EP4698564A2

EP4698564A2 - Antigen-binding domains and methods of use thereof

Info

Publication number: EP4698564A2
Application number: EP24793372.4A
Authority: EP
Inventors: Nicholas FRANKEL; Frances Deen LIU; Russell Morrison GORDLEY
Original assignee: Senti Biosciences Inc
Current assignee: Senti Biosciences Inc
Priority date: 2023-04-17
Filing date: 2024-04-17
Publication date: 2026-02-25
Also published as: AU2024256204C1; WO2024220498A3; WO2024220498A2; CN121127497A; TW202506715A; AU2024256204A1; KR20260005287A

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 THEREOF

CROSS 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 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. [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 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 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 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 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 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 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 intracellular signaling 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-4 transmembrane domain, a LAX transmembrane domain, a LAT transmembrane domain, a PD- 1 transmembrane domain, a LAG-3 transmembrane domain, a TIM3 transmembrane domain, a KIR3DS1 transmembrane domain, a KIR3DL1 transmembrane domain, an NKG2D transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain. In some 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 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. 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 of an 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 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. 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, optionally wherein 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 of

VSIG2+ 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, 3^rd Edition, 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 (3^rd Edition, 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 kits and 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. Without being 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, the portion 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 10 ⁶M. 10 ⁷M. 10 ^SM.

10 ⁹M. or 10 ^l0M. 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 intact immunoglobulins, 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 binding molecule 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 10 ⁶M. 10 ⁷M. 10 ^SM.

10 ⁹M. or 10 ^l0M. 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 by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al, (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues 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 acid sequence, 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 a defined 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 acid sequence (Gly-Gly-Gly-Gly-Ser)_n or (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.

[00100] Other interpretational conventions

[00101] 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.

[00102] 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

[00103] 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 and cells 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.

[00104] 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.

[00105] 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).

[00106] 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.

[00107] 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 the group 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.

[00108] 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

[00110] 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 some embodiments a chimeric receptor of the present invention comprises a single antigen-binding domain.

Antibodies and Antigen-binding fragments

[00111] 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.

[00112] 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.

[00113] In some embodiments, a chimeric receptor comprises an antigen-binding domain that specifically binds CEA.

[00114] 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 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 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.

[00115] 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.

[00116] 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.

[00117] 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. [00118] 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.

[00119] 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. [00120] 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.

[00121] 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. [00122] 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.

[00123] 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-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 CEACAM6 and one or more additional antigens.

[00124] 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.

[00125] 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 10'⁶ M, at most about 10'⁷ M, at most about 10'⁸ M, at most about 10'⁹ M, at most about IO'¹⁰ M, at most about 10'¹¹ M, or at most about 10'¹² M.

V-set and Immunoglobulin Domain Containing 2 (VSIG2)-Specific Antigen-binding Domains

[00126] 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”).

[00127] 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

[00128] 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.

[00129] 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 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.

[00130] 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 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 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. [00131] 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.

[00132] 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.

[00133] 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.

[00134] 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.

[00135] 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.

[00136] 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, the VSIG2-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.

[00137] 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.

[00138] hi some embodiments, the scFv has an ammo acid sequence selected from SEQ ID NOs: 108-132.

[00139] 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.

[00140] 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)

[00141] 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, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or combinations thereof.

[00142] 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.

[00143] 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.

[00144] 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. "Third generation" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4- IBB) and an intracellular activation signaling domain (CD3 .

[00145] 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.

[00146] 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.

[00147] 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 10'⁷ M or less, about 1 x 10'⁷ M or less, about 9 x 10'⁸ M or less, about 1 x 10'⁸ M or less, about 9 x 10'⁹ M or less, about 5 x 10'⁹ M or less, about 4 x 10'⁹ M or less, about 3 x 10'⁹ M or less, about 2 x 10'⁹ M or less, or about 1 x 10'⁹ M or less. In some embodiments, the Ka ranges from about is about 2 x 10'⁷ M to about 1 x 10'⁹ M. 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 in iCAR formats can be selected based on affinity, including selected based on higher affinity or lower affinity relative to other VSIG2-specific antigen-binding domains.

[00148] 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). [00149] 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

[00150] 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.

[00151] 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, a recombinant 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.

[00152] 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.

[00153] 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.

[00154] 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). [00155] 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 more scFvs 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

[00156] 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 of EAAAKEAAAKEAAAKEAAAK. In some embodiments, the peptide linker comprises an amino acid sequence of GGSGSGGSGSGGSGS.

[00157] 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).

[00158] 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.

[00159] 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.

[00160] 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).

[00161] 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 antibody molecule, 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.

[00162] 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.

[00163] 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

[00164] 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 that spans 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.

[00165] 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, without limitation, 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.

[00166] 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.

[00167] 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.

[00168] 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

[00169] 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 Sequences

Table 6. Spacer Nucleic Acid Sequences

[00170] 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.

[00171] 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). [00172] In some aspects, the transmembrane domain further comprises at least a portion of an extracellular domain of the same protein.

Intracellular Signaling Domains

[00173] 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.

[00174] 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.

[00175] 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.

[00176] 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 sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE 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.

[00177] 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 signaling domains 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.

[00178] 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.

[00179] 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. [00180] 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.

[00181] Non-limiting examples of intracellular signaling domains (I CDs) are provided in Table 7.

Table 7

[00182] 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.

[00183] 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.

