAM6) and a second chimeric receptor comprising an antigen binding domain that binds VSIG2.

[0107] In certain embodiments, the present disclosure relates to solid tumor antigens and combinations of solid tumor antigens that are suitable for use in chimeric receptors (e.g., chimeric TCRs or CARs) to increase efficacy and / or reduce off-tumor toxicity in the treatment of the solid tumor. In certain embodiments, a first solid tumor antigen is a CEA-family member. In certain embodiments, a first solid tumor antigen is a CEA-family member selected from thegroup consisting of CEA, CEACAM1, CEACAM5, and CEACAM6. As used herein, “CEA” refers to a family of highly related proteins (CD66 proteins), including, without limitation CEACAM1 (CD66a), CEACAM5 (CD66e), and CEACAM6 (CD66c). In certain embodiments, an antibody or antigen-binding fragment that binds CEA binds more than one CD66 protein. Table 1 provides CEA-family antigens suitable for use in chimeric receptors described in the methods and compositions presented herein.

[0108] In some embodiments, the first solid tumor antigen is a CEACAM1 antigen. CEACAM1 is also known in the art as BGP, BGP1, BGPI, or CD66a. In some embodiments, the first solid tumor antigen is a CEACAM5 antigen. CEACAM5, was previously known in the art as CEA. At present CEACAM5 is also known as Meconium Antigen 100, Carcinoembryonic Antigen, or CD66e. In some embodiments, the first solid tumor antigen is a CEACAM6 antigen. CEACAM6 is also known in the art as CEAL, NCA, Normal Cross-Reacting Antigen, Non-Specific Crossreacting Antigen, or CD66c. 0109] In certain embodiments, a second antigen is a VSIG2 antigen. VSIG2 is encoded by the VSIG2 gene, is known in the art as V-Set and Immunoglobulin Domain Containing 2, CTXL, CTH, Cortical Thymocyte-Like Protein, CT-Like Protein, Cortical Thymocyte Receptor (X. Laevis CTX) Like, and is represented by the UniProt Accession No. Q96IQ7.Chimeric receptors

[0110] Certain aspects of the present disclosure relate to chimeric receptors and nucleic acids that encode such chimeric receptors that bind to an antigen of interest. In certain embodiments, a 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 someembodiments a chimeric receptor of the present invention comprises a single antigen-binding domain.Antibodies and Antigen-binding fragments

[0111] In some embodiments, chimeric receptors comprise one or more antigen binding domains capable of binding a solid tumor antigen, e.g., a CEA-family member antigen (such as listed in Table 1). Antigen binding domains of the chimeric receptors can comprise antibody sequences, or antigen-binding fragments thereof, of the representative anti-CEA antibodies provided in Table 2. In some embodiments, the antigen-binding domains comprise the CDR sequences of an antibody or antigen-binding fragment thereof of Table 2.

[0112] In some embodiments, commercially available antibodies may be used for binding to a solid tumor antigen. The CDRs of the commercially available antibodies are readily accessible by one skilled in the art using conventional sequencing technology. Further, one skilled in the art is able to construct nucleic acids encoding scFvs and chimeric receptors (e.g., CARs and TCRs) based on the CDRs of such commercially available antibodies.

[0113] In some embodiments, a chimeric receptor comprises an antigen-binding domain that specifically binds CEA.

[0114] In some embodiments, a chimeric receptor comprises an antigen-binding domain that specifically binds CEACAM1. In some embodiments, the CEACAM1 -specific antigen-binding domain is derived from an anti-CEACAMl antibody, such as the MRG1 antibody or an antigenbinding fragment thereof. In certain embodiments, the CEACAM1 -specific antigen-binding domain comprises a heavy chain variable domain (VH) that comprises 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 MRG1 disclosedin Table 2, and a light chain variable domain (VL) that comprises 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 MRG1 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of MRG1 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigenbinding domain. For example, the chimeric receptor may be specific for CEACAM1 and one or more additional antigens. In some embodiments, the chimeric receptor may be specific for CEACAM1 and CEACAM5. In some embodiments, the chimeric receptor may be specific for CEACAM1 and CEACAM6. In some embodiments, the chimeric receptor may be specific for CEACAM5 and CEACAM6.

[0115] In some embodiments, a chimeric receptor comprises an antigen-binding domain that specifically binds CEACAM5. In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as labetuzimab (i.e., hMN14) or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigenbinding domain comprises a heavy chain variable domain (VH) that comprises 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) that comprises 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 the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of hMN14 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0116] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as cibisatamab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain(HC) that comprises 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 HC of cibisatamab disclosed in Table 2, and a light chain (LC) that comprises 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 LC of cibisatamab disclosed in Table 2. In certain embodiments, the second antigenbinding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) of the HC and LC sequences of cibisatamab 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, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the HC and LC sequences of cibisatamab disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises a light chain variable domain and a 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 for CEACAM5 and one or more additional antigens.

[0117] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as tusamitamab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain (HC) that comprises 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 HC of tusamitamab disclosed in Table 2, and a light chain (LC) that comprises 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 LC of tusamitamab disclosed in Table 2. In certain embodiments, the second antigenbinding site comprises a heavy chain variable domain (VH) and a 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, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the HC and LC sequences of tusamitamab disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises a light chain variable domain and a 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 for CEACAM5 and one or more additional antigens.

[0118] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as BW431 / 26 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain variable domain (VH) that comprises 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 BW431 / 26 disclosed in Table 2, and a light chain variable domain (VL) that comprises 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 BW431 / 26 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of BW431 / 26 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0119] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as A5B7 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain variable domain (VH) that comprises 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 A5B7 disclosed in Table 2, and a light chain variable domain (VL) that comprises 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 A5B7 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of A5B7 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0120] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as MFE23 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain variable domain (VH) that comprises 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 MFE23 disclosed in Table 2, and a light chain variable domain (VL) that comprises 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 MFE23 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of MFE23 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0121] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as hMFE23 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigen-binding domain comprises a heavy chain variable domain (VH) that comprises 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 hMFE23 disclosed in Table 2, and a light chain variable domain (VL) that comprises 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 hMFE23 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of hMFE23 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0122] In some embodiments, the CEACAM5 -specific antigen-binding domain is derived from an anti-CEACAM5 antibody capable of specifically binding 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 an antigen-binding fragment thereof. In certain embodiments, the CEACAM5 -specific antigenbinding domain comprises a heavy chain variable domain (VH) that comprises 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 FM4 disclosed in Table 2, and a light chain variable domain (VL) that comprises 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 FM4 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of FM4 disclosed in Table 2, respectively. The antigen-binding domain may be an scFv that comprises 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 for CEACAM5 and one or more additional antigens.

[0123] In some embodiments, a chimeric receptor comprises an antigen-binding domain that specifically binds CEACAM6. In some embodiments, the CEACAM6-specific antigen-binding domain is derived from an anti-CEACAM6 antibody, such as tinurilimab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM6-specific antigen-binding domain comprises a heavy chain (HC) that comprises 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 HC of tinurilimab disclosed in Table 2, and a light chain (LC) that comprises 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 LC of tinurilimab disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) of the HC and LC sequences of tinurilimab 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, determined under Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the HC and LC sequences of tinurilimab disclosed in Table 2, respectively. The antigen-bindingdomain may be an scFv that comprises a light chain variable domain and a 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 for CEACAM6 and one or more additional antigens.

[0124] Certain aspects of the present disclosure relate to chimeric receptors (e.g., CAR or chimeric TCR) comprising an extracellular antigen-binding domain that binds to one or more antigens of the present disclosure. In some embodiments, the antigen-binding domains are derived from an antibody, or antigen-binding fragment thereof CDR sequences and known systems for defining them, e.g., Kabat, are discussed in detail above.

[0125] Suitable antibodies of the present disclosure include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to a solid tumor antigen, e.g., CEA, CEACAM1, CEACAM5, or CEACAM6. In some embodiments, the antibody may have a KD of at most about at most 10'6M, at most about 10'7M, at most about 10'8M, at most about 10'9M, at most about IO'10M, at most about 10'11M, or at most about 10'12M.V-set and Immunoglobulin Domain Containing 2 (VSIG2)-Specific Antigen-binding Domains

[0126] The present disclosure provides chimeric proteins, and polynucleotides that encode such chimeric proteins, that bind to V-set and immunoglobulin domain-containing protein 2 (VSIG2). In some embodiments, VSIG2-specific chimeric proteins bind to human VSIG2 (e.g., Uniprot Q96IQ7, herein incorporated by reference for all purposes) or an epitope fragment thereof. VSIG2 can be expressed on epithelial cells. VSIG2 can be expressed on cells generally considered to be 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 “5AI0”).

[0127] The present disclosure provides an VSIG2-specific antigen-binding domain including one or more of the complementarity determining region (CDR) amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3. In general, the VSIG2-specific antigen-binding domains provided herein include a CDRL3 light chain amino acid sequence QHHAVIPWT (SEQ ID NO:9) and / or a CDRH3 heavy chain amino acid variant sequence of any one of SEQ ID NOs: 67-87 shown in Table 22.Table 3. Parental VSIG2-specific Antigen-binding Domains Sequences

[0128] The present In some embodiments, the VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH includes a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, and a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3; wherein the VL includes a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, and a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7; and wherein: (i) the VH includes a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and the VL includes a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) the VH includes a VH complementarity region 3 (CDRH3) having the amino acid sequence of any one of SEQ ID NO: 67-87, and the VL includes a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0129] In some embodiments, the VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH includes a VH complementarity region 1 (CDRH1) having an amino acid sequence of SEQ ID NO: 2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4; and wherein the VL includes a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7; wherein: (i) the VH includes a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and the VL includes a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or (ii) the VH includes a VH complementarity region 3 (CDRH3) having the amino acid sequence ofany one of SEQ ID NO: 67-87, and the VL includes a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9.

[0130] In some embodiments, the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 88-107. In some embodiments, the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, 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) the CDRH3 has the amino acid sequence of SEQ ID NO: 79, and the CDRL3 has 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 ofSEQ 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 CDRH3 has the amino acid sequence of SEQ ID NO: 87, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8. In some embodiments, 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.

[0131] In some embodiments, the VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) the VH region includes the amino acid sequence of SEQ ID NO: 16, and the VL region includes the amino acid sequence selected of SEQ ID NO: 15; or (ii) the VH region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107, and the VL region includes the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0132] In some embodiments, the VSIG2-specific antigen-binding domain has a variable heavy (VH) region and a variable light (VL) region, wherein the VL has an 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.

[0133] In some embodiments, the VSIG2-specific antigen-binding domain has a variable heavy (VH) region and a variable light (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107. In some embodiments, the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0134] In some embodiments, the VSIG2-specific antigen-binding domain has a VH region including an amino acid sequence with 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 to the amino acid sequence listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3.

[0135] In some embodiments, the VSIG2-specific antigen-binding domain has a VL region including an amino acid sequence with 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 to the amino acid sequence listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3.

[0136] The VSIG2-specific antigen-binding domain can be in any of the formats described herein, such as a Fab, Fab', F(ab')2, Fv, scFv, linear antibody, single domain antibody such as sdAb (either VL or VH), camelid VHH, and multi-specific formats. In some embodiments, theVSIG2-specific antigen-binding domain is in a F(ab) format. In some embodiments, the VSIG2- specific antigen-binding domain is in a F(ab') format.

[0137] In some embodiments, the VSIG2-specific antigen-binding domain is in a single chain variable fragment (scFv) format, including scFv formats having any of the peptide linkers described herein (e.g., see 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.

[0138] hi some embodiments, the scFv has an ammo acid sequence selected from SEQ ID NOs: 108-132.

[0139] In some embodiments, the VSIG2-specific antigen-binding domain is humanized, wherein CDRs are non-human mammalian sequences including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, that are grafted onto a scaffold having human framework region (FR) sequences, typically with the CDRs interspersed in a FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus). FR sequences for a light chain can include FR1 sequence DIQMTQSPSTLSASVGDRVTITC; FR2 sequence WYQQKPGKAPKLLIY; FR3 sequence GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC; and / or FR4 sequence FGQGTKVEIK. FR sequences for a heavy chain can include FR1 sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFS; FR2 sequence WVRQAPGKGLEWVA; FR3 sequence RFTISRDNAKSSLYLQMNSLRAEDTAVYYCAR; and / or FR4 sequence WGQGTLVTVSS.

[0140] The present disclosure also provides chimeric proteins, and nucleic acids that encode such chimeric proteins, that include an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3. The chimeric proteins may include any of the VSIG2-specific antigen-binding domains as previously described.Chimeric Antigen Receptors (CARs)

[0141] Certain aspects of the present disclosure relate to chimeric receptors that have any one of the VSIG2-specific antigen-binding domain described herein and are capable of specifically binding to an VSIG2 protein, an VSIG2-derived antigen, or an VSIG2-derived epitope. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, CARs are chimeric proteins that include an antigen-binding domain and polypeptide molecules that are heterologous to the antigen-binding domain, such as peptides heterologous to an antibody that an antigen-binding domain may be derived from. Polypeptide molecules that are heterologous to the antigen-binding domain can include, but are not limited to, atransmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or combinations thereof.

[0142] In some embodiments, CARs are engineered receptors that graft or confer a specificity of interest (e.g., VSIG2) onto an immune effector cell. In certain embodiments, CARs can be used to graft the specificity of an antibody onto an immunoresponsive cell, such as a T cell. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, fused to one or more intracellular signaling domains.

[0143] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR and also generally referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of the immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of the immunoresponsive cell. In some embodiments, activation of an immunoresponsive cell results in killing of target cells. In some embodiments, activation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of the immunoresponsive cell can be combinations of the above responses.

[0144] A CAR of the present disclosure may be a first, second, or third generation CAR."First generation" CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. "First generation" CARs generally have the intracellular signaling domain from the CD3-zeta (CD3Q chain, which is the primary transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. "Second generation" CARs provide both co-stimulation (e.g., CD28 or 4- 1BB) and activation (CD3Q. Preclinical studies have indicated that "Second Generation" CARs can improve the anti-tumor activity of immunoresponsive cell, such as a T cell. "Thirdgeneration" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4- IBB) and an intracellular activation signaling domain (CD3 .