[00184] 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 FLAG tag, 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).

[00185] 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.

[00186] 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.

[00187] 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.

[00188] 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)

[00189] 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.

[00190] 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

[00191] 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.

[00192] 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 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 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)

[00193] 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 the present 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).

[00194] 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).

[00195] 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. [00196] 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 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.

Chimeric TCRs

[00197] 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

[00198] 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.

[00199] 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 of the nucleic acid sequences listed in Table 5. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[00200] 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.

[00201] 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.

[00202] 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.

[00203] 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.

[00204] 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.

[00205] 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. [00206] 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.

[00207] 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.

[00208] 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 mammalian expression plasmids and has been shown to be effective in driving chimeric receptor expression from nucleic acids cloned into a lentiviral vector.

[00209] 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.

[00210] 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.

[00211] 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.

[00212] 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.

[00213] 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 a polypeptide 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.

[00214] 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. [00215] 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.

[00216] 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 nucleic acid 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.

[00217] 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 can entrap 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. [00218] 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. [00219] 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.

[00220] 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.

[00221] 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).

[00222] 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

[00223] 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). [00224] 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).

[00225] 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.

[00226] 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: [00227] 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”) [00228] 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)

[00229] 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.

[00230] 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 the biological 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.

[00231] 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.

[00232] 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).

[00233] 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.

[00234] 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 tumor microenvironment, is relative to the immunostimulatory and/or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s).

[00235] 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.

[00236] 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).

[00237] 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).

[00238] 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.

[00239] 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).

[00240] 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.

[00241] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and/or modify tumor microenvironments may be any of the cytokines described herein. [00242] 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.

[00243] 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 (T_reg) 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.

[00244] 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 Molecules

Table 10: Sequences of exemplary effector molecules [00245] 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.

[00246] 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.

[00247] 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.

[00248] 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.

[00249] 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.

[00250] 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.

[00251] 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.

[00252] 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.

[00253] 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.

[00254] 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.

[00255] 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.

[00256] 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.

[00257] 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.

[00258] 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.

[00259] 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.

[00260] 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.

[00261] 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. [00262] 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.

[00263] 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.

[00264] 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.

[00265] 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. [00266] 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.

[00267] 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.

[00268] 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.

[00269] 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.

[00270] 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. [00271] 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.

[00272] 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.

[00273] 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. [00274] 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. [00275] 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

[00276] 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.

[00277] 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. [00278] The secretion signal peptide operably associated with an effector molecule can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated 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 Peptides

Protease Cleavage Site

[00279] 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 GPI anchored 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.

[00280] 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.

[00281] 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, and cleavage 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 [00282] 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.

[00283] 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.

[00284] 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 comprise TCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTT 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. [00285] 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. [00286] 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.

[00287] 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 tethering domain (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 .

[00288] In the Membrane-Cleavable system, 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 of a cell.

[00289] 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”).

[00290] 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. [00291] 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.

[00292] 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.

[00293] 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 [00294] 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 l peptidase 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-specific peptidase 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), Atp23 peptidase (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 (Homo sapiens)-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 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 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 (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 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). [00295] 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. [00296] 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

[00297] 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.

[00298] 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 Issued

U.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. [00299] 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

[00300] 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.

[00301] 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 FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRD

AL. 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.

[00302] 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 IMiDs include, but are not limited to, a thalidomide, a lenalidomide, or a pomalidomide. The IMiD can be an FDA-approved drug.

[00303] Proteins described herein can contain a degron domain (e.g., referred to as “D” in the formula S - C - MT - D or D - MT - C - S for membrane -cleavable chimeric proteins described herein). In the absence of an IMiD, degron/ubiquitin-mediated degradation of the chimeric protein does not occur. 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. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein such that secretion of the effector molecule is reduced or eliminated. In general, for membrane -cleavable chimeric proteins fused to a degron domain, the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering domain, e.g., the most C-terminal domain in the formula S - C - MT - D or the most N-terminal domain in the formula D - MT - C - S . The degron domain 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 the degron domain. 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 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. In general, the degron is oriented in relation to the cell membrane tethering domain such that the degron is exposed to the cytosol following localization to the cell membrane such that the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosol) and is capable of mediating ubiquitin-mediated degradation.

[00304] For degron-fusion proteins, the degron domain can be N-terminal or C-terminal of the protein of interest, e.g., the effector molecule. The degron domain can be connected to the protein of interest by a polypeptide linker, i. e. , a polypeptide sequence not generally considered to be part of the protein of interest or the degron domain. 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 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. A polypeptide linker can be cleavable, e.g., any of the protease cleavage sites described herein.

Engineered Nucleic Acids

[00305] Provided herein are engineered nucleic acids (e.g., an expression cassette) encoding at least one protein of the present disclosure, such as the cytokines, CARs, ACPs, and/or membrane -cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. Provided herein are engineered nucleic acids (e.g., an expression cassette) encoding two or more proteins, such as two or more of the cytokines, CARs, ACPs, and/or membrane -cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.

[00306] In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric protein, oriented from N- terminal to C-terminal, having the formula: S - C - MT or MT - C - S. S refers to a secretable effector molecule. C refers to a protease cleavage site. MT refers to a cell membrane tethering domain. The promoter is operably linked to the exogenous polynucleotide sequence and S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

[00307] In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine. In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a CAR. In certain embodiments described herein, the engineered nucleic acids encode an expression cassete containing a promoter and an exogenous polynucleotide sequence encoding a membrane -cleavable chimeric protein having a protein of interest (e.g. , any of the effector molecules described herein). The promoter is operably linked to the exogenous polynucleotide sequence and the membrane-cleavable chimeric protein is configured to be expressed as a single polypeptide.