[0145] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, the one or more chimeric inhibitory receptors bind antigens that are expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0146] In some embodiments, a chimeric inhibitory receptor (e.g., an VSIG2-specific chimeric inhibitory receptor) may be used, for example, with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell of the present disclosure (e.g., an immunoresponsive cell) as NOT-logic gates to control, modulate, or otherwise inhibit one or more activities of the one or more activating chimeric receptors. For instance, if a healthy cell expresses both an antigen that is recognized by a tumor-targeting chimeric receptor and an antigen that is recognized by an chimeric inhibitory receptor, an immunoresponsive cell expressing the tumor antigen may bind to the healthy cell. In such a case, the inhibitory chimeric antigen will also bind its cognate ligand on the healthy cell and the inhibitory function of the chimeric inhibitory receptor will reduce, decrease, prevent, or inhibit the activation of the immunoresponsive cell via the tumor-targeting chimeric receptor (“NOT-logic gating”). In some embodiments, a chimeric inhibitory receptor of the present disclosure may inhibit one or more activities of a cell of the present disclosure (e.g., an immunoresponsive cell). In some embodiments, an immunoresponsive cell may comprise one or more tumor-targeting chimeric receptors and one or more chimeric inhibitory receptors that targets an antigen that is not expressed, or generally considered to be expressed, on the tumor (e.g., VSIG2). Combinations of tumor-targeting chimeric receptors and chimeric inhibitory receptors in the same immunoresponsive cell may be used to reduce on-target off-tumor toxicity.

[0147] In some embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure binds to one or more antigens (e.g., VSIG2) with a dissociation constant (Kd) of about 2 x 10'7M or less, about 1 x 10'7M or less, about 9 x 10'8M or less, about 1 x 10'8M or less, about 9 x 10'9M or less, about 5 x 10'9M or less, about 4 x 10'9M or less, about 3 x 10'9M or less, about 2 x 10'9M or less, or about 1 x 10'9M or less. In some embodiments, the Ka ranges from about is about 2 x 10'7M to about 1 x 10'9M. In some embodiments, VSIG2- specific antigen-binding domains in aCAR formats can be selected based on affinity, including selected based on having a higher affinity or lower affinity relative to other VSIG2-specific antigen-binding domains. In some embodiments, VSIG2-specific antigen-binding domains iniCAR formats can be selected based on affinity, including selected based on higher affinity or lower affinity relative to other VSIG2-specific antigen-binding domains.

[0148] Binding of the extracellular antigen-binding domain of a CAR of the present disclosure can be determined by, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), bio-layer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or a Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in an RIA assay. The radioactive isotope can be detected by such means as the use of a y counter or a 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 for the extracellular antigen-binding domain and wherein the secondary antibody is labeled (e.g., radioactively or with a fluorescent marker).

[0149] In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain that binds to VSIG2 (e.g., an VSIG2 protein, an VSIG2-derived antigen, or an 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 Fab fragment, which may be crosslinked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment.Extracellular Antigen-binding Domain

[0150] The extracellular antigen-binding domain of a CAR of the present disclosure specifically binds to VSIG2 (e.g., an VSIG2 protein, an VSIG2 -derived antigen, or an VSIG2- derived epitope). In certain embodiments, the extracellular antigen-binding domain binds to VSIG2 expressed on a hematopoietic stem cell. In certain embodiments, the extracellular antigen-binding domain binds to V SIG2 expressed on cells generally considered to be healthy, such as healthy HSCPs. In some embodiments, VSIG2 is human VSIG2.

[0151] Antigen-binding domains of the present disclosure can include any domain that binds to the antigen including, without limitation, a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a bispecific antibody, a conjugated antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a singledomain antibody (sdAb) such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues for the antigen-binding domain of an antibody of antibody fragment.

[0152] In some embodiments, the extracellular antigen-binding domain comprises 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 comprises an antigenbinding fragment of an antibody.

[0153] In some embodiments, the extracellular antigen-binding domain comprises a F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises a F(ab') fragment.

[0154] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single chain variable fragments (scFvs). 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 comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind 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 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0155] In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises any of the amino acid sequences shown in Table 4. In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein 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 the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When there are two or more scFv linked together, each scFv can be linked to the next scFv with a peptide linked. In some embodiments, each of the one or morescFvs is separated by a peptide linker. In some embodiments, the peptide linker separating each of the scFvs comprises an amino acid sequence as shown in Table 4.Table 4. Peptide Linkers

[0156] In some embodiments, the peptide linker comprises an amino acid sequence of GGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGGSGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGGSGGSGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGGSGGSGGSGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGSGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGSGGGSGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGSGGGSGGGSGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGSGGGSGGGSGGGSGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGGSGGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGGSGGGGSGGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS. In some embodiments, the peptide linker comprises an amino acid sequence of GSTSGSGKPGSGEGSTKG. In some embodiments, the peptide linker comprises an amino acid sequence ofEAAAKEAAAKEAAAKEAAAK. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGSGGSGSGGSGS.

[0157] In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domain of the first chimeric receptor and the antigen-binding domain of the second chimeric receptor can be an appropriate antigen biding domain described herein or known in the art. For example, the first or second antigen-binding domain can be one or more antibodies, antigen-binding fragments of an antibody, F(ab) fragments, F(ab') fragments, single chain variable fragments (scFvs), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises two single chain variable fragments (scFvs). 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 can be specific for VSIG2 and the chimeric receptor can be specific for a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on a CRC tumor cell).

[0158] In some embodiments, the extracellular antigen-binding domain comprises a singledomain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0159] In some embodiments, a CAR of the present 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 antigenbinding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different antigen.

[0160] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are attached to one another via a flexible 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, an scFv, a sdAb, a recombinant fibronectin domain, a 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).

[0161] In certain embodiments, the bispecific CAR or tanCAR comprises an antigen-binding domain comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibodymolecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHi) upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLi) upstream of its VH (VHi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VLi VH1-VH2-VL2. In some embodiments, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VLi and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VHi and VH2 if the construct is arranged as VL1-VH1-VH2- VL2. The linker may be a linker as described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1 , 2, 3, 4, 5, or 6. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, a linker is disposed between the VL and VH of the first scFv. In some embodiments, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers may be the same or different. Accordingly, in some embodiments, a bispecific CAR or tanCAR comprises VLs, VHs, and may further comprise one or more linkers in an arrangement as described herein.

[0162] In some embodiments, chimeric receptors comprise a bivalent CAR. In some embodiments, the bivalent CAR is an VSIG2 bivalent CAR. In some embodiments, the bivalent VSIG2 CAR comprises one or more of the anti-VSIG2 sequences shown in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3. In some embodiments, the ABDs of the bivalent VSIG2 CAR each comprises the same ABD.

[0163] In some embodiments, chimeric receptors comprise a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises an VSIG2 CAR. In some embodiments, the bicistronic VSIG2 CAR comprises one or more of the anti- VSIG2 sequences shown in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3.Transmembrane Domain

[0164] In some embodiments, the transmembrane domain of a CAR of the present disclosure (e.g., the VSIG2-specific CARs described herein) comprises a hydrophobic alpha helix thatspans at least a portion of a cell membrane. It has been shown that different transmembrane domains can result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of a CAR of the present disclosure can comprise the transmembrane domain of a CD 8 polypeptide, a CD28 polypeptide, a SIRPa polypeptide, CD25 polypeptide, a CD7 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a LAX polypeptide, a LAT polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a TIM3 polypeptide, a KIR3DS1 polypeptide, a KIR3DL1 polypeptide, a NKG2D polypeptide, a NKG2A polypeptide, a TIGIT polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0165] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, without limitation, NCBI Reference Nos. 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, without limitation, NCBI Reference Nos. 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, without limitation, NCBI Reference Nos. 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, without limitation, NCBI Reference Nos. 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 may be used. Exemplary 4-1BB polypeptides include, without limitation, NCBI Reference Nos. NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an 0X40 polypeptide. Any suitable 0X40 polypeptide may be used. Exemplary 0X40 polypeptides include, without limitation, NCBI Reference Nos. NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, without limitation, NCBI Reference Nos. NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, without limitation, NCBI Reference Nos. 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, withoutlimitation, NCBI Reference Nos. 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, without limitation, NCBI Reference Nos. 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. Exemplary 2B4 polypeptides include, without limitation, NCBI Reference Nos. 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. Exemplary BTLA polypeptides include, without limitation, NCBI Reference Nos. NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, without limitation, NCBI Reference Nos. NP_001075106.2 and NP_001075107.2.

[0166] In some embodiments, the transmembrane domain comprises a polypeptide comprising an amino acid sequence 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 No. NP_001139345, AAA92533.1, NP 006I30.I, NP 03I668.3, NP 932I70.I, NP 00I 106862. 1, NP_000607.1, NP_038516.1, NP_001552.2, NP 00I070977.I, 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, or fragments thereof. In some embodiments, the homology may be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may comprise 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 can have an amino acid sequence that is a consecutive portion of NCBI Reference No. NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932I70.I, NP_001106862. l, NP_000607.I, NP_0385I6.I, NP_00I552.2, NP 00I070977.I, 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 that is 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 amino acids in length.

[0167] Further examples of suitable polypeptides from which a transmembrane domain may be derived include, without limitation, the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CDl la, CD18),GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, and NG2C.

[0168] In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 38). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 39). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:40).Spacer Region

[0169] In some embodiments, a CAR of the present disclosure (e.g., the VSIG2-specific CARs described herein) can also comprise a spacer region that links the extracellular antigenbinding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge from a human protein. For example, the spacer (also referred to herein as “hinge”) may be a human Ig (immunoglobulin) hinge, including without limitation an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may comprise 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, a spacer region may comprise any of the amino acid sequences listed in Table 5, 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%, or at least 99% identical to any of the amino acid sequences listed in Table 5. In some embodiments, nucleic acids encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table 6, 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 to any of the nucleic acid sequences listed in Table 6.Table 5. Spacer Amino Acid SequencesTable 6. Spacer Nucleic Acid Sequences

[0170] In some embodiments, the spacer region comprises the sequence for the CD28 hinge shown in SEQ ID NO:41. In some embodiments, the spacer region comprises the sequence for the IgG4 minimal hinge shown in SEQ ID NO:42. In some embodiments, the spacer region comprises the sequence for the IgG4 minimal hinge, no disulfides shown in SEQ ID NO:43. In some embodiments, the spacer region comprises the sequence for the IgG4 S228P minimal hinge, enhanced disulfide formation shown in SEQ ID NO:44. In some embodiments, the spacer region comprises the sequence shown for the IgGl minimal hinge in SEQ ID NO:45. In some embodiments, the spacer region comprises the sequence shown for the extended CD8a hinge in SEQ ID NO:46. In some embodiments, the spacer region comprises the sequence shown LNGFR hinge in SEQ ID NO:47. In some embodiments, the spacer region comprises the sequence shown forthe truncated LNGFR hinge (TNFR-Cysl) in SEQ ID NO:48. In some embodiments, the spacer region comprises the sequence shown for the PDGFR-beta extracellular linker in SEQ ID NO:49. In some embodiments, the spacer region comprises the sequence shown forthe example spacer (CD8 hinge) in SEQ ID NO:50. In some embodiments, the spacer region comprises the sequence shown for the example spacer in SEQ ID NO:51. In some embodiments, the spacer region comprises the sequence shown for the example spacer in SEQ ID NO:52.

[0171] In some embodiments, a CAR of the present disclosure may further include a short oligopeptide or polypeptide linker that is between 2 amino acid residues and 10 amino acid residues in length, and that may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine -serine doublet. In some embodiments, the linker comprises the ammo acid sequence of GGCKJSGGCKJS (SEQ ID NO:62).

[0172] In some aspects, the transmembrane domain further comprises at least a portion of an extracellular domain of the same protein.Intracellular Signaling Domains

[0173] In some embodiments, a CAR of the present disclosure (e.g., the VSIG2-specific CARs described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain.

[0174] Examples of suitable intracellular signaling domains that may be used in CARs of the present disclosure include, without limitation, cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to modulate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0175] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or costimulatory signal is thus also typically required for full activation. Accordingly, T cell activation may be mediated by two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic domain, e.g., a co-stimulatory domain). In addition, T cell signaling and function (e.g., an activating signaling cascade) can be negatively regulated by inhibitory receptors present in a T cell through intracellular inhibitory co-signaling domains.

[0176] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can include an inhibitory intracellular signaling domains. Examples of inhibitory intracellular domains (ICD) that can 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, SIRPa, 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 with 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 to one or more of the amino acid sequences shown in Table 25. In some embodiments, the inhibitory intracellular signaling domain includes the amino acid sequenceVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139). In some embodiments, the inhibitory intracellular signaling domain includes an amino acid sequence with 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 to one or more of the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139). In some embodiments, the inhibitory intracellular signaling domain includes one or more intracellular inhibitory co-signaling domains. In some embodiments, the one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., a transmembrane domain) through a peptide linker (e.g. , see T able 4) or a spacer or hinge sequence (e.g. , see Table 5). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains can be linked through 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, the one or more intracellular inhibitory co-signaling domains of a chimeric protein comprises one or more ITIM-containing protein, or fragment(s) thereof. ITIMs are conserved amino acid sequences found in cytoplasmic tails of many inhibitory immune receptors. In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or a fragment(s) thereof. The inhibitory intracellular signaling domain can further include an enzymatic inhibitory domain. In some embodiments, the enzymatic inhibitory domain comprises an enzyme catalytic domain. In some embodiments, the enzyme catalytic 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 signaling is described in more detail in 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.

[0177] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way or in an inhibitory way. Primary intracellular signalingdomains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of suitable ITAM- containing primary intracellular signaling domains that that may be used in the CARs of the present disclosure include, without limitation, those of CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FCERI, DAP 10, DAP 12, and CD66d.

[0178] In some embodiments, a CAR of the present disclosure (e.g., the VSIG2-specific CARs described herein) comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta polypeptide. A CD3-zeta polypeptide of the present disclosure may have an amino acid sequence 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 ofNCBI Reference No. NP_932170 or NP_001106864.2, or fragments thereof. In some embodiments, the CD3-zeta polypeptide may comprise 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 can have an amino acid sequence that is a consecutive portion ofNCBI Reference No. NP_932170 or NP_001106864.2 that is 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 in length.

[0179] In other embodiments, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0180] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise the CD3-zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the present disclosure. For example, the intracellular signaling domain of the CAR can comprise a CD3-zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain may refer to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule of the present disclosure is a cell surface molecule other than an antigen receptor or its ligands that may be required for an efficient response of lymphocytes to an antigen. Examples of suitable costimulatory molecules include, without limitation, CD97, CD2, ICOS, CD27, CD154, CD8, 0X40, 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, and a ligand that specifically binds with CD83, an MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, CDS, ICAM-1, (CD1 la / CD18), BAFFR, KIRD3S1, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and the like.

[0181] Non-limiting examples of intracellular signaling domains (I CDs) are provided in Table 7.Table 7

[0182] In some embodiments, the intracellular signaling sequences within the cytoplasmic portion of a CAR of the present disclosure may be linked to each other in a random or specified order. In some embodiments, a short oligopeptide or polypeptide linker, for example, between 2 amino acids and 10 amino acids (e.g., 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In one embodiment, a glycineserine doublet can be used as a suitable linker. In one embodiment, a single ammo acid, e.g., an alanine or a glycine, can be used as a suitable linker.

[0183] In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains, e.g., two costimulatory signaling domains, three costimulatory signaling domains, four costimulatory signaling domains, five costimulatory signaling domains, six costimulatory signaling domains, seven costimulatory signaling domains, eight costimulatory signaling domains, nine costimulatory signaling domains, 10 costimulatory signaling domains, or more costimulatory signaling domains. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the two or more costimulatory signaling domains are separated by a linker of the present disclosure (e.g., 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.

[0184] In some embodiments, a CAR of the present disclosure further includes an epitope tag. An epitope tag is a polypeptide sequence included within a polypeptide as a label that can be detected, for example, by a monoclonal antibody. Examples of epitope tags include a FLAGtag, a strep tag, an HA tag, a V5 tag, and a myc tag. An exemplary epitope tag is a myc tag of amino acid sequence EQKLISEEDLNGAA (SEQ ID NO: 20).

[0185] In some embodiments, a cell of the present disclosure expresses a CAR that includes an antigen-binding domain that binds VSIG2, a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0186] In some embodiments, a cell of the present disclosure expresses an iCARthat includes an antigen-binding domain that binds VSIG2 (e.g., an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3 ), a transmembrane domain of the present disclosure, and one or more intracellular inhibitory co-signaling domains. In some embodiments, VSIG2-specific antigen-binding domains in iCAR formats can be selected based on higher affinity to VSIG2 relative to other VSIG2-specific antigen-binding domains. In some embodiments, VSIG2-specific antigen-binding domains in iCAR formats can be selected based on lower affinity to VSIG2 relative to other VSIG2-specific antigen-binding domains. In some embodiments, a cell of the present disclosure expresses a CAR that includes an antigen-binding domain that binds VSIG2 (e.g., an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3 ), a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains. In some embodiments, a cell of the present disclosure expresses a CAR that includes an antigen-binding domain that binds VSIG2 (e.g., an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3), a transmembrane domain of the present disclosure, a hinge positioned between the antigen-binding domain and the transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0187] In some embodiments, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR and includes a transmembrane domain and at least one intracellular inhibitory co-signaling domain each derived from a protein selected from PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A , LAG3, LAIR1, SIRPa, 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.

[0188] In some embodiments, the transmembrane domain is derived from a first protein and the one or more intracellular signaling domains are derived from a second protein that are distinct from the first protein.Natural killer CARs (NK CARs)

[0189] In some embodiments, a CAR of the present disclosure comprises one or more components of a natural killer (NK) cell, thereby forming an NK CAR. The NK component may be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any suitable natural killer cell receptor, including without limitation, a killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS DPI; a natural cytotoxicity receptor (NCR), such as NKp30, NKp44, NKp46; a signaling lymphocyte activation molecule (SLAM) family of immune cell receptor, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; an Fc receptor (FcR), such as CD 16, and CD64; and an Ly49 receptor, such as LY49A and LY49C. In some embodiments, the NK-CAR may interact with an adaptor molecule or intracellular signaling domain, such as DAP12. The structural components as described above of a CAR are also applicable to the structure of an NK CAR.

[0190] Exemplary configurations and sequences of CARs comprising NK receptor components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.Additional Chimeric Receptor Targets

[0191] Certain aspects of the present disclosure relate to chimeric receptors and nucleic acids that encode such chimeric receptors that bind to an antigen of interest in addition to VSIG2. Certain aspects of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, that have been genetically modified to express one or more of such chimeric receptors that bind to an antigen of interest in addition to VSIG2, and to methods of using such receptors and cells to treat and / or prevent myeloid malignancies, such as CRC, and other pathologies where an antigen-specific immune response is desired. Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination. The present disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0192] In some embodiments, a first chimeric receptor includes an antigen-binding domain that binds VSIG2 (e.g. , an VSIG2-specific antigen-binding domain having one or more of theamino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3) and a second chimeric receptor includes an additional antigen-binding domain that binds a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, a cell can express a first chimeric receptor specific for VSIG2 (e.g., a CAR including an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3) and a second chimeric receptor specific for a second antigen, such as a tumor-associated antigen (e.g. , a CRC-associated antigen). In some embodiments, a cell can express a first chimeric inhibitory receptor specific for VSIG2 (e.g. , an inhibitory CAR including an VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23) and a second chimeric receptor specific for a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). For example, a cell (e.g., an immunoresponsive cell) can be engineered to co-expresses or capable of co-expressing an iCARthat includes an antigenbinding domain that binds VSIG2 (e.g., a VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 3, Table 21, Table 22, and / or Table 23, with at least one CDR amino acid sequence including a sequence mutation with respect to the parental CDR amino acid sequences shown in Table 3) and an aCAR that targets a tumor- associated antigen (e.g., a CRC-associated antigen). Suitable antibodies that bind to an antigen in addition to VSIG2 include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to a second antigen, such a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, commercially available antibodies may be used for binding to a second antigen, such a tumor-associated antigen (e.g., an CRC-associated antigen). The CDRs of the commercially available antibodies are readily accessible by one skilled in the art using conventional sequencing technology. Further, one skilled in the art is able to construct nucleic acids encoding scFvs and chimeric receptors (e.g., CARs and TCRs) based on the CDRs of such commercially available antibodies.T cell receptor (TCR)

[0193] Certain aspects of the present disclosure relate to chimeric receptors that specifically bind to a second antigen, such a tumor-associated antigen (e.g., a CRC-associated antigen) and the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). TCRs of thepresent disclosure are disulfide-linked heterodimeric proteins containing two variable chains expressed as part of a complex with the invariant CD3 chain molecules. TCRs are found on the surface of T cells, and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR of the present disclosure comprises an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0194] Each chain of a TCR is composed 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 the peptide / MHC complex. Each of the variable regions has three complementarity determining regions (CDRs).

[0195] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules CD35 / E, CDSy / e. and CD247 / or CD247^ / r|. When a TCR complex engages with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0196] In some embodiments, a TCR of the present disclosure is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from a naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from a naturally occurring TCR by at least 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 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 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residues.Chimeric TCRs

[0197] In some embodiments, a TCR of the present disclosure comprises one or more antigen-binding domains that may be grafted to one or more constant domain of a TCR chain, for example a TCR alpha chain or TCR beta chain, to create a chimeric TCR that binds specifically to a second antigen of interest, such a tumor-associated antigen (e.g., a CRC- associated antigen). Without wishing to be bound by theory, it is believed that chimeric TCRs may signal through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., scFv) can be grafted to the constant domain, e.g., at least a portion of the extracellular constant domain, the transmembrane domain and the cytoplasmic domain, of a TCR chain, such as the TCR alpha chain and / or the TCR beta chain. As another example, the CDRs of an antibody or antibody fragment may be grafted into a TCR alpha chain and / or beta chain to create a chimeric TCR that binds specifically to a second antigen, such a tumor- associated antigen (e.g., a CRC-associated antigen). Such chimeric TCRs may be produced by methods known in the art (e.g., 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).VSIG2-Specific Protein-Encoding Nucleic Acid Constructs

[0198] Certain aspects of the present disclosure relate to nucleic acids (e.g., isolated nucleic acids) encoding one or more VSIG2-specific proteins of the present disclosure (e.g., the VSIG2- specific CARs described herein). In some embodiments, the nucleic acid is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the nucleic acid is a DNA construct.

[0199] In some embodiments, a nucleic acid of the present disclosure encodes a chimeric receptor that comprises one or more antigen-binding domain, where each domain binds to a target antigen (e.g., VSIG2), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the nucleic acid encodes a chimeric receptor that comprises an antigen-binding domain, a transmembrane domain, a primary signaling domain (e.g., CD3-zeta domain), and one or more costimulatory signaling domains. In some embodiments, the nucleic acid further comprises 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 ofthe nucleic acid sequences listed in Table 5. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0200] The nucleic acids of the present disclosure may be obtained using any suitable recombinant methods known in the art, including, without limitation, by screening libraries from cells expressing the gene of interest, by deriving the gene of interest from a vector known to include the gene, or by isolating the gene of interest directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

[0201] In some embodiments, a nucleic acid of the present disclosure in comprised within a vector. In some embodiments, a nucleic acid of the present disclosure is expressed in a cell via transposons, a CRISPR / Cas9 system, a TALEN, or a zinc finger nuclease.

[0202] In some embodiments, expression of a nucleic acid encoding a chimeric receptor of the present disclosure may be achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. A suitable vector can replicate and integrate in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid.

[0203] In some embodiments, expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols (e.g., US5399346, US5580859, and US5589466). In some embodiments, a vector of the present disclosure is a gene therapy vector.

[0204] A nucleic acid of the present disclosure can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, without limitation, a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid. In some embodiments, the vector may be an expression vector, a replication vector, a probe generation vector, or a sequencing vector.

[0205] In some embodiments, the plasmid vector comprises a transposon / transposase system to incorporate the nucleic acids of the present disclosure into the host cell genome. Methods of expressing proteins in immune cells using a transposon and transposase plasmid system 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 are hereby incorporated by reference in their entirety. In some embodiments, the transposon system is the Sleeping Beauty transposon / transposase or the piggyBac transposon / transposase.

[0206] In some embodiments, an expression vector of the present disclosure may be provided to a cell in the form of a viral vector. Suitable viral vector systems are well known in the art. For example, viral vectors may be derived from retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In some embodiments, a vector of the present disclosure is a lentiviral vector. Lentiviral vectors are suitable for long-term gene transfer as such vectors allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors are also advantageous over vectors derived from onco- retroviruses (e.g., murine leukemia viruses) in that lentiviral vectors can transduce nonproliferating cells. In some embodiments, a vector of the present disclosure is an adenoviral vector (A5 / 35). In some embodiments, a vector of the present disclosure contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WOOl / 96584; W001 / 29058; and US6326193). A number of viral based systems have been developed for gene transfer into mammalian cells. A selected gene can be inserted into a vector and packaged in 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. A number of retroviral systems are known in the art.

[0207] In some embodiments, vectors of the present disclosure include additional promoter elements, such as enhancers that regulate the frequency of transcriptional initiation. Enhancers are typically located in a region that is 30 bp to 110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements may be flexible, so that promoter function is preserved 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 apart before activity begins to decline. Depending on the promoter, individual elements may function either cooperatively or independently to activate transcription. Exemplary promoters may include, without limitation, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EFla promoter, the ubiquitin C promoter, and the phosphoglycerokinase (PGK) promoter.

[0208] In some embodiments, a promoter that is capable of expressing a nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been widely used in mammalianexpression plasmids and has been shown to be effective in driving chimeric receptor expression from nucleic acids cloned into a lentiviral vector.

[0209] In some embodiments, a promoter that is capable of expressing a nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is a constitutive promoter. For example, a suitable constitutive promoter is the immediate early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter that is capable of driving high levels of expression of any polynucleotide sequence operatively linked to the promoter. Other suitable constitutive promoters include, without limitation, a ubiquitin C (UbiC) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, an elongation factor-la promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0210] In some embodiments, a promoter that is capable of expressing a nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is an inducible promoter. Use of an inducible promoter may provide a molecular switch that is capable of inducing or repressing expression of a nucleic acid of the present disclosure when the promoter is operatively linked to the nucleic acid. Examples of inducible promoters include, without limitation, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0211] In some embodiments, a vector of the present disclosure may further comprise a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator, an element allowing episomal replication, and / or elements allowing for selection.

[0212] In some embodiments, a vector of the present disclosure can further comprise a selectable marker gene and / or reporter gene to facilitate identification and selection of chimeric receptor-expressing cells from a population of cells that have been transduced with the vector. In some embodiments, the selectable marker may be encoded by a nucleic acid that is separate from the vector and used in a co-transfection procedure. Either selectable marker or reporter gene may be flanked with appropriate regulator sequences to allow expression in host cells. Examples of selectable markers include, without limitation, antibiotic-resistance genes, such as neo and the like.

[0213] In some embodiments, reporter genes may be used for identifying transduced cells and for evaluating the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes apolypeptide whose expression results in an easily detectable property, such as enzymatic activity. 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, without limitation, genes encoding for luciferase, genes encoding for beta- galactosidase, genes encoding for chloramphenicol acetyl transferase, genes encoding for secreted alkaline phosphatase, and genes encoding for green fluorescent protein. Suitable expression systems are well known in the art and may be prepared using known techniques or obtained commercially. In some embodiments, a construct with a minimal 5' flanking region showing the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0214] In some embodiments, a vector comprising a nucleic acid sequence encoding an VSIG2-specific protein (e.g., chimeric receptor) of the present disclosure further comprises a second nucleic acid encoding a polypeptide that increases the activity of the chimeric receptor.

[0215] In embodiments where an VSIG2-specific protein-expressing cell comprises two or more heterologous proteins (e.g., two or more chimeric receptors), a single nucleic acid may encode the two or more proteins under a single regulatory control element (e.g., promoter) or under separate regulatory control elements for each protein-encoding nucleotide sequence comprised in the nucleic acid. In some embodiments where an VSIG2-specific proteinexpressing cell comprises two or more heterologous proteins, each heterologous protein may be encoded by a separate nucleic acid. In some embodiments, each separate nucleic acid comprises its own control element (e.g., promoter). In some embodiments, a single nucleic acid encodes the two or more chimeric receptors and the chimeric receptor-encoding nucleotide sequences are in the same reading frame and are expressed as a single polypeptide chain. In such embodiments, the two or more chimeric receptors may be separated by one or more peptide cleavage sites, such as auto-cleavage sites or substrates for an intracellular protease. Suitable peptide cleavage sites may include, without limitation, a T2A peptide cleavage site, a P2A peptide cleavage site, an E2A peptide cleavage sire, and an F2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise a T2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise an E2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise a T2A and an E2A peptide cleavage site.

[0216] Methods of introducing and expressing genes into a cell are well known in the art. For example, in some embodiments, an expression vector can be transferred into a host cell by physical, chemical, or biological means. Examples of physical means for introducing a nucleicacid into a host cell include, without limitation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Examples of chemical means for introducing a nucleic acid into a host cell include, without limitation, colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. Examples of biological means for introducing a nucleic acid into a host cell include, without limitation, the use of DNA and RNA vectors.