[00308] In certain embodiments described herein, the engineered nucleic acids encode an expression cassete containing a promoter and an exogenous polynucleotide sequence encoding a combination of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins described herein. In certain embodiments described herein, the engineered nucleic acids encode an expression cassete containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and CAR. In certain embodiments described herein, the engineered nucleic acids encode an expression cassete containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and an ACP.

[00309] In certain embodiments described herein, the engineered nucleic acids encode two or more expression cassetes each containing a promoter and an exogenous polynucleotide sequence encoding a cytokine, CAR, ACP, and/or membrane -cleavable chimeric protein described herein. In certain embodiments described herein, the engineered nucleic acids encode two or more expression cassetes each containing a promoter and each separately encoding an exogenous polynucleotide sequence encoding a cytokine and CAR, respectively. In certain embodiments described herein, the engineered nucleic acids encode two or more expression cassetes each containing a promoter and each separately encoding an exogenous polynucleotide sequence encoding a cytokine and an ACP, respectively. In certain embodiments, the two or more expression cassetes are oriented in a head-to-tail orientation. In certain embodiments, the two or more expression cassetes are oriented in a head-to-head orientation. In certain embodiments, the two or more expression cassetes are oriented in a tail-to-tail orientation. In some cases, each expression cassete contains its own promoter to drive expression of the polynucleotide sequence encoding a cytokine and/or CAR. In certain embodiments, the cytokine and CAR are organized as such: 5’-cytokine-CAR-3’ or 5’-CAR-cytokine-3’.

[00310] An “engineered nucleic acid” is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally- occurring, it may include nucleotide sequences that occur in nature. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and/or a viral nucleotide sequence. The term “engineered nucleic acids” includes recombinant nucleic acids and synthetic nucleic acids. A “recombinant nucleic acid” refers to a molecule that is constructed by joining nucleic acid molecules and, in some embodiments, can replicate in a live cell. A “synthetic nucleic acid” refers to a molecule that is amplified or chemically, or by other means, synthesized. Synthetic nucleic acids include those that are chemically modified, or otherwise modified, but can base pair with naturally- occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified intemucleotide linkages and non-natural nucleic acids. Modifications are described in further detail in U.S. Pat. No. 6,673,611 and U.S. Application Publication 2004/0019001 and, each of which is incorporated by reference in their entirety. Modified intemucleotide linkages can be a phosphorodithioate or phosphorothioate linkage. Non-natural nucleic acids can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), glycol nucleic acid (GNA), a phosphorodiamidate morpholino oligomer (PMO or “morpholino”), and threose nucleic acid (TNA). Non-natural nucleic acids are described in further detail in International Application WO 1998/039352, U.S. Application Pub. No. 2013/0156849, and U.S. Pat. Nos. 6,670,461;

5,539,082; 5,185,444, each herein incorporated by reference in their entirety. Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing. Engineered nucleic acid of the present disclosure may be encoded by a single molecule (e.g., included in the same plasmid or other vector) or by multiple different molecules (e.g, multiple different independently-replicating molecules). Engineered nucleic acids can be an isolated nucleic acid. Isolated nucleic acids include, but are not limited to a cDNA polynucleotide, an RNA polynucleotide, an RNAi oligonucleotide (e.g., siRNAs, miRNAs, antisense oligonucleotides, shRNAs, etc.), an mRNA polynucleotide, a circular plasmid, a linear DNA fragment, a vector, a minicircle, a ssDNA, a bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC), and an oligonucleotide.

[00311] Engineered nucleic acid of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, the Y extension activity of a DNA polymerase and DNA ligase activity. The 5 ' exonuclease activity chews back the 5 ' end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. In some embodiments, engineered nucleic acid constructs are produced using INFUSION® cloning (Clontech).

Promoters

[00312] In general, in all embodiments described herein, the engineered nucleic acids encoding the proteins herein (e.g., a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein) encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the protein. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 distinct proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 distinct proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct proteins. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g. , an exogenous polynucleotide sequence) encoding at least 2 cytokines. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 cytokines. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytokines. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 membrane-cleavable chimeric proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 membrane -cleavable chimeric proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more membrane-cleavable chimeric proteins.

[00313] A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

[00314] A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.” In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

[00315] Promoters of an engineered nucleic acid may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal. The signal may be endogenous or a normally exogenous condition (e.g, light), compound (e.g., chemical or nonchemical compound) or protein (e.g., cytokine) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

[00316] A promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, transcription from the promoter is activated, deactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin-adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon-gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3): 121-34, incorporated herein by reference), an interferon-stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein by reference), aNF- kappaB response element (Wang, V. et al. Cell Reports. 2012; 2(4): 824-839, incorporated herein by reference), and a STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

[00317] Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 15, which shows the design of the promoter and transcription factor, as well as the effect of the inducer molecule towards the transcription factor (TF) and transgene transcription (T) is shown (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, deactivation; DR, derepression) (see Homer, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of inducible promoters include those presented in Table 16.

Table 15. Examples of Responsive Promoters

Table 16. Exemplary Components of Inducible Promoters

[00318] Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1 -alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (see Table 17).

Table 17. Exemplary Constitutive Promoters

[00319] The promoter can be a tissue-specific promoter. In general, a tissue-specific promoter directs transcription of a nucleic acid, (e.g, the engineered nucleic acids encoding the proteins herein (e.g, a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein) such that expression is limited to a specific cell type, organelle, or tissue. Tissue-specific promoters include, but are not limited to, albumin (liver specific, Pinkert et al., (1987)), lymphoid specific promoters (Calame and Eaton, 1988), particular promoters of T-cell receptors (Winoto and Baltimore, (1989)) and immunoglobulins; Bancrji et al., (1983); Queen and Baltimore, 1983), neuron specific promoters (e.g. the neurofilament promoter; Byrne and Ruddle, 1989), pancreas specific promoters (Edlund et al., (1985)) or mammary gland specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166) as well as developmentally regulated promoters such as the murine hox promoters (Kessel and Gruss, Science 249:374-379 (1990)) or the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are fully incorporated by reference herein. The promoter can be constitutive in the respective specific cell type, organelle, or tissue. Tissue-specific promoters and/or regulatory elements can also include promoters from the liver fatty acid binding (FAB) protein gene, specific for colon epithelial cells; the insulin gene, specific for pancreatic cells; the transphyretin, .alpha.1- antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), apolipoprotein Al and LDL receptor genes, specific for liver cells; the myelin basic protein (MBP) gene, specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene, specific for glial cells; OPSIN, specific for targeting to the eye; and the neural-specific enolase (NSE) promoter that is specific for nerve cells. Examples of tissue-specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac tissue and immunoglobulin heavy or light chain promoters for expression in B cells. Other tissue specific promoters include the human smooth muscle alpha-actin promoter. Exemplary tissue-specific expression elements for the liver include but are not limited to HMG-COA reductase promoter, sterol regulatory element 1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, human C- reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-alpha hydroylase (CYP-7) promoter, beta- galactosidase alpha-2,6 sialylkansferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter. Exemplary tissue-specific expression elements for the prostate include but are not limited to the prostatic acid phosphatase (PAP) promoter, prostatic secretory protein of 94 (PSP 94) promoter, prostate specific antigen complex promoter, and human glandular kallikrein gene promoter (hgt-1). Exemplary tissue- specific expression elements for gastric tissue include but are not limited to the human H+/K+-ATPase alpha subunit promoter. Exemplary tissue-specific expression elements for the pancreas include but are not limited to pancreatitis associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, gamma-gamma enolase (neuron- specific enolase, NSE) promoter. Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-l/granzyme B promoter, the terminal deoxy transferase (TdT), lambda 5, VpreB, and lek (lymphocyte specific tyrosine protein kinase p561ck) promoter, the humans CD2 promoter and its 3 ' transcriptional enhancer, and the human NK and T cell specific activation (NKG5) promoter. Exemplary tissue-specific expression elements for the colon include, but are not limited to, pp60c-src tyrosine kinase promoter, organ-specific neoantigens (OSNs) promoter, and colon specific antigen-P promoter. Tissue-specific expression elements for breast cells are for example, but are not limited to, the human alpha-lactalbumin promoter. Exemplary tissue-specific expression elements for the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[00320] In some embodiments, a promoter of the present disclosure is modulated by signals within a tumor microenvironment. A tumor microenvironment is considered to modulate a promoter if, in the presence of the tumor microenvironment, the activity of the promoter is increased or decreased by at least 10%, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased 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%, relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased by 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%, relative to activity of the promoter in the absence of the tumor microenvironment.

[00321] In some embodiments, the activity of the promoter is increased or decreased by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold), relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased 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, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold, relative to activity of the promoter in the absence of the tumor microenvironment.

[00322] In some embodiments, a promoter of the present disclosure is activated under a hypoxic condition. A “hypoxic condition” is a condition where the body or a region of the body is deprived of adequate oxygen supply at the tissue level. Hypoxic conditions can cause inflammation (e.g., the level of inflammatory cytokines increase under hypoxic conditions). In some embodiments, the promoter that is activated under hypoxic condition is operably linked to a nucleotide encoding a protein that decreases the expression of activity of inflammatory cytokines, thus reducing the inflammation caused by the hypoxic condition. In some embodiments, the promoter that is activated under hypoxic conditions comprises a hypoxia responsive element (HRE). A “hypoxia responsive element (HRE)” is a response element that responds to hypoxia-inducible factor (HIF). The HRE, in some embodiments, comprises a consensus motif NCGTG (where N is either A or G).