[0217] In some embodiments, liposomes may be used as a non-viral delivery system to introduce a nucleic acid or vector of the present disclosure into a host cell in vitro, ex vivo, or in vivo. In some embodiments, the nucleic acid may be associated with a lipid, for example by being encapsulated in the aqueous interior of a liposome, being interspersed within the lipid bilayer of a liposome, being attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, being entrapped in a liposome, being complexed with a liposome, being dispersed in a solution containing a lipid, being mixed with a lipid, being combined with a lipid, being contained as a suspension in a lipid, being contained or complexed with a micelle, or otherwise being associated with a lipid. 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 be present in a bilayer structure, as micelles or with a "collapsed" structure. Such compositions may also be interspersed in a solution, forming aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that may be naturally occurring or synthetic. In some embodiments, lipids can include the fatty droplets that naturally occur in the cytoplasm or the class of compounds that contain 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, without limitation, dimyristyl phosphatidylcholine ("DMPC"), dicetylphosphate ("DCP"), cholesterol, and dimyristylphosphatidylglycerol ("DMPG"). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about - 20°C. Chloroform is used as the solvent, as it is more readily evaporated than methanol. As used herein, a "liposome" may encompass a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. In some embodiments, liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, multilamellar liposomes may have multiple lipid layers separated by aqueous medium. Multilamellar liposomes can form spontaneously when phospholipids are suspended in an excess of aqueous solution. In some embodiments, lipid components may undergo self-rearrangement before the formation of closed structures and canentrap water and dissolved solutes between the lipid bilayers. In some embodiments, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0218] In some embodiments, a nucleic acid or vector of the present disclosure is introduced into a mammalian host cell, such as an immunoresponsive cell of the present disclosure. In some embodiments, the presence of a nucleic acid or vector of the present disclosure in a host cell may be confirmed by any suitable assay known in the art, including without limitation Southern blot assays, Northern blot assays, RT-PCR, PCR, ELISA assays, and Western blot assays.

[0219] In some embodiments, a nucleic acid or vector of the present disclosure is stably transduced into an immunoresponsive cell of the present disclosure. In some embodiments, cells that exhibit stable expression of the nucleic acid or vector 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.

[0220] In embodiments where an VSIG2-specific protein (e.g., chimeric receptor) of the present disclosure is transiently expressed in a cell, an VSIG2-specific protein-encoding nucleic acid or vector of the present disclosure is transfected into an immunoresponsive cell of the present disclosure. In some embodiments the immunoresponsive cell expresses the VSIG2- specific protein for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after transfection.

[0221] In some embodiments, the nucleic acid construct encodes a bicistronically encoded chimeric antigen receptors. In some embodiments, the encoded bicistronic chimeric antigen receptors comprise an VSIG2 CAR (such as an VSIG2 inhibitory CAR) and a CAR specific for a second antigen (such as a tumor-targeting chimeric receptor).

[0222] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor. In some embodiments, the encoded bivalent chimeric antigen receptor comprises an VSIG2 antigen-binding domain and a second antigen-binding domain.Multicistronic Expression Systems

[0223] Provided herein, in various embodiments, are multicistronic expression systems. In some embodiments, the multicistronic expression system comprises: (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). In certain embodiments, the multicistronic expression system comprises an activating CAR (aCAR) and an inhibitory CAR (iCAR).

[0224] Also provided herein, in various embodiments, are immunoresponsive cells engineered to have the following: (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).

[0225] The multicistronic expression system or immunoresponsive cells disclosed herein can include an activation-control polypeptide. The ACP can include a synthetic transcription factor. A synthetic transcription factor is a non-naturally occurring protein that includes a DNA-binding domain and a transcriptional effector domain and is capable of modulating (i.e., activating or repressing) transcription through binding to a cognate promoter recognized by the DNA-binding domain (an ACP-responsive promoter). In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator.

[0226] The membrane-cleavable chimeric protein can be engineered such that secretion of the effector molecule can be regulated in a protease -dependent manner. Specifically, the membrane -cleavable chimeric protein can be engineered such that secretion of the effector molecule can be regulated as part of a “Membrane-Cleavable” system, where incorporation of a protease cleavage site (“C”) and a cell membrane tethering domain (“MT”) allow for regulated secretion of an effector molecule in a protease-dependent manner. Without wishing to be bound by theory, the components of the Membrane-Cleavable system present in the membrane- cleavable chimeric protein generally regulate secretion through the below cellular processes:

[0227] MT: The cell membrane tethering domain contains a transmembrane domain (or a transmembrane -intracellular domain) that directs cellular-trafficking of the chimeric protein such that the protein is inserted into, or otherwise associated with, a cell membrane (“tethered”)

[0228] C: Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space. Generally, the protease cleavage site is protease-specific, including sites engineered to be protease-specific. The protease cleavage site can be selected or engineered to achieve optimal protein expression, celltype specific cleavage, cell-state specific cleavage, and / or cleavage and release of the payload at desired kinetics (e.g., ratio of membrane-bound to secreted chimeric protein levels)

[0229] In some aspects, membrane -cleavable chimeric proteins (or engineered nucleic acids encoding the membrane-cleavable chimeric proteins) are provided for herein having a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane tethering domain.

[0230] An “effector molecule,” refers to a molecule (e.g., a nucleic acid such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates thebiological activity of that molecule to which it binds. For example, an effector molecule may act as a ligand to increase or decrease enzymatic 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 a molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0231] In general, for all membrane -cleavable chimeric proteins described herein, an effector molecule is a cytokine or active fragment thereof (the secretable effector molecule referred to as “S” in the formula S - C - MT or MT - C - S) that includes a cytokine or active fragments thereof.

[0232] The term “modulate” encompasses maintenance of a biological activity, inhibition (partial or complete) of a biological activity, and stimulation / activation (partial or complete) of a biological activity. The term also encompasses decreasing or increasing (e.g., enhancing) a biological activity. Two different effector molecules are considered to “modulate different tumor-mediated immunosuppressive mechanisms” when one effector molecule modulates a tumor-mediated immunosuppressive mechanism (e.g., stimulates T cell signaling) that is different from the tumor-mediated immunosuppressive mechanism modulated by the other effector molecule (e.g., stimulates antigen presentation and / or processing).

[0233] Modulation by an effector molecule may be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates activity of that molecule, and as a result of that modulation, the activity of yet another molecule (to which the effector molecule is not bound) is modulated.

[0234] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immunostimulatory and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immunostimulatory and / or anti-tumor immune response by 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%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immunostimulatory and / or anti-tumor immune response 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%. It should be understood that “an increase” in an immunostimulatory and / or anti-tumor immune response, for example, systemically or in a tumormicroenvironment, is relative to the immunostimulatory and / or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s).

[0235] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immunostimulatory and / or anti-tumor immune response (e.g, systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor- mediated immunosuppressive mechanism may result in an increase in an immunostimulatory and / or anti -tumor immune response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor- mediated immunosuppressive mechanism results in an increase in an immunostimulatory and / or anti-tumor immune response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

[0236] Non-limiting examples of immunostimulatory and / or anti-tumor 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, stroma degradation, immunostimulatory metabolite production, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Thl polarization, promoting an anti -tumor immune response), and / or Type I interferon signaling. An effector molecule may stimulate at least one (one or more) of the foregoing immunostimulatory mechanisms, thus resulting in an increase in an immunostimulatory response. Changes in the foregoing immunostimulatory and / or anti -tumor immune mechanisms may be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g, of particular markers), and / or cell secretion assays (e.g, of cytokines).

[0237] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor- mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by 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%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response 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%. It should be understood that “a decrease” in an immunosuppressive response, for example, systemically or in a tumor microenvironment, is relative to the immunosuppressive response that would otherwise occur, in the absence of the effector molecule(s).

[0238] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g, 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2- 70, 2-80, 2-90, or 2- 100 fold.

[0239] Non-limiting examples of immunosuppressive mechanisms include negative costimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), T regulatory (Treg) cell signaling, tumor checkpoint molecule production / maintenance, myeloid-derived suppressor cell signaling, activity and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. An effector molecule may inhibit at least one (one or more) of the foregoing immunosuppressive mechanisms, thus resulting in a decrease in an immunosuppressive response. Changes in the foregoing immunosuppressive mechanisms may be assessed, for example, by assaying for an increase in T cell proliferation and / or an increase in IFNy production (negative co-stimulatory signaling, Treg cell signaling and / or MDSC); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (tumor checkpoint molecule production / maintenance); ELISA, LUMINEX®, RNA via qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling).

[0240] In some embodiments, effector molecules function additively: the effect of two effector molecules, for example, may be equal to the sum of the effect of the two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules, for example, may be greater than the combined function of the two effector molecules.

[0241] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or modify tumor microenvironments may be any of the cytokines described herein.

[0242] 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.

[0243] In some embodiments, at least one of the effector molecules (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 stroma degradation, (h) stimulates immunostimulatory metabolite production, (i) stimulates Type I interferon signaling, (j) inhibits negative costimulatory signaling, (k) inhibits pro- apoptotic signaling of anti-tumor immune cells, (1) inhibits T regulatory (Treg) cell signaling, activity and / or recruitment, (m) inhibits tumor checkpoint molecules, (n) stimulates stimulator of interferon genes (STING) signaling, (o) inhibits myeloid-derived suppressor cell signaling, activity and / or recruitment, (p) degrades immunosuppressive factors / metabolites, (q) inhibits vascular endothelial growth factor signaling, and / or (r) directly kills tumor cells.

[0244] Non-limiting examples of cytokines are listed in Table 8. Effector molecules can be human or human equivalents of murine effector molecules listed in Table 8. Effector molecules can be human-derived, such as the endogenous human effector molecule or an effector molecule modified and / or optimized for function, e.g., codon optimized to improve expression, modified to improve stability, or modified at its 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 the improvement desired, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).Table 8. Exemplary Effector MoleculesTable 10: Sequences of exemplary effector molecules

[0245] The first engineered nucleic acid can include a nucleotide sequence 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 a sequence provided in Table 10. The first engineered nucleic acid can include a nucleotide sequence having a sequence provided in Table 10.

[0246] The first engineered nucleic acid can include a nucleotide sequence 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 a sequence of human IL15. The first engineered nucleic acid can include a nucleotide sequence having a sequence of human IL15.

[0247] The second engineered nucleic acid can include a nucleotide sequence 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 a sequence provided in Table 10. The second engineered nucleic acid can include a nucleotide sequence having a sequence provided in Table 10.

[0248] The second engineered nucleic acid can include a nucleotide sequence 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 a sequence of human IL21. The second engineered nucleic acid can include a nucleotide sequence having a sequence of human IL21.

[0249] The first engineered nucleic acid can include a nucleotide sequence having a first sequence provided in Table 10; and (b) the second engineered nucleic acid can include a nucleotide sequence having a sequence provided in Table 10.

[0250] The first engineered nucleic acid can include a nucleotide sequence having a first sequence of human IL15; and (b) the second engineered nucleic acid can include a nucleotide sequence having a sequence of human IL21.

[0251] Immunoresponsive cells provided for herein can include any one of the engineered nucleic acids described herein. Immunoresponsive cells provided for herein can include combinations of any one of the engineered nucleic acids described herein. Immunoresponsive cells provided for herein can include two or more of any one of the engineered nucleic acids described herein.

[0252] Immunoresponsive cells provided for herein can include a nucleotide sequence 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 a sequence provided in Table 10. Immunoresponsive cells provided for herein can include a nucleotide sequence having a sequence provided in Table 10.

[0253] Immunoresponsive cells provided for herein can include a nucleotide sequence 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 a sequence of human IL15.Immunoresponsive cells provided for herein can include a nucleotide sequence having a sequence of human IL15.

[0254] Immunoresponsive cells provided for herein can include a nucleotide sequence 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 a sequence of human IL21. Immunoresponsive cells provided for herein can include a nucleotide sequence having a sequence of human IL21.

[0255] Immunoresponsive cells provided for herein can include a nucleotide sequence having a first sequence provided in Table 10; and (b) a second engineered nucleic acid including a nucleotide sequence having a sequence provided in Table 10.

[0256] Immunoresponsive cells provided for herein can include a nucleotide sequence having a first sequence of human IL15; and (b) a second engineered nucleic acid including a nucleotide sequence having a sequence of human IL15.

[0257] Expression vectors provided for herein can include any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include combinations of any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include two or more of any one of the engineered nucleic acids described herein.

[0258] Expression vectors provided for herein can include a nucleotide sequence 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 a sequence provided in Table 10. Expression vectors provided for herein can include a nucleotide sequence having a sequence provided in Table 10.

[0259] Expression vectors provided for herein can include a nucleotide sequence 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 a sequence of human IL15. Expression vectors provided for herein can include a nucleotide sequence having a sequence of human IL15.

[0260] Expression vectors provided for herein can include a nucleotide sequence 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 a sequence of human IL21. Expression vectors provided for herein can include nucleotide sequence having a sequence of human IL21.

[0261] Expression vectors provided for herein can include a nucleotide sequence having a first sequence provided in Table 10; and (b) a second engineered nucleic acid including a nucleotide sequence having a sequence provided in Table 10.

[0262] Expression vectors provided for herein can include a nucleotide sequence having a first sequence of human IL15; and (b) a second engineered nucleic acid including a nucleotide sequence having a sequence of human IL21.

[0263] In various embodiments, a first cytokine and / or a second cytokine of a multicistronic expression system disclosed herein is a calibrated release cytokine. As used herein, the terms “membrane-cleavable,” “controlled release,” and “calibrated release” are used interchangeably. In certain embodiments, the cytokine is membrane cleavable. In certain embodiments, the cytokine is a calibrated release (cr) cytokine. In certain embodiments, the calibrated release cytokine comprises a B7-1 transmembrane domain. In certain embodiments, the B7-1 transmembrane domain comprises the amino acid sequence of a B7-1 transmembrane domain disclosed in Table 14. In certain embodiments, the calibrated release cytokine comprises a “slow” protease cleavage site comprising the amino acid sequence of VTPEPIFSLI. In certain embodiments, the calibrated release cytokine comprises a “fast protease cleavage site comprising the amino acid sequence of PRAEALKGG.

[0264] In some embodiments, the cytokine is a calibrated release IL15 (crIL15). In some embodiments the crIL15 comprises the “slow” protease cleavage site. In certain embodiments, the crIL15 comprising the “slow” protease cleavage site comprises the amino acid sequence of crIL15 - “slow” protease cleavage site disclosed in Table 10. An exemplary nucleic acid sequence encoding the crIL15 comprising the “slow” protease cleavage site is disclosed in Table 10. In certain embodiments, a nucleic acid encoding crIL15 comprising the “slow” 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 crIL15 comprising the “slow” protease cleavage site disclosed in Table 10.

[0265] In some embodiments, the crIL15 comprising the “slow” protease cleavage site also comprises a furin cleavage site. The crIL15 comprising the “slow” protease cleavage site and the furin cleavage site may comprise the amino acid sequence of crIL15 “slow” protease cleavage site and furin cleavage site disclosed in Table 10. An exemplary nucleic acid sequence encoding crIL15 comprising the “slow” protease cleavage site and the furin cleavage site is disclosed in Table 10. In certain embodiments, a nucleic acid encoding crIL15 comprising the “slow” protease cleavage site and the 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 sequence of crIL15 “slow” protease cleavage site and furin cleavage site disclosed in Table 10.

[0266] In certain embodiments, the crIL15 comprises the “fast” protease cleavage site comprising the amino acid sequence of PRAEALKGG. In certain embodiments, the crIL15 comprising the “fast” protease cleavage site comprises the amino acid sequence of crIL15 - “fast” protease cleavage site disclosed in Table 10. An exemplary nucleic acid sequence encoding crIL15 comprising the “fast” protease cleavage site is disclosed in Table 10. In certain embodiments, a nucleic acid encoding crIL15 comprising the “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 crIL15 - “fast” protease cleavage site disclosed in Table 10.