Activation-Conditional Control Polypeptide (ACP) Promoter Systems

[00323] In some embodiments, a synthetic promoter is a promoter system including an activation-conditional control polypeptide- (ACP-) binding domain sequence and a promoter sequence. Such a system is also referred to herein as an “ACP-responsive promoter.” In general, an ACP promoter system includes a first expression cassette encoding an activation-conditional control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous polynucleotide sequence, such as the exogenous polynucleotide sequence encoding the cytokines, including membrane-cleavable chimeric proteins versions of cytokines, described herein or any other protein of interest (e.g. , a protease or CAR). In some embodiments, the first expression cassette and second expression cassette are each encoded by a separate engineered nucleic acid. In other embodiments, the first expression cassete and the second expression cassete are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest. In some embodiments, a synthetic promoter comprises the nucleic acid sequence of AATTAACGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTTGAAGCAGTCG ACGCCGAAGTCCCGTCTCAGTAAAGGTTGAAGCAGTCGACGCCGAAGAATCGGACT GCCTTCGTATGAAGCAGTCGACGCCGAAGGTATCAGTCGCCTCGGAATGAAGCAGT CGACGCCGAAGATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGT TCTAGAGGGTATATAATGGGGGCCAACGCGTACCGGTGTC or 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 thereto. In some embodiments, a synthetic promoter comprises the nucleic acid sequence of CGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTCGGCGTAGCCGATGTCG CGCTCCCGTCTCAGTAAAGGTCGGCGTAGCCGATGTCGCGCAATCGGACTGCCTTCG TACGGCGTAGCCGATGTCGCGCGTATCAGTCGCCTCGGAACGGCGTAGCCGATGTC GCGCATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAG GGTATATAATGGGGGCCA or 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 thereto.

[00324] The promoters of the ACP promoter system, e.g., either a promoter driving expression of the ACP or the promoter sequence of the ACP-responsive promoter, can include any of the promoter sequences described herein (see “Promoters” above). The ACP-responsive promoter can be derived from minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof. In some embodiments, the ACP-responsive promoter includes a minimal promoter.

[00325] In some embodiments, the ACP -binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP-responsive promoter includes a minimal promoter and the ACP -binding domain includes one or more zinc finger binding sites. The ACP -binding domain can include 1, 2, 3, 4,5 ,6 7, 8, 9, 10, or more zinc finger binding sites. In some embodiments, the transcription factor is a zinc-finger-containing transcription factor. In some embodiments, the zinc-finger-containing transcription factor is a synthetic transcription factor. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). A zinc finger array comprises multiple zinc finger protein motifs that are linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence, e.g. , a ZFA with desired specificity to an ACP-binding domain having a specific zinc finger binding site composition and/or configuration. The ZF motifs can be directly adjacent to each other, or separated by a flexible linker sequence. In some embodiments, a ZFA is an array, string, or chain ofZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,1 3,

14, or 15 zinc finger motifs. The ZFA can have from 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4- 9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 zinc finger motifs. The ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. The ZF domain can have from 1-10, 1-

15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 ZFAs. In some embodiments, the ZF protein domain comprises one to ten ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs.

[00326] In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain.

[00327] The ACP can also further include an effector domain, such as a transcriptional effector domain. For instance, a transcriptional effector domain can be the effector or activator domain of a transcription factor. Transcription factor activation domains are also known as transactivation domains, and act as scaffold domains for proteins such as transcription coregulators that act to activate or repress transcription of genes. Any suitable transcriptional effector domains can be used in the ACP including, but not limited to, a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300, known as a p300 HAT core activation domain; a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain, or any combination thereof.

[00328] In some embodiments, the effector domain is s transcription effector domain selected from: a Herpes Simplex Virus Protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain ofNFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human ElA- associated protein p300, known as a p300 HAT core activation domain; a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

[00329] In some embodiments, the ACP is a small molecule (e.g., drug) inducible polypeptide. For example, in some embodiments, the ACP may be induced by tetracycline (or derivative thereof), and comprises a TetR domain and a VP 16 effector domain. In some embodiments, the ACP includes an estrogen receptor variant, such as ERT2, and may be regulated by tamoxifen, or a metabolite thereof (such as 4-hydroxy-tamoxifen [4-OHT], N- desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen), through tamoxifen-controlled nuclear localization. In some embodiments, the ACP comprises a nuclear-localization signal (NLS). In certain embodiments, the NLS comprises the amino acid sequence of MPKKKRKV. An exemplary nucleic acid sequence encoding MPKKKRKV is ATGCCCAAGAAGAAGCGGAAGGTT or ATGCCCAAGAAAAAGCGGAAGGTG. In some embodiments, a nucleic acid sequence encoding MPKKKRKV may comprise ATGCCCAAGAAGAAGCGGAAGGTT or ATGCCCAAGAAAAAGCGGAAGGTG, or 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 ATGCCCAAGAAGAAGCGGAAGGTT or ATGCCCAAGAAAAAGCGGAAGGTG.

[00330] In some embodiments, the ACP is a small molecule (e.g., drug) inducible polypeptide that includes a repressible protease and one or more cognate cleavage sites of the repressible protease. In some embodiments, a repressible protease is active (cleaves a cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave a cognate cleavage site) in the presence of the specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given repressible protease of the present disclosure. The repressible protease can be any of the proteases described herein that is capable of inactivation by the presence or absence of a specific agent (see “Protease Cleavage Site” above for exemplary repressible proteases, cognate cleavage sites, and protease inhibitors).

[00331] In some embodiments, the ACP has a degron domain (see “Degron Systems and Domains” above for exemplary degron sequences). The degron domain can be in any order or position relative to the individual domains of the ACP. For example, the degron domain can be N-terminal of the repressible protease, C-terminal of the repressible protease, N-terminal of the ZF protein domain, C-terminal of the ZF protein domain, N-terminal of the effector domain, or C-terminal of the effector domain.

[00332] Exemplary sequences of components of ACPs and exemplary ACPs of the present disclosure are provided in Table 18. In some embodiments, nucleic acids may comprise a sequence in Table 18, 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 a sequence in Table 18.

Table 18.

Multicistronic and Multiple Promoter Systems

[00333] In some embodiments, engineered nucleic acids (e.g., an engineered nucleic acid comprising an expression cassette) are configured to produce multiple proteins (e.g., a cytokine, CAR, ACP, membrane -cleavable chimeric protein, and/or combinations thereof). For example, nucleic acids may be configured to produce 2-20 different proteins. In some embodiments, nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2- 11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3- 11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11,

4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9,

5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20,

7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16,

8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9- 11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12- 14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18- 20, 18-19, or 19-20 proteins. In some embodiments, nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins.

[00334] In some embodiments, engineered nucleic acids can be multicistronic, i. e. , more than one separate polypeptide (e.g., multiple proteins, such as a cytokine, CAR, ACP, and/or membrane -cleavable chimeric protein described herein) can be produced from a single mRNA transcript. Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first protein can be linked to a nucleotide sequence encoding a second protein, such as in a first gene linker: second gene 5’ to 3’ orientation. A linker can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence, such as such a flexible linker (e.g., a Gly-Ser-Gly sequence), that further promotes cleavage. In some embodiments, an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element. In certain embodiments, the E2A/T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP. An exemplary nucleic acid encoding the E2A/T2A ribosome skipping element is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT. In certain embodiments, a nucleic acid encoding E2A/T2A ribosome skipping element 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

GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT. In some embodiments, an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element. In certain embodiments, the E2A/T2A ribosome skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP. An exemplary nucleic acid encoding the E2A/T2A ribosome skipping element is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT. In certain embodiments, a nucleic acid encoding an E2A/T2A ribosome skipping element 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 CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT.

[00335] A linker can encode an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a splice acceptor, such as a viral splice acceptor.

[00336] A linker can be a combination of linkers, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[00337] In general, a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and a third protein, each separated by linkers such that separate polypeptides encoded by the first, second, and third proteins are produced).

[00338] Engineered nucleic acids can use multiple promoters to express genes from multiple ORFs, z.e., more than one separate mRNA transcript can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second protein. In general, any number of promoters can be used to express any number of proteins. In some embodiments, at least one of the ORFs expressed from the multiple promoters can be multicistronic.

[00339] Expression cassettes encoded on the same engineered nucleic acid can be oriented in any manner suitable for expression of the encoded exogenous polynucleotide sequences. Expression cassettes encoded on the same engineered nucleic acid can be oriented in the same direction, z.e., transcription of separate cassettes proceeds in the same direction. Constructs oriented in the same direction can be organized in a head-to-tail format referring to the 5' end (head) of the first gene being adjacent to the 3' end (tail) of the upstream gene. Expression cassettes encoded on the same engineered nucleic acid can be oriented in an opposite direction, i. e. , transcription of separate cassettes proceeds in the opposite direction (also referred to herein as “bidirectional”). Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “head-to-head” directionality. As used herein, head- to-head refers to the 5' end (head) of a first gene of a bidirectional construct being adjacent to the 5' end (head) of an upstream gene of the bidirectional construct. Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “tail-to-tail” directionality. As used herein, tail-to-tail refers to the 3' end (tail) of a first gene of a bidirectional construct being adjacent to the 3' end (tail) of an upstream gene of the bidirectional construct.

[00340] ‘ ‘Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence, the multicistronic linkers described above, or the additional promoters that are operably linked to additional ORFs described above. [00341] Exogenous polynucleotide sequences encoded by the expression cassette can include a 3 ’ untranslated region (UTR) comprising an mRNA-destabilizing element that is operably linked to the exogenous polynucleotide sequence, such as exogenous polynucleotide sequences encoding a cytokine (e.g., IL12 or IL12p70). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element and/or a stem-loop destabilizing element (SLDE). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element. In some embodiments, the AU-rich element includes at least two overlapping motifs of the sequence ATTTA. In some embodiments, the AU-rich element comprises ATTTATTTATTTATTTATTTA. In some embodiments, the mRNA-destabilizing element comprises a stem-loop destabilizing element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG. In some embodiments, the mRNA-destabilizing element comprises at least one AU-rich element and at least one SLDE. “AuSLDE” as used herein refers to an AU-rich element operably linked to a stem-loop destabilizing element (SLDE). An exemplary AuSLDE sequence comprises

ATTTAT TATTTATTTATTTAacatcggttccCTGTTTAATATT AAACAG. In some embodiments, the mRNA-destabilizing element comprises a 2X AuSLDE. An exemplary AuSLDE sequence is provided as AITTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaagcATTTA TTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG.

[00342] In certain embodiments, an engineered nucleic acid described herein comprises an insulator sequence. Such insulator sequences function to prevent inappropriate interactions between adjacent regions of a construct. In certain embodiments, an insulator sequence comprises the nucleic acid sequence of ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACG TCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCAT CACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACT ACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCAT TTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCTor 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 thereto.

Immunoresponsive cells

[00343] Certain aspects of the present disclosure relate to a cell, such as an immunoresponsive cell, that has been genetically engineered to comprise one or more chimeric receptors of the present disclosure or one or more nucleic acids encoding such chimeric receptors, and to methods of using such cells for treating malignancies (e.g., CRC). [00344] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primary cell. In some embodiments, the mammalian cell is a cell line. In some embodiments, the mammalian cell a bone marrow cell, a blood cell, a skin cell, bone cell, a muscle cell, a neuronal cell, a fat cell, a liver cell, or a heart cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, without limitation embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSC), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell that is derived or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. Immune cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immune cells include, without limitation, T cells (e.g, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary neuronal cells include, without limitation, neural progenitor cells, neurons (e.g., sensory neurons, motor neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, or serotonergic neurons), astrocytes, oligodendrocytes, and microglia.