[0267] In certain embodiments, the crIL15 comprises the amino acid sequence of crIL15 disclosed in Table 10. An exemplary nucleic acid sequence encoding crIL15 is disclosed in Table 10. In certain embodiments, a 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.

[0268] In certain embodiments, the crIL15 comprises a sushi domain. In certain embodiments the crIL15 comprises an IgE leader sequence. In certain embodiments, the crIL15 comprises a sushi domain and an IgE leader sequence. In certain embodiments, the crIL15 comprises the amino acid sequence of crIL15 - sushi domain and IgE leader sequence disclosed in Table 10. An exemplary nucleic acid sequence encoding crIL15 comprising a sushi domain and an IgE leader sequence is disclosed in Table 10. In certain embodiments, a nucleic acid encoding crIL15 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 crIL15 provided in Table 10.

[0269] In certain embodiments, the chimeric IL 15 comprises a sushi domain. In certain embodiments the chimeric IL15 comprises an IgE leader sequence. In certain embodiments, the chimeric IL 15 comprises a sushi domain and an IgE leader sequence. In certain embodiments, the chimeric IL 15 comprises the amino acid sequence of chimeric IL 15 - sushi domain and IgE leader sequence disclosed in Table 10. An exemplary nucleic acid sequence encoding chimeric IL 15 comprising a sushi domain and an IgE leader sequence is disclosed in Table 10. In certain embodiments, a nucleic acid encoding chimeric IL 15 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 chimeric IL 15 - sushi domain and IgE leader sequence disclosed in Table 10.

[0270] In certain embodiments, the IL15 is a membrane-bound IL15 (mbIL15). In certain embodiments, the 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, a 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.

[0271] In certain embodiments, the 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, a 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.

[0272] In certain embodiments, the IL21 comprises a codon-optimized IL21 leader sequence. In certain embodiments, the IL21 comprises the amino acid sequence of IL21 - codon- optimized leader sequence disclosed in Table 10. Two exemplary nucleic acid sequence encoding IL21 comprising a codon-optimized IL21 leader sequence are provided in Table 10. In certain embodiments, a nucleic acid encoding IL21 comprising a codon-optimized IL21 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 IL21 - codon-optimized leader sequence - 1. In certain embodiments, a nucleic acid encoding IL21 comprising a codon-optimized IL21 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 IL21 - codon-optimized leader sequence - 2.

[0273] In some embodiments, the IL21 comprises a furin cleavage site. In certain embodiments, the IL21 comprises the amino acid sequence of IL21 - furin cleavage sequence disclosed in Table 10. An exemplary nucleic acid sequence encoding IL21 comprising a furin cleavage site is provided in Table 10. In certain embodiments, a nucleic acid encoding IL21 comprising 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 sequence of IL21 - furin cleavage sequence disclosed in Table 10.

[0274] In certain embodiments, the IL7 comprises the amino acid sequence of IL7 disclosed in Table 10. An exemplary nucleic acid sequence encoding IL7 is disclosed in Table 10. In certain embodiments, a 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.

[0275] In certain embodiments, the IL12p70 comprises the amino acid sequence of IL12p70. An exemplary nucleic acid sequence encoding IL12p70 is disclosed in Table 10. In certain embodiments, a 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 sequence of IL12p70 disclosed in Table 10.Secretion Signals and Signal- Anchors

[0276] The one or more effector molecules (e.g., any of the cytokines described herein) of the membrane-cleavable chimeric proteins provided for herein are in general secretable effector molecules having a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the chimeric protein’s N-terminus (e.g., an effector molecule’s N-terminus for S - C - MT) that direct newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the proper protein processing pathways. For chimeric proteins having the formula MT - C - S, a membrane tethering domain generally has a signalanchor sequence (e.g., signal-anchor sequences of a Type II transmembrane protein) that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways. For chimeric proteins having the formula S - C - MT, a membrane tethering domain having a reverse signal-anchor sequence (e.g., signal-anchor sequences of certain Type III transmembrane proteins) can be used, generally without a separate secretion signal peptide, that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways.

[0277] In general, for all membrane-cleavable chimeric proteins described herein, the one or more effector molecules are secretable effector molecules (referred to as “S” in the formula S - C - MT or MT - C - S). In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal such that each effector molecule is capable of secretion from an engineered cell following cleavage of the protease cleavage site.

[0278] The secretion signal peptide operably associated with an effector molecule can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenouslyassociated with the given effector molecule, such as a cytokine’s endogenous secretion signal peptide). The secretion signal peptide operably associated with an effector molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments. Non-limiting examples of non- native secretion signal peptide are shown in Table 11.Table 11. Exemplary Signal Secretion PeptidesProtease Cleavage Site

[0279] In general, all membrane -cleavable chimeric proteins described herein contain a protease cleavage site (referred to as “C” in the formula S - C - MT or MT - C - S). In general, the protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease. Examples of protease cleavage sites include, but are not limited to, a Type 1 transmembrane protease cleavage site, a Type II transmembrane protease cleavage site, a GPIanchored protease cleavage site, an ADAM8 protease cleavage site, an ADAM9 protease cleavage site, an ADAM 10 protease cleavage site, an ADAM 12 protease cleavage site, an ADAM 15 protease cleavage site, an ADAM 17 protease cleavage site, an ADAM 19 protease cleavage site, an ADAM20 protease cleavage site, an ADAM21 protease cleavage site, an ADAM28 protease cleavage site, an ADAM30 protease cleavage site, an ADAM33 protease cleavage site, a BACE1 protease cleavage site, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, or an NS3 protease cleavage site.

[0280] One example of a protease cleavage site is a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including, but not limited to, a NS3 / NS4A, a NS4A / NS4B, aNS4B / NS5A, or aNS5A / NS5B cleavage site. For a description ofNS3 protease and representative sequences of its cleavage sites for various strains of HCV, see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; herein incorporated by reference in its entirety. For example, the sequences of HCV NS4A / 4B protease cleavage site; HCV NS5A / 5B protease cleavage site; C-terminal degron with NS4A / 4B protease cleavage site; N-terminal degron with HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. 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, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference.

[0281] Another example of a protease cleavage site is an ADAM17-specific protease (also referred to as Tumor Necrosis Factor-a Converting Enzyme [TACE]) cleavage site. An ADAM17-specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM 17. An ADAM17-specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM17. An engineered ADAM17-specific protease cleavage site can be an engineered for specific desired properties including, but not limited to, optimal expression of the chimeric proteins, specificity for ADAM17, rate-of- cleavage by ADAM17, ratio of secreted and membrane -bound chimeric protein levels, andcleavage in different cell states. A protease cleavage site can be selected for specific cleavage by ADAMI 7. For example, certain protease cleavage sites capable of being cleaved by ADAM 17 are also capable of cleavage by additional ADAM family proteases, such as ADAM10. Accordingly, an ADAM17-specific protease cleavage site can be selected and / or engineered such that cleavage by other proteases, such as ADAM10, is reduced or eliminated. A protease cleavage site can be selected for rate-of-cleavage by ADAM 17. For example, it can be desirable to select a protease cleavage site demonstrating a specific rate-of-cleavage by ADAM 17, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by ADAM17. In such cases, in general, a specific rate-of-cleavage can be selected to regulate the rate of processing of the chimeric protein, which in turn regulates the rate of release / secretion of the payload effector molecule. Accordingly, an ADAM17-specific protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage by ADAMI 7. A protease cleavage site can be selected for both specific cleavage by ADAM 17 and rate-of-cleavage by ADAMI 7. Exemplary ADAM17-specific protease cleavage sites, including those demonstrating particular specificity and rate-of-cleavage kinetics, are shown in Table 12 below with reference to the site of cleavage (P5-P1: N-terminal; Pl’-P5’: C-terminal). Further details of ADAM17 and ADAM10, including 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 herein incorporated by reference for purposes.Table 12 - Potential ADAM17 Protease Cleavage Site Sequences

[0282] In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE. In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG. In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG, wherein Xi is A, Y, P, S, or F, and wherein X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG. In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR. In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG. In some embodiments, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS. In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD. In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI.

[0283] In certain embodiments, a cleavage site comprises a linker sequence. A cleavage site may be flanked on the N terminal and / or C terminal sides by a linker sequence. For example and without limitation, the cleavage site may be flanked on both the N terminal and C terminal sides by a partial glycine -serine (GS) linker sequence. Upon cleavage, the N terminal partial GS linker, and C terminal partial GS linker, join to form a GS linker sequence, such as the amino acid sequence of SGGGGSGGGGSGGGGSGGGGSGGGSLQ.

[0284] In certain embodiments, the cleavage site and linker comprise the amino acid sequence of SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ. An exemplary nucleic acid sequence encoding SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ is TCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTT CAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA. In some embodiments, nucleic acids encoding SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ may compriseTCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTT CAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA, 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.

[0285] In certain embodiments, the protease cleavage site is N-terminal to a linker. In certain embodiments, the protease cleavage site and linker comprise the amino acid sequence of PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ. An exemplary nucleic acid sequence encoding PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAG GCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT. In some embodiments, nucleic acids encoding PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ may comprise CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAG GCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT, 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.

[0286] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF, which is a cleavage site that is native to CD 16 and is cleavable by ADAM17. In certain embodiments, ITQGLAVSTISSFF is comprised within a linker. In certain embodiments, the linker comprises the amino acid sequence of SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ. An exemplary nucleic acid sequence encoding SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ is AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCG TGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTG GATCTCTTCAA. In some embodiments, nucleic acids encoding SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ may comprise AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCG TGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTG GATCTCTTCAA, 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.

[0287] The protease cleavage site can be C-terminal of the secretable effector molecule. The protease cleavage site can be N-terminal of the secretable effector molecule. In general, for all membrane -cleavable chimeric proteins described herein, the protease cleavage site is either: (1) C-terminal of the secretable effector molecule and N-terminal of the cell membrane tetheringdomain (in other words, the protease cleavage site is in between the secretable effector molecule and the cell membrane tethering domain); or (2) N-terminal of the secretable effector molecule and C-terminal of the cell membrane tethering domain (also between the secretable effector molecule and the cell membrane tethering domain with domain orientation inverted). The protease cleavage site can be connected to the secretable effector molecule by a polypeptide linker, i. e. , a polypeptide sequence not generally considered to be part of the effector molecule or protease cleavage site. The protease cleavage site can be connected to the cell membrane tethering domain by a polypeptide linker, i. e. , a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or protease cleavage site. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GSJrGG), A(EAAAK)SA, and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD, an eGK linker such as the amino acid sequence EGKSSGSGSESKST, an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ, and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional exemplary polypeptide linkers include SGGGGSGGGGSG,TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, and GGGSGGGGSGGGSLQ. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. An exemplary nucleic acid sequence encoding TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCC TCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAA GAGGACTGGATTTCGCCTGCGAC. In certain embodiments, a nucleic acid encoding TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 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 ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCC TCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAA GAGGACTGGATTTCGCCTGCGAC .

[0288] In the Membrane-Cleavable system, following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of thechimeric protein such that the effector molecule is released (“secreted”) into the extracellular space of a cell.

[0289] In general, a protease that cleaves the protease cleavage site is a protease specific for that specific protease cleavage site. For example, in the case of a disintegrin and metalloproteinase (“ADAM”) family protease, the protease that cleaves a specific ADAM protease cleavage site is generally limited to the ADAM protease(s) that specifically recognize the specific ADAM protease cleavage site motif. A protease cleavage site can be selected and / or engineered such that cleavage by undesired proteases is reduced or eliminated. Proteases can be membrane -bound or membrane-associated. Proteases can be secreted, e.g., secreted in a specific cellular environment, such as a tumor microenvironment (“TME”).

[0290] A protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to a cell expressing the chimeric protein. In other words, a cell engineered to express the chimeric protein can endogenously express the protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of the protease refers to both expression under generally homeostatic conditions (e.g., a cell generally considered to be healthy), and also to differential expression under non-homeostatic conditions (e.g. , upregulated expression in a tumor cell). The protease cleavage site can be selected based on the known proteases endogenously expressed by a desired cell population. In such cases, in general, the cleavage of the protease cleavage site (and thus release / secretion of a payload) can be restricted to only those cells of interest due to the cell-restricted protease needing to come in contact with the protease cleavage site of chimeric protein expressed in the same cell. For example, and without wishing to be bound by theory, ADAM 17 is believed to be restricted in its endogenous expression to NK cell and T cells. Thus, selection of an ADAM17-specific protease cleavage site may restrict the cleavage of the protease cleavage site to NK cell and T cells co-expressing the chimeric protein. In other examples, a protease cleavage site can be selected for a specific tumor-associated protease known to be expressed in a particular tumor population of interest (e.g., in a specific tumor cell engineered to express the chimeric protein). Protease and / or expression databases can be used to select an appropriate protease cleavage site, such as selecting a protease cleavage site cleaved by a tumor-associated proteases through consulting Oncomine (www.oncomine.org), the European Bioinformatic Institute (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.bumham.org), each of which is incorporated by reference for all purposes.

[0291] A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to a cell expressing the chimeric protein. For example, a cell engineered to express the chimeric protein can also be engineered to express a protease not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein. A cell engineered to express both the chimeric protein and the protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic systems (multicistronic and multi-promoter systems are described in greater detail in the Section herein titled “Multicistronic and Multiple Promoter Systems”). Heterologous proteases and their corresponding protease cleavage site can be selected as described above with reference to endogenous proteases.

[0292] A protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a separate distinct cell than the cell that expresses the chimeric protein. For example, the protease can be generally expressed in a specific cellular environment, such as a tumor microenvironment. In such cases, in general, the cleavage of the protease cleavage site can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. In embodiments having membrane-cleavable chimeric proteins, in general, the secretion of the effector molecule can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the separate distinct cell. A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the separate distinct cell. For example, the separate distinct cell can be engineered to express a protease not generally expressed by the separate distinct cell.