[00345] In some embodiments, the cell is an immunoresponsive cell. Immunoresponsive cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immunoresponsive cells of the present disclosure include, without limitation, cells of the lymphoid lineage. The lymphoid lineage, comprising B cells, T cells, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Examples of immunoresponsive cells of the lymphoid lineage include, without limitation, T cells, Natural Killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those from which lymphoid cells may be derived or differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, T cells of the present disclosure can be any type of T cells, including, without limitation, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal associated invariant T cells, and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells may be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as a chimeric TCRs or CARs.

[00346] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[00347] In some embodiments, an immunoresponsive cell of the present disclosure is a T cell. T cells of the present disclosure may be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

[00348] In some embodiments, an immunoresponsive cell of the present disclosure is a universal T cell with deficient TCR-a[3. Methods of developing universal T cells are described in the art, for example, in Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[00349] In some embodiments, an immunoresponsive cell of the present disclosure is an isolated immunoresponsive cell comprising one or more chimeric receptors of the present disclosure. In some embodiments, the immunoresponsive cell comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more chimeric receptors of the present disclosure.

[00350] In some embodiments, an immunoresponsive cell is a T cell. In some embodiments, an immunoresponsive cell is a Natural Killer (NK) cell.

[00351] In some embodiments, an immunoresponsive cell express or is capable of expressing an immune receptor. Immune receptors generally are capable of inducing signal transduction or changes in protein expression in the immune receptor-expressing cell that results in the modulation of an immune response upon binding to a cognate ligand (e.g., regulate, activate, initiate, stimulate, increase, prevent, attenuate, inhibit, reduce, decrease, inhibit, or suppress an immune response). For example, when CD3 chains present in a TCR/CAR cluster in response to ligand binding, an immunoreceptor tyrosine-based activation motifs (ITAMs)-meditated signal transduction cascade is produced. Specifically, in certain embodiments, when an endogenous TCR, exogenous TCR, chimeric TCR, or a CAR (specifically an activating CAR) binds their respective antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3y/6/e/^. etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated that in turn can initiate a T cell activation pathway and ultimately activates transcription factors, such as NF-KB and AP-1. These transcription factors are capable of inducing 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, such as cytokine production and/or T cell mediated killing.

Cells Expressing Multiple Chimeric Receptors

[00352] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises two or more chimeric receptors of the present disclosure. In some embodiments, the cell comprises two or more chimeric receptors, wherein one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, wherein at least one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors, wherein at least one of the four or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises five or more chimeric receptors, wherein at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[00353] In some embodiments, each of the two or more chimeric receptors comprise a different antigen-binding domain, e.g., that binds to the same antigen or to a different antigen. In some embodiments each antigen bound by the two or more chimeric receptors are expressed on the same cell, such as a tumor cell (e.g., same CRC tumor cell).

[00354] In embodiments where a cell of the present disclosure (e.g., an immunoresponsive cell) expresses two or more distinct chimeric receptors, the antigen-binding domain of each of the different chimeric receptors may be designed such that the antigen-binding domains do not interact with one another. For example, a cell of the present disclosure (e.g., an immunoresponsive cell) expressing a first chimeric receptor (e.g., an VSIG2-specific chimeric receptor) and a second chimeric receptor may comprise a first chimeric receptor that comprises an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor may comprise an antibody fragment, such as an scFv, while the antigen-binding domain of the second chimeric receptor may comprise a VHH.

[00355] Without wishing to be bound by theory, it is believed that in cells having a plurality of chimeric membrane embedded receptors that each comprise an antigen-binding domain, interactions between the antigen-binding domains of each of the receptors can be undesirable, because such interactions may inhibit the ability of one or more of the antigen-binding domains to bind their cognate antigens. Accordingly, in embodiments where cells of the present disclosure (e.g., immunoresponsive cells) express two or more chimeric receptors, the chimeric receptors comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv and the antigen-binding domain of the second chimeric receptor comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[00356] In some embodiments, when present on the surface of a cell, binding of the antigenbinding domain of the first chimeric receptor to its cognate antigen (e.g. , an VSIG2-specific chimeric receptor binding to VSIG2) is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen in the absence of the second chimeric receptor. In some embodiments, when present on the surface of a cell, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with one another less than if both were scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with one another 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen-binding domains.

Chimeric Inhibitory Receptors

[00357] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chimeric inhibitory receptors of the present disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigenbinding domain that binds an antigen generally expressed on normal cells (e.g., cells generally considered to be healthy) but not on tumor cells, such as CRC cells. In some embodiments, a chimeric inhibitory receptor 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).

[00358] 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. [00359] 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. 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).

[00360] In some embodiments, a cell of the present disclosure comprises one or more chimeric inhibitory receptors of the present disclosure and further comprises a tumor-targeting chimeric receptor that binds to one or more tumor-associated antigens. In some embodiments, the one or more tumor-associated antigens include a CRC-associated antigen.

[00361] In some embodiments, the inhibitory chimeric receptor binds an antigen that is expressed on a non-tumor cell. Exemplary antigens for use in a chimeric inhibitory receptor are described in Table 19.