[0293] Proteases include, but are not limited to, a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease. A protease can be an NS3 protease. A protease can be an ADAM17 protease. Proteases can be tumor associated proteases, such as, a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a 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, CD 10 (CALLA), or prostate specific antigen. Proteases can also include, but are not limited to, proteases listed in Table 13 below. Exemplary cognate protease cleavage sites for certain proteases are also listed in Table 13.Table 13: Exemplary Proteases with Cognate Cleavage Sites and Inhibitors

[0294] A protease can be any of the following human proteases (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. & Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320-325; herein incorporated by reference for all purposes): pepsin A (MER000885), gastricsin (MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005534), napsin A (MER004981), Memame-AA034 peptidase (MERO 14038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens)-type putative peptidase (MER107386), napsin B pseudogene (MER004982), CYMP g.p. (Homo sapiens) (MER002929), subfamily A1A unassigned peptidases (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retrovirus endopeptidase (MER043650), S71 -related human endogenous retropepsin (MER001812), RTVL-H-type putative peptidase (MER047117), RTVL-H-type putative peptidase (MER047133), RTVL-H-type putative peptidase (MER047160), RTVL-H-type putative peptidase (MER047206), RTVL-H-type putative peptidase (MER047253), RTVL-H-type putative peptidase (MER047260), RTVL-H- type putative peptidase (MER047291), RTVL-H-type putative peptidase (MER047418), RTVL- H-type putative peptidase (MER047440), RTVL-H-type putative peptidase (MER047479), RTVL-H-type putative peptidase (MER047559), RTVL-H-type putative peptidase (MER047583), RTVL-H-type putative peptidase (MER015446), human endogenous retrovirus retropepsin homologue 1 (MERO 15479), human endogenous retrovirus retropepsin homologue 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 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 retrovirus 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 retrovirus retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retrovirus 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) (MER005305), 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 non-peptidase homologues (MER047046), subfamily A2A non-peptidase homologues (MER047052), subfamily A2A non-peptidase homologues (MER047076), subfamily A2A non-peptidase homologues (MER047080), subfamily A2A non- peptidase homologues (MER047088), subfamily A2A non-peptidase homologues (MER047089), subfamily A2A non-peptidase homologues (MER047091), subfamily A2A non- peptidase homologues (MER047092), subfamily A2A non-peptidase homologues (MER047093), subfamily A2A non-peptidase homologues (MER047094), subfamily A2A non- peptidase homologues (MER047097), subfamily A2A non-peptidase homologues (MER047099), subfamily A2A non-peptidase homologues MER047101), subfamily A2A non- peptidase homologues (MER047102), subfamily A2A non-peptidase homologues (MER047107), subfamily A2A non-peptidase homologues (MER047108), subfamily A2A non- peptidase homologues (MER047109), subfamily A2A non-peptidase homologues(MER047110), subfamily A2A non-peptidase homologues MER047111), subfamily A2A non- peptidase homologues (MER047114), subfamily A2A non-peptidase homologues(MER047118), subfamily A2A non-peptidase homologues (MER047121), subfamily A2A non- peptidase homologues (MER047122), subfamily A2A non-peptidase homologues (MER047126), subfamily A2A non-peptidase homologues (MER047129), subfamily A2A non-I l lpeptidase homologues (MER047130), subfamily A2A non-peptidase homologues (MER047134), subfamily A2A non-peptidase homologues (MER047135), subfamily A2A non- peptidase homologues (MER047137), subfamily A2A non-peptidase homologues(MER047140), subfamily A2A non-peptidase homologues (MER047141), subfamily A2A non- peptidase homologues (MER047142), subfamily A2A non-peptidase homologues(MER047148), subfamily A2A non-peptidase homologues (MER047149), subfamily A2A non- peptidase homologues (MER047151), subfamily A2A non-peptidase homologues(MER047154), subfamily A2A non-peptidase homologues (MER047155), subfamily A2A non- peptidase homologues (MER047156), subfamily A2A non-peptidase homologues(MER047157), subfamily A2A non-peptidase homologues (MER047159), subfamily A2A non- peptidase homologues (MER047161), subfamily A2A non-peptidase homologues(MER047163), subfamily A2A non-peptidase homologues (MER047166), subfamily A2A non- peptidase homologues (MER047171), subfamily A2A non-peptidase homologues(MER047173), subfamily A2A non-peptidase homologues (MER047174), subfamily A2A non- peptidase homologues (MER047179), subfamily A2A non-peptidase homologues(MER047183), subfamily A2A non-peptidase homologues (MER047186), subfamily A2A non- peptidase homologues (MER047190), subfamily A2A non-peptidase homologues(MER047191), subfamily A2A non-peptidase homologues (MER047196), subfamily A2A non- peptidase homologues (MER047198), subfamily A2A non-peptidase homologues(MER047199), subfamily A2A non-peptidase homologues (MER047201), subfamily A2A non- peptidase homologues (MER047202), subfamily A2A non-peptidase homologues(MER047203), subfamily A2A non-peptidase homologues (MER047204), subfamily A2A non- peptidase homologues (MER047205), subfamily A2A non-peptidase homologues(MER047207), subfamily A2A non-peptidase homologues (MER047208), subfamily A2A non- peptidase homologues (MER047210), subfamily A2A non-peptidase homologues(MER047211), subfamily A2A non-peptidase homologues (MER047212), subfamily A2A non- peptidase homologues (MER047213), subfamily A2A non-peptidase homologues(MER047215), subfamily A2A non-peptidase homologues (MER047216), subfamily A2A non- peptidase homologues (MER047218), subfamily A2A non-peptidase homologues(MER047219), subfamily A2A non-peptidase homologues (MER047221), subfamily A2A non- peptidase homologues (MER047224), subfamily A2A non-peptidase homologues(MER047225), subfamily A2A non-peptidase homologues (MER047226), subfamily A2A non- peptidase homologues (MER047227), subfamily A2A non-peptidase homologues(MER047230), subfamily A2A non-peptidase homologues (MER047232), subfamily A2A non- peptidase homologues (MER047233), subfamily A2A non-peptidase homologues(MER047234), subfamily A2A non-peptidase homologues (MER047236), subfamily A2A nonpeptidase homologues (MER047238), subfamily A2A non-peptidase homologues(MER047239), subfamily A2A non-peptidase homologues (MER047240), subfamily A2A non- peptidase homologues (MER047242), subfamily A2A non-peptidase homologues(MER047243), subfamily A2A non-peptidase homologues (MER047249), subfamily A2A non- peptidase homologues (MER047251), subfamily A2A non-peptidase homologues(MER047252), subfamily A2A non-peptidase homologues (MER047254), subfamily A2A non- peptidase homologues (MER047255), subfamily A2A non-peptidase homologues(MER047263), subfamily A2A non-peptidase homologues (MER047265), subfamily A2A non- peptidase homologues (MER047266), subfamily A2A non-peptidase homologues(MER047267), subfamily A2A non-peptidase homologues (MER047268), subfamily A2A non- peptidase homologues (MER047269), subfamily A2A non-peptidase homologues(MER047272), subfamily A2A non-peptidase homologues (MER047273), subfamily A2A non- peptidase homologues (MER047274), subfamily A2A non-peptidase homologues(MER047275), subfamily A2A non-peptidase homologues (MER047276), subfamily A2A non- peptidase homologues (MER047279), subfamily A2A non-peptidase homologues(MER047280), subfamily A2A non-peptidase homologues (MER047281), subfamily A2A non- peptidase homologues (MER047282), subfamily A2A non-peptidase homologues(MER047284), subfamily A2A non-peptidase homologues (MER047285), subfamily A2A non- peptidase homologues (MER047289), subfamily A2A non-peptidase homologues(MER047290), subfamily A2A non-peptidase homologues (MER047294), subfamily A2A non- peptidase homologues (MER047295), subfamily A2A non-peptidase homologues(MER047298), subfamily A2A non-peptidase homologues (MER047300), subfamily A2A non- peptidase homologues (MER047302), subfamily A2A non-peptidase homologues(MER047304), subfamily A2A non-peptidase homologues (MER047305), subfamily A2A non- peptidase homologues (MER047306), subfamily A2A non-peptidase homologues(MER047307), subfamily A2A non-peptidase homologues (MER047310), subfamily A2A non- peptidase homologues (MER047311), subfamily A2A non-peptidase homologues(MER047314), subfamily A2A non-peptidase homologues (MER047318), subfamily A2A non- peptidase homologues (MER047320), subfamily A2A non-peptidase homologues(MER047321), subfamily A2A non-peptidase homologues (MER047322), subfamily A2A non- peptidase homologues (MER047326), subfamily A2A non-peptidase homologues(MER047327), subfamily A2A non-peptidase homologues (MER047330), subfamily A2A non- peptidase homologues (MER047333), subfamily A2A non-peptidase homologues(MER047362), subfamily A2A non-peptidase homologues (MER047366), subfamily A2A non-peptidase homologues (MER047369), subfamily A2A non-peptidase homologues (MER047370), subfamily A2A non-peptidase homologues (MER047371), subfamily A2A non- peptidase homologues (MER047375), subfamily A2A non-peptidase homologues(MER047376), subfamily A2A non-peptidase homologues (MER047381), subfamily A2A non- peptidase homologues (MER047383), subfamily A2A non-peptidase homologues(MER047384), subfamily A2A non-peptidase homologues (MER047385), subfamily A2A non- peptidase homologues (MER047388), subfamily A2A non-peptidase homologues(MER047389), subfamily A2A non-peptidase homologues (MER047391), subfamily A2A non- peptidase homologues (MER047394), subfamily A2A non-peptidase homologues(MER047396), subfamily A2A non-peptidase homologues (MER047400), subfamily A2A non- peptidase homologues (MER047401), subfamily A2A non-peptidase homologues(MER047403), subfamily A2A non-peptidase homologues (MER047406), subfamily A2A non- peptidase homologues (MER047407), subfamily A2A non-peptidase homologues(MER047410), subfamily A2A non-peptidase homologues (MER047411), subfamily A2A non- peptidase homologues (MER047413), subfamily A2A non-peptidase homologues(MER047414), subfamily A2A non-peptidase homologues (MER047416), subfamily A2A non- peptidase homologues (MER047417), subfamily A2A non-peptidase homologues(MER047420), subfamily A2A non-peptidase homologues (MER047423), subfamily A2A non- peptidase homologues (MER047424), subfamily A2A non-peptidase homologues(MER047428), subfamily A2A non-peptidase homologues (MER047429), subfamily A2A non- peptidase homologues (MER047431), subfamily A2A non-peptidase homologues(MER047434), subfamily A2A non-peptidase homologues (MER047439), subfamily A2A non- peptidase homologues (MER047442), subfamily A2A non-peptidase homologues(MER047445), subfamily A2A non-peptidase homologues (MER047449), subfamily A2A non- peptidase homologues (MER047450), subfamily A2A non-peptidase homologues(MER047452), subfamily A2A non-peptidase homologues (MER047455), subfamily A2A non- peptidase homologues (MER047457), subfamily A2A non-peptidase homologues(MER047458), subfamily A2A non-peptidase homologues (MER047459), subfamily A2A non- peptidase homologues (MER047463), subfamily A2A non-peptidase homologues(MER047468), subfamily A2A non-peptidase homologues (MER047469), subfamily A2A non- peptidase homologues (MER047470), subfamily A2A non-peptidase homologues(MER047476), subfamily A2A non-peptidase homologues (MER047478), subfamily A2A non- peptidase homologues (MER047483), subfamily A2A non-peptidase homologues(MER047488), subfamily A2A non-peptidase homologues (MER047489), subfamily A2A non- peptidase homologues (MER047490), subfamily A2A non-peptidase homologues(MER047493), subfamily A2A non-peptidase homologues (MER047494), subfamily A2A nonpeptidase homologues (MER047495), subfamily A2A non-peptidase homologues (MER047496), subfamily A2A non-peptidase homologues (MER047497), subfamily A2A non- peptidase homologues (MER047499), subfamily A2A non-peptidase homologues (MER047502), subfamily A2A non-peptidase homologues (MER047504), subfamily A2A non- peptidase homologues (MER047511), subfamily A2A non-peptidase homologues (MER047513), subfamily A2A non-peptidase homologues (MER047514), subfamily A2A non- peptidase homologues (MER047515), subfamily A2A non-peptidase homologues (MER047516), subfamily A2A non-peptidase homologues (MER047520), subfamily A2A non- peptidase homologues (MER047533), subfamily A2A non-peptidase homologues (MER047537), subfamily A2A non-peptidase homologues (MER047569), subfamily A2A non- peptidase homologues (MER047570), subfamily A2A non-peptidase homologues (MER047584), subfamily A2A non-peptidase homologues (MER047603), subfamily A2A non- peptidase homologues (MER047604), subfamily A2A non-peptidase homologues (MER047606), subfamily A2A non-peptidase homologues (MER047609), subfamily A2A non- peptidase homologues (MER047616), subfamily A2A non-peptidase homologues(MER047619), subfamily A2A non-peptidase homologues (MER047648), subfamily A2A non- peptidase homologues (MER047649), subfamily A2A non-peptidase homologues (MER047662), subfamily A2A non-peptidase homologues (MER048004), subfamily A2A non- peptidase homologues (MER048018), subfamily A2A non-peptidase homologues (MER048019), subfamily A2A non-peptidase homologues (MER048023), subfamily A2A non- peptidase homologues (MER048037), subfamily A2A unassigned peptidases (MER047164), subfamily A2A unassigned peptidases (MER047231), subfamily A2A unassigned peptidases (MER047386), skin aspartic protease (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas 1 peptidase (MERO 19701), impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase (MERO 19711), 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), 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 g.p. (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 (MER029744), Memame-AA253 peptidase (MER005537), calpamodulin (MER000718), hypothetical protein 940251 (MER003201), ubiquitinyl hydrolase- L1 (MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinyl hydrolase-BAP 1 (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 (MERO 14033), ubiquitin-specific peptidase 32 (MERO 14290), ubiquitin-specific peptidase 26 (Homo sapiens-type) (MERO 14292), ubiquitin-specific peptidase 24 (MER005706), ubiquitinspecific peptidase 42 (MERO 11852), ubiquitin-specific peptidase 46 (MERO 14629), ubiquitinspecific peptidase 37 (MERO 14633), ubiquitin-specific peptidase 28 (MERO 14634), ubiquitinspecific peptidase 47 (MER014636), ubiquitin-specific peptidase 38 (MER014637), ubiquitinspecific peptidase 44 (MER014638), ubiquitin-specific peptidase 50 (MER030315), ubiquitinspecific peptidase 35 (MERO 14646), ubiquitin-specific peptidase 30 (MERO 14649), Memame- AA091 peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitinspecific peptidase 51 (MERO 14769), ubiquitin-specific peptidase 34 (MERO 14780), ubiquitinspecific peptidase 48 (MER064620), ubiquitin-specific peptidase 40 (MERO 15483), ubiquitinspecific peptidase 41 (MER045268), ubiquitin-specific peptidase 31 (MER015493), Memame- AA129 peptidase (MER016485), ubiquitin-specific peptidase 49 (MER016486), Memame- AA187 peptidase (MER052579), USP17-like peptidase (MER030192), ubiquitin-specificpeptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER027329), ubiquitin-specific endopeptidase 39 [misleading] (MER064621), Memame-AA090 non-peptidase homologue (MERO 14739), ubiquitin-specific peptidase 43 [misleading] (MER030140), ubiquitin-specific peptidase 52 [misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Memame-AA088 peptidase (MERO 14750), autophagin-2 (MERO 13564), autophagin-1 (MERO 13561), autophagin-3 (MERO 14316), autophagin-4 (MER064622), Cezanne deubiquitinylating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor alpha-induced protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), otubain-1 (MER029056), otubain-2 (MER029061), CylD protein (MER030104), UfSPl peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinylating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otudl protein (MER125457), glycosyltransferase 28 domain containing 1, isoform CRA_c (Homo sapiens)- like (MER123606), hinlL g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain containing 1 (Homo sapiens) (MER125334), Josephin domain containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumain (plant alpha form) (MER044591), legumain (MER001800), glycosylphosphatidylinositol: protein transamidase (MER002479), legumain pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidases (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), caspase-14 (MER012083), paracaspase (MER019325), Memame-AA143 peptidase (MER021304), Memame-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Memame-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Memame-AA093 caspase pseudogene (MER014766), subfamily C14A non-peptidase homologues (MER185329), subfamily C14A non-peptidase homologues (MER179956), separase (Homo sapiens-type) (MER011775), separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MERO 12183), SENP5 peptidase (MERO 14032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl-peptidase I (chordate) (MER011032), Memame-AA073 peptidase (MER029978), Sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Memame- AA164 protein (MER020410), LOCI 38971 g.p. (Homo sapiens) (MER020074), Atp23peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamylpeptidase II (MERO 12221), cytosol alanyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase -like 1 (MERO 12271), leukocyte -derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata binding protein associated factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (MER011061), Memame-AA153 protein (MER020514), thimet oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Memame-AA154 protein (MER021317), leishmanolysin- 2 (MER014492), leishmanolysin-3 (MER180031), matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8 (MER001084), matrix 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 (MER004766), membranetype matrix metallopeptidase-5 (MER005638), membrane-type matrix metallopeptidase-6 (MERO 12071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MERO 14098), matrix metallopeptidase-26 (MERO 12072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-23A (MER037217), macrophage elastase homologue (chromosome 8, Homo sapiens) (MER030035), Memame-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), subfamily M10A non-peptidase homologues (MER175912), subfamily M10A non-peptidase homologues (MER187997), subfamily M10A non-peptidase homologues (MER187998), subfamily M10A non-peptidase homologues (MER180000), meprin alpha subunit (MER001111), meprin beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian-type tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MERO 16700), ADAMTS15 peptidase (MERO 17029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 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), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MEROO51OO), ADAMTS4 peptidase (MEROO51O1), AD AMTS 1 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), AD AMTS 10 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), nonpeptidase homologue (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homologue (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homologue (Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homo sapiens- type) (MER005199), ADAMI 1 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Memame-AA225 peptidase homologue (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAMI g.p. (Homo sapiens) (MER003912), subfamily M12B non-peptidase homologues (MER188210), subfamily M12B non-peptidase homologues (MER188211), subfamily M12B non-peptidase homologues (MER188212), subfamily M12B non-peptidase homologues (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER005197), neprilysin-2 (MER013406), Kell blood-group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), paraplegin (MER004454), Afg3-like protein 2 (MER005496), Afg3- like protein 1A (MER014306), pappalysin-1 (MER002217), pappalysin-2 (MER014521), famesylated-protein converting enzyme 1 (MER002646), metalloprotease-related protein- 1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase Al (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 XI (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homologues (MER199530), insulysin (MER001214), mitochondrial processing peptidase beta-subunit (MER004497), nardilysin (MER003883), eupitrilysin (MER004877), mitochondrial 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), insulysin unit 2 (MER046821), nardilysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), nardilysin unit 3 (MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homologues (MER188757), leucyl aminopeptidase (animal) (MER003100), Memame-AA040 peptidase (MER003919), leucyl aminopeptidase- 1 (Caenorhabditis-type) (MERO 13416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote) (MER001248), aminopeptidase Pl (MER004321), mitochondrial intermediate cleaving peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MERO 14055), Memame-AA020 peptidase homologue (MERO 10972), proliferation-association protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1 -like (Homo sapiens chromosome X) (MER029983), Memame-AA226 peptidase homologue (Homo sapiens) (MER056262), Memame-AA227 peptidase homologue (Homo sapiens) (MER047299), subfamily M24A non-peptidase homologues (MER179893), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), camosine dipeptidase II (MER014551), camosine dipeptidase I (MER015142), Memame-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), glutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Memame-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclise (MER015095), glutamate carboxypeptidase II (Homosapiens)-type non-peptidase homologue (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane -bound dipeptidase-3 (MERO 13496), dihydro-orotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase related protein- 1 (MER030143), dihydropyrimidinase related protein-2 (MER030155), dihydropyrimidinase related protein-3 (MER030151), dihydropyrimidinase related protein-4 (MER030149), dihydropyrimidinase related protein-5 (MER030136), hypothetical protein like 5730457F1 IRIK (MER033184), 1300019j 08rik protein (MER033186)), guanine aminohydrolase (MER037714), Kael putative peptidase (MER001577), OSGEPLl-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homologues (MER199845), subfamily M23B non-peptidase homologues (MER199846), subfamily M23B non-peptidase homologues (MER199847), subfamily M23B non-peptidase homologues (MER137320), subfamily M23B non-peptidase homologues (MER201557), subfamily M23B non-peptidase homologues (MER199417), subfamily M23B non-peptidase homologues (MER199418), subfamily M23B non-peptidase homologues (MER199419), subfamily M23B non-peptidase homologues (MER199420), subfamily M23B non-peptidase homologues (MER175932), subfamily M23B non-peptidase homologues (MER199665), Pohl peptidase (MER020382), Jabl / MPN domain metalloenzyme (MER022057), Memame-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinase MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Memame- AA168 protein (MER021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MER030133), IFP38 peptidase homologue (MER030132), subfamily M67A non- peptidase homologues (MER191181), subfamily M67A unassigned peptidases (MER191144), granzyme B (Homo sapiens-type) (MER000168), testisin (MER005212), tryptase beta (MER000136), kallikrein-related peptidase 5 (MER005544), corin (MER005881), kallikrein- related peptidase 12 (MER006038), DESCI peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronan-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Memame-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Memame-AA038 peptidase (MER062848), Memame-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens-type) (MER000020), elastase-2 (MEROOO 118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase (Homo sapiens-type) (MER000123), tryptase alpha (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase-1 (MER003733), pancreatic endopeptidase E (MEROOO 149), pancreatic elastase II (MEROOO 146), enteropeptidase (MER002068), chymotrypsin C (MER000761), prostasin (MER002460), kallikrein 1 (MER000093), kallikrein- related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MEROOO 115), mesotrypsin (MER000022), complement component Clr-like peptidase (MER016352), complement factor D (MEROOO 130), complement component activated Clr (MER000238), complement component activated Cis (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228), pancreatic endopeptidase E form B (MEROOO 150), pancreatic elastase IIB (MER000147), coagulation factor Xlla (MER000187), plasma kallikrein (MER000203) coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MEROOO 188), protein C (activated) (MER000222), acrosin (MER000078), hepsin (MEROOO 156), hepatocyte growth factor activator (MEROOO 186), mannan-binding lectin-associated 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), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2 type A (MER000021), HtrAl peptidase (Homo sapiens-type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysndl peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), matriptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matriptase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovochymase-2 (MER022410), HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testis serine peptidase 5 (MER029901), Memame-AA258 peptidase (MER000285), polyserase-IA unit 1 (MER030879),polyserase-IA unit 2 (MER030880), testis serine peptidase 2 (human-type) (MER033187), hypothetical acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2 (MER061748), peptidase similar to tryptophan / serine protease (MER056263), polyserase-2 unit 1 (MER061777), Memame-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452- like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain-rescue-factor- 1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polyserase-2 unit 2 (MER061760), polyserase-2 unit 3 (MER065694), Memame-AA201 (peptidase homologue) MER099175, secreted trypsin-like serine peptidase homologue (MER030000), polyserase-IA unit 3 (MER029880), azurocidin (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 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), psi-KLKl pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unassigned peptidases (MER216982), subfamily S1A unassigned peptidases (MER216148), amidophosphoribosyltransferase precursor (MER003314), glutamine-fructose-6- phosphate transaminase 1 (MER003322), glutamine :fructose-6-phosphate amidotransferase (MER012158), Memame-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 non-peptidase homologues (MER159286), family C44 unassigned peptidases (MER185625) family C44 unassigned peptidases (MER185626), secemin 1 (MER045376), secemin 2 (MER064573), secemin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamine acid amidase precursor (MER141667), proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit li (MER000552), proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase cl7 (MER026497), proteasome subunit alpha 6 (MER000557), proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4 (MER000554), 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), Memame-AA230 peptidase homologue (Homo sapiens) (MER047329), Memame-AA231 pseudogene (Homo sapiens)(MER047172), Memame-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspase-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), similar to gamma- glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Memame-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homologue (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1 -like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5 "-monophosphate synthetase (MER043387), carbamoyl-phosphate synthase (Homo sapiens-type) (MER078640), dihydro-orotase (N-terminal unit) (Homo sapiens-type) (MER060647), DJ-1 putative peptidase (MER003390), Memame-AAIOO putative peptidase (MER014802), Memame-AAlOl nonpeptidase homologue (MERO 14803), KIAA0361 protein (Homo sapiens-type) (MER042827), Fl 134283 protein (Homo sapiens) (MER044553), non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homologues (MER177016), family C56 non-peptidase homologues (MER176613), family C56 non- peptidase homologues (MER176918), EGF-like module containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), EGF-like module containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus)-type protein (MER123205), GPR56 (Homo sapiens)-type protein (MER122057), latrophilin 2 (MER122199), latrophilin-1 (MER126380), latrophilin 3 (MER124612), protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gprl 14 protein (MER159320), GPR126 vascular inducible 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 homologue (MER166269), GPR113 protein (MER159352),brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001), PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A nonpeptidase homologues (MER201339), subfamily S8A non-peptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily S8A unassigned peptidases (MER191614), tripeptidyl- peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MERO 13484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MERO 17240), Memame-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER017367), Memame-AA196 putative peptidase (MER017368), Memame-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase DI (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile saltdependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues (MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER209181), family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase -like protein (MER005492), RISC peptidase (MERO 10960), family S15 unassigned peptidases (MER199442), family S15unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase -like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccgl -interacting factor b (MER210738), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622). taspase-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). similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205). Memame-AA211 putative peptidase (MER026207). gamma- glutamyltransferase 6 (MER159283). gamma-glutamyl transpeptidase homologue (chromosome 2, Homo sapiens) (MER037241). polycystin-1 (MER126824), KIAA1879 protein (MER159329). polycystic kidney disease 1 -like 3 (MER172554). gamma-glutamyl hydrolase (MER002963). guanine 5 "-monophosphate synthetase (MER043387). carbamoyl -phosphate synthase (Homo sapiens-type) (MER078640). dihydro-orotase (N-terminal unit) (Homo sapiens- type) (MER060647). DJ-1 putative peptidase (MER003390). Memame-AAIOO putative peptidase (MER014802). Memame-AAlOl non-peptidase homologue (MER014803). KIAA0361 protein (Homo sapiens-type) (MER042827). Fl 134283 protein (Homo sapiens) (MER044553). non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094). family C56 non-peptidase homologues (MER177016), family C56 non- peptidase homologues (MER176613). family C56 non-peptidase homologues (MER176918). EGF-like module containing mucin-like hormone receptor-like 2 (MER037230). CD97 antigen (human type) (MER037286). EGF-like module containing mucin-like hormone receptor-like 3 (MER037288). EGF-like module containing mucin-like hormone receptor-like 1 (MER037278). EGF-like module containing mucin-like hormone receptor-like 4 (MER037294). cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Musmusculus)-type protein (MER123205). GPR56 (Homo sapiens)-type protein (MER122057). latrophilin 2 (MER122199). latrophilin- 1 (MER126380). latrophilin 3 (MER124612). protocadherin Flamingo 2 (MER124239). ETL protein (MER126267). G protein-coupled receptor 112 (MER126114). seven transmembrane helix receptor (MER125448). Gprl 14 protein (MER159320). GPR126 vascular inducible 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 homologue (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001), PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidase homologues (MER201339), subfamily S8A nonpeptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily S8A unassigned peptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MERO 13484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Memame-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER017367), Memame-AA196 putative peptidase (MER017368), Memame-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MERO 11604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase DI (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2(MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues(MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER209181), family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MERO 10960), family S15 unassigned peptidases (MER199442), family S15 unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase -like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLI22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccgl -interacting factor b (MER210738).

[0295] Protease enzymatic activity can be regulated. For example, certain proteases can be inactivated by the presence or absence of a specific agent (e.g., that binds to the protease, such as specific small molecule inhibitors). Such proteases can be referred to as a “repressible protease.” Exemplary inhibitors for certain proteases are listed in Table 13. For example, an NS3 protease can be repressed by a protease inhibitor including, but not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In another example, protease activity can be regulated through regulating expression of the protease itself, such as engineering a cell to express a protease using an inducible promoter system (e.g., Tet On / Off systems) or cell-specific promoters (promoters that can be used to express a heterologous protease are described in more detail in the Section herein titled “Promoters”). A protease can also contain a degron, such as any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0296] Protease enzymatic activity can also be regulated through selection of a specific protease cleavage site. For example, a protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage by a desired protease, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage in a cell-state specific manner. For example, various cell states (e.g., following cellular signaling, such as immune cell activation) can influence the expression and / or localization of certain proteases. As an illustrative example, ADAM17 protein levels and localization is known to be influenced by signaling, such as through Protein kinase C (PKC) signaling pathways (e.g, activation by the PKC activator Phorbol-12-myristat-13-acetat [PMA]). Accordingly, a protease cleavage site can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of an effector molecule is increased or decreased, as desired, depending on the protease properties (e.g., expression and / or localization) in a specific cell state. As another example, a protease cleavage site (particularly in combination with a specific membrane tethering domain) can be selected and / or engineered for optimal protein expression of the chimeric protein.Cell Membrane Tethering Domain

[0297] The membrane-cleavable chimeric proteins provided for herein include a cellmembrane tethering domain (referred to as “MT” in the formula S - C - MT or MT - C - S). In general, the cell-membrane tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to be localized to (e.g., inserted into), or otherwise associated with, the cell membrane of the cell expressing the chimeric protein. The cell-membrane tethering domain can be a transmembrane -intracellular domain. The cell-membrane tethering domain can be a transmembrane domain. The cell-membrane tethering domain can be an integral membrane protein domain (e.g., a transmembrane domain). The cell -membrane tethering domain can be derived from a Type I, Type II, or Type III transmembrane protein. The cell-membrane tethering domain can include post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post- translational modification tag, where the post-translational modification tag allows association with a cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid-anchor domains (e.g., a GPI lipid-anchor, a myristoylation tag, or palmitoylation tag). Examples of cell-membrane tethering domains include, but are not limited to, a transmembrane -intracellular domain and / or transmembrane domain derived from PDGFR-beta,CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethering domain can be a cell surface receptor or a cell membrane-bound portion thereof. Sequences of exemplary cell membrane tethering domains are provided in Table 14.Table 14.