Co-stimulatory ligands

[00362] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) can further include one or more recombinant or exogenous co-stimulatory ligands. For example, the cell can be further transduced with one or more co-stimulatory ligands, such that the cell co-expresses or is induced to co-express one or more chimeric receptors of the present disclosure (e.g., the VSIG2-specific CARs described herein) and one or more co-stimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between the one or more chimeric receptors and the one or more co-stimulatory ligands may provide a non- antigen-specific signal important for full activation of the cell. Examples of suitable co- stimulatory ligands include, without limitation, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L)/CD 154, CD137L/4-1BBL, TNF-a, CD134L/OX40L/CD252, CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumor necrosis factor beta (TNFP)/lymphotoxin- alpha (LTa), lymphotoxin-beta (LTP), CD257/B cellactivating factor (B AFF)/Bly s/THANK/Tall- 1, glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF -related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins and possess an immunoglobulin domain (fold). Examples of suitable immunoglobulin superfamily ligands include, without limitation, CD80 and CD86, both ligands for CD28, PD-L1/(B7-H1) that ligands for PD-1. In certain embodiments, the one or more co-stimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

Chemokine receptor

[00363] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chimeric receptors (e.g., the VSIG2-specific CARs described herein) and may further include one or more chemokine receptors. For example, transgenic expression of chemokine receptor CCR2b or CXCR2 in cells, such as T cells, enhances trafficking to CCL2-secreting or CXCL1 -secreting solid tumors (Craddock et al, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw et al. Hum Gene Ther. 2002 Nov 1; 13(16): 1971 -80). Without wishing to be bound by theory, it is believed that chemokine receptors expressed on chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by tumors and improve targeting of the cell to the tumor, which may facilitate the infiltration of the cell to the tumor and enhance the antitumor efficacy of the cell. Chemokine receptors of the present disclosure may include a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment thereof. Examples of suitable chemokine receptors that may expressed on a cell of the present disclosure include, without limitation, a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; a CC chemokine receptor, such as CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; a CX3C chemokine receptor, such as CX3CR1; an XC chemokine receptor, such as XCR1; and chemokine-binding fragments thereof. In some embodiments, the chemokine receptor to be expressed on the cell is chosen based on the chemokines secreted by the tumor.

Chimeric Receptor Regulation

[00364] Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities of chimeric receptor-expressing cells of the present disclosure (e.g., the VSIG2-specific CARs described herein). There are several ways chimeric receptor activities can be regulated. In some embodiments, a regulatable chimeric receptor, wherein one or more chimeric receptor activities can be controlled, may be desirable to optimize the safety and/or efficacy of the chimeric receptor therapy. For example, inducing apoptosis using a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18): 1673-1683) can be used as a safety switch in the chimeric receptor therapy. In some embodiments, a chimeric receptor-expressing cell of the present disclosure can also express an inducible Caspase-9 (iCaspase-9) that, upon administration of a dimerizer drug, such as rimiducid (IUPAC name: [(lR)-3-(3,4-dimethoxyphenyl)-l-[3-[2-[2-[[2-[3-[(lR)-3-(3,4-dimethoxyphenyl)-l-[(2S)- l-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2- carbonyl] oxypropyl] phenoxy] acetyl] amino] ethylamino] -2-oxoethoxy]phenyl]propyl] (2 S) - 1 - [(2S)-2-(3, 4, 5 -trimethoxyphenyl)butanoyl] piperidine -2 -carboxylate), induces activation of the Caspase-9 and results in apoptosis of the cells. In some embodiments, the iCaspase-9 contains a binding domain that comprises a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, which results in inducible and selective depletion of the chimeric receptor-expressing cells.

[00365] Alternatively, in some embodiments a chimeric receptor of the present disclosure may be regulated by utilizing a small molecule or an antibody that deactivates or otherwise inhibits chimeric receptor activity. For example, an antibody may delete the chimeric receptorexpressing cells by inducing antibody dependent cell-mediated cytotoxicity (ADCC). In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express an antigen that is recognized by a molecule that is capable of inducing cell death by ADCC or complement-induced cell death. For example, a chimeric receptor-expressing cell of the present disclosure may further express a receptor capable of being targeted by an antibody or antibody fragment. Examples of suitable receptors that may be targeted by an antibody or antibody fragment include, without limitation, EpCAM, VEGFR, integrins (e.g., av[33, a4, al%p3, a4[37, a5pi, avP3, av), members of the TNF receptor superfamily (e.g., TRAIL-R1 and TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HLA-DR, CEA, CA- 125, MUC1, TAG-67, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CDl la/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, and EGFR, and truncated versions thereof.

[00366] In some embodiments, a chimeric receptor-expressing cell of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capacity but retains an epitope that is recognized by molecules capable of inducing ADCC (e.g., WO2011/056894).

[00367] In some embodiments, a chimeric receptor-expressing cell of the present disclosure further includes a highly expressing compact marker/suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor-expressing cell, which binds an anti-CD20 antibody (e.g., rituximab) resulting in selective depletion of the chimeric receptorexpressing cell by ADCC. Other methods for depleting chimeric receptor-expressing cells of the present disclosure my include, without limitation, administration of a monoclonal anti-CD52 antibody that selectively binds and targets the chimeric receptor-expressing cell for destruction by inducing ADCC. In some embodiments, the