[0298] In general, for all membrane -cleavable chimeric proteins described herein, the cell membrane tethering domain is either: (1) C-terminal of the protease cleavage site and N- terminal of any intracellular domain, if present (in other words, the cell membrane tethering domain is in between the protease cleavage site and, if present, an intracellular domain); or (2) N-terminal of the protease cleavage site and C-terminal of any intracellular domain, if present (also between the protease cleavage site and, if present, an intracellular domain with domain orientation inverted). In embodiments featuring a degron associated with the chimeric protein, the degron domain is the terminal cytoplasmic -oriented domain, specifically relative to the cell membrane tethering (in other words, the cell membrane tethering domain is in between the protease cleavage site and the degron). The cell membrane tethering domain can be connected to the protease cleavage site by a polypeptide linker, z.e., a polypeptide sequence not generally considered to be part of cell membrane tethering domain or protease cleavage site. The cell membrane tethering domain can be connected to an intracellular domain, if present, by a polypeptide linker, i. e. , a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the intracellular domain. The cell membrane tethering domain can be connected to the degron, if present, by a polypeptide linker, z.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or degron. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible 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)SA, and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD, an eGK linker such as the amino acid sequence EGKSSGSGSESKST, an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ, and linkers described in more detail in IssuedU.S. Pat. No. 5,990,275 herein incorporated by reference). Additional polypeptide linkers include SGGGGSGGGGSG,TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, and GGGSGGGGSGGGSLQ. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.

[0299] In general, the cell-membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are extracellularly exposed following insertion into, or association with, the cell membrane, such that the protease cleavage site is capable of being cleaved by its respective protease and releasing (“secreting”) the effector molecule into the extracellular space.Degron Systems and Domains

[0300] In some embodiments, any of the proteins described herein can include a degron domain including, but not limited to, a cytokine, a CAR, a protease, a transcription factor, a promoter or constituent of a promoter system (e.g. , an ACP), and / or any of the membrane- cleavable chimeric protein described herein. In general, the degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation through a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of a degron-fusion protein.

[0301] The degron domain can be a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD) including, but not limited to, IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof that is capable of drug-inducible binding of CRBN. The CRBN polypeptide substrate domain can be a chimeric fusion product of native CRBN polypeptide sequences, such as a IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL. Degron domains, and in particular CRBN degron systems, are described in more detail in International Application Pub. No. WO2019 / 089592A1, herein incorporated 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 ItcBa;LQMLPESEDEESYDTESEFTEFTEDELPYDDGSLQMLPESEDEESYDTESEFTEFTEDELP YDD), GRR (residues 352-408 of human pl05;EIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPH), DRR (residues 210-295 of yeast Cdc34;IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDND SVMDRKQPHKAEDESEDVEDVERVSKKD), SNS (tandem repeat of SP2 and NB (SP2-NB- SP2 of influenza A or influenza B; e.g., IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDND SVMDRKQPHKAEDESEDVEDVERVSKKD), RPB (four copies of residues 1688-1702 of yeast RPB;IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDND SVMDRKQPHKAEDESEDVEDVERVSKKD), SPmix (tandem repeat of SP1 and SP2 (SP2- SP1-SP2-SP1-SP2 of influenza A virus M2 protein;PESMREEYRKEGSSLLTEVETPGSPESMREEYRKEGSSLLTEVETPGSPESMREEYRKE), NS2 (three copies of residues 79-93 of influenza A virus NS protein;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), an APC / C degron, a COP1 E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfdin-binding degron, a KEAP 1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a phytohormone- dependent SCF-LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, an Siah binding motif, an SPOP SBC docking motif, or a PCNA binding PIP box.

[0302] Regulated degradation can be drug -inducible. Drugs capable of mediating / regulating degradation can be small-molecule compounds. Drugs capable of mediating / regulating degradation can include an “immunomodulatory drug” (IMiD). In general, as used herein, IMiDs refer to a class of small -molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is 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 degradation of proteins having a CRBN polypeptide substrate domain (e.g., any of secretable effector molecules or other proteins of interest described herein). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDsinclude, but are not limited to, a thalidomide, a lenalidomide, or a pomalidomide. The IMiD can be an FDA-approved drug.

[0303] Proteins described herein can contain a degron domain (e.g., referred to as “D” in the formula ...

Claims

CLAIMSWhat is claimed is:

1. An isolated antibody or antigen binding fragment thereof that specifically binds to human V- set Immunoglobulin domain containing 2 (VSIG2) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1 or 2, and a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3 or 4; wherein the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, and a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7; and wherein:(i) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and the VL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 9, or(ii) the VH comprises a VH complementarity region 3 (CDRH3) having the amino acid sequence of any one of SEQ ID NO: 67-87, and the VL comprises a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8 or 9, optionally wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 88-107, optionally wherein the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

2. The antibody or antigen binding fragment thereof of claim 1, wherein: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; orB) 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; orC) 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; orD) 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; orE) 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; orF) 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; orG) 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; orH) 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; orI) 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; orJ) 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; orK) 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; orL) 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; orM) 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; orN) 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; orO) 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; orP) 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; orQ) the CDRH3 has the amino acid sequence of SEQ ID NO: 79, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8; orR) 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; orS) 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; orT) 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; orU) 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; orV) 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; orW) 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; orX) 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; orY) the CDRH3 has the amino acid sequence of SEQ ID NO: 87, and the CDRL3 has the amino acid sequence of SEQ ID NO: 8.

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

4. A chimeric protein comprising an antibody or antigen binding fragment thereof of any one of claims 1-3 and a heterologous molecule or moiety, optionally wherein the chimeric protein isan antibody-drug conjugate, and wherein the heterologous molecule or moiety comprises a therapeutic agent, optionally wherein the chimeric protein is a chimeric antigen receptor (CAR), and wherein 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, optionally wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response, optionally wherein the one or more intracellular inhibitory domains comprise an ICD derived from PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A , LAG3, LAIR1, SIRPa, 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, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139) or an amino acid sequence with 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 to one or more of the amino acid sequenceVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO: 139), optionally wherein the intracellular inhibitory domain further comprises an enzymatic inhibitory domain and / or an additional intracellular inhibitory co-signaling domain, optionally wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, optionally wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52.

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

6. The engineered expression system of claim 5, wherein the first CAR comprises a first spacer between the first extracellular antigen-binding domain and the first transmembrane domain, optionally wherein the first spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, optionally wherein the first spacer comprises the amino acid sequence of SEQ ID NO: 5, and / or wherein the second CAR comprises a second spacer between the second extracellular antigen-binding domain and the second transmembrane domain, optionally wherein the second spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, optionally wherein the second spacer comprises the amino acid sequence of SEQ ID NO: 50.

7. The engineered expression system of claim 5 or claim 6, wherein the one or more intracellular signaling domains of the first CAR are selected from the group consisting of: a CD3zeta-chain intracellular signaling domain, a CD3epsilon-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD1 la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, an NKp46 intracellular signaling domain, an NKp30 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKG2D intracellular signaling domain, a CD226 intracellular signaling domain, and a CD 160 intracellular signaling domain, optionally wherein the first CAR comprises a CD28 intracellular signaling domain and a CD3zeta-chain intracellular signaling domain; and / or optionally wherein the first transmembrane domain are selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- IBB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, aLAX 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, optionally wherein the first CAR comprises a CD28 transmembrane domain.

8. The engineered expression system of any one of claims 5-7, wherein the first and second nucleic acid sequences are comprised within a single expression vector; or wherein the first nucleic acid sequence is comprised within a first expression vector and the second nucleic acid sequence is comprised within a second expression vector.

9. The engineered expression system of any one of claims 5-8, wherein the first antigenbinding domain binds to any one of CEACAM5, CEACAM6, or CEACAM1, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), optionally wherein: i) the VH comprises a VH complementarity region 1 (CDRH1) , a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an hMN14 VH, and wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an hMN14 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of an hMN14 VH, and 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 to the amino acid sequence of an hMN14 VL, optionally wherein the VH comprises the amino acid sequence of an hMN 14 VH, and the VL comprises the amino acid sequence of an hMN 14 VL, optionally wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL); or ii) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a BW431 / 26 VH; optionally wherein the VL comprises a VLcomplementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a BW431 / 26 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of a BW431 / 26 VH, and 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 to the amino acid sequence of a BW431 / 26 VL, optionally wherein the VH comprises the amino acid sequence of a BW431 / 26 VH, and the VL comprises the amino acid sequence of a 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 a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an A5B7 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an A5B7 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of an A5B7 VH, and 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 to the amino acid sequence of an A5B7 VL, optionally wherein the VH comprises the amino acid sequence of an A5B7 VH, and the VL comprises the amino acid sequence of an A5B7 VL, iv) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an MFE23 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an MFE23 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein the VHcomprises 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 to the amino acid sequence of an MFE23 VH, and 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 to the amino acid sequence of an MFE23 VL, optionally wherein the VH comprises the amino acid sequence of an MFE23 VH, and the VL comprises the amino acid sequence of an MFE23 VH; or v) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an hMFE23 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an hMFE23 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of an hMFE23 VH, and 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 to the amino acid sequence of an hMFE23 VL, optionally wherein the VH comprises the amino acid sequence of an hMFE23 VH, and the VL comprises the amino acid sequence of an hMFE23 VL; or vi) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an FM4 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an FM4 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of an FM4 VH, and 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 to the amino acid sequence of an FM4 VL,optionally wherein the VH comprises the amino acid sequence of an FM4 VH, and the VL comprises the amino acid sequence of an FM4 VL; or vii) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a cibisatamab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a cibisatamab LC, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of the VH of a cibisatamab HC, and 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 to the amino acid sequence of the VL of a cibisatamab LC, optionally wherein the VH comprises the amino acid sequence of the VH of a cibisatamab HC and the VL comprises the amino acid sequence of the VL of a cibisatamab LC; or viii) the VH comprises a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a tusamitamab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a tusamitamab LC, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of the VH of a tusamitamab HC, and the 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 to the amino acid sequence of the VL of a tusamitamab LC, optionally wherein the HC comprises the amino acid sequence of the VH of a tusamitamab HC, and the VL comprises the amino acid sequence of the VL of a 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 a VHcomplementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of an MRG1 VH; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of an MRG1 VL, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of an MRG1 VH, and 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 to the amino acid sequence of an MRG1 VL, optionally wherein wherein the VH comprises the amino acid sequence of an MRG1 VH, and the VL comprises the amino acid sequence of an 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 a VH complementarity region 1 (CDRH1), a VH complementarity region 2 (CDRH2), and a VH complementarity region 3 (CDRH3) of a tinurilimab HC; wherein the VL comprises a VL complementarity region 1 (CDRL1), a VL complementarity region 2 (CDRL2), and a VL complementarity region 3 (CDRL3) of a tinurilimab LC, and wherein the antibody or antigen binding fragment thereof is humanized, optionally wherein 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 to the amino acid sequence of the VH of a a tinurilimab HC and 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 to the amino acid sequence of the VL of a tinurilimab LC, optionally wherein the VH comprises the amino acid sequence of the VH of a tinurilimab HC and the VL comprises the amino acid sequence of the VL of a tinurilimab LC.

10. The engineered expression system of any one of claims 5-9, further comprising: a. A fourth nucleotide sequence encoding a first cytokine; andb. A fifth nucleotide sequence encoding a second cytokine.

11. The engineered expression system of any one of claims 5-10, wherein at least one of the first and the second cytokines is a controlled release cytokine, optionally wherein the controlled release cytokine has the formula:S - C - MT or MT - C - S whereinS comprises a secretable effector molecule;C comprises a protease cleavage site; andMT comprises a cell membrane tethering domain. optionally wherein the protease cleavage site is cleaved by ADAM10 and / or ADAM17, optionally wherein the protease cleavage site comprises the amino acid sequence of PRAEALKGG or VTPEPIFSLI, optionally wherein the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA, optionally wherein the cell membrane tethering domain comprises a B7-1 transmembrane domain comprising the B7-1 transmembrane domain amino acid sequence set forth in Table 14.

12. The engineered expression system of any one of claims 5-11, wherein the first cytokine is IL15, optionally wherein the IL15 comprises the amino acid sequence of IL15 set forth in Table 10, optionally wherein the IL15 is controlled-release IL15 (crIL15).

13. The engineered expression system of any one of claims 5-12, wherein the second cytokine is IL21, optionally wherein the IL21 comprises the amino acid sequence set forth in Table 10, optionally wherein the IL21 is controlled-release IL21 (crIL21).

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

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

16. An isolated cell or population of engineered cells comprising the engineered nucleic acid of claim 14, the composition of claim 15, the engineered expression system of any one of claims 5- 13, the antigen binding fragment of any one of claims 1-3, the chimeric protein of claim 4, optionally wherein the cell or population of cells is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the cell is autologous or wherein the cell is allogeneic, optionally wherein the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface, optionally wherein 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 comprising: a. an effective amount of the cell or population of engineered cells of claim 16 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof; or b. an effective amount of genetically modified cells expressing the antigen binding fragment of any one of claims 1-3 or the chimeric protein of claim 4 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof, optionally wherein the pharmaceutical composition is for treating and / or preventing a tumor.

18. A method of inhibiting a cell-mediated immune response to a normal cell in a subject, the method comprising administering to a subject a. a therapeutically effective dose of the composition of claim 15,b. any of the cells of claim 16, optionally wherein the isolated cell or population of cells express the chimeric protein comprising the inhibitory CAR of claim 4, c. the composition of claim 17; optionally wherein the method further comprises stimulating a cell-mediated immune response to a tumor cell in the subject, and wherein the isolated cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface, optionally wherein at least one of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor, optionally wherein the normal cell comprises a human VSIG2, optionally wherein the human VSIG2 is expressed on a surface of the normal cell, optionally wherein the normal cell is a healthy (e.g., non-tumor) epithelial cell.

19. A method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of the composition of claim 15, or any of the cells of claim 16, or the composition of claim 17.

20. A kit for treating and / or preventing a tumor, comprising: a. the chimeric protein of claim 4, optionally wherein the kit further comprises written instructions for using the chimeric protein for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject. b. the cell or population of cells of claim 16, optionally wherein the kit further comprises written instructions for using the cell for treating and / or preventing a tumor in a subject c. the vector of claim 14, optionally wherein the kit further comprises written instructions for using the vector for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject; d. the composition of claim 15 or claim 17, optionally wherein the kit further comprises written instructions for using the composition for treating and / or preventing a tumor in a subject;e. the engineered nucleic acid of claim 14, optionally wherein the kit further comprises written instructions for using the nucleic acid for producing one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

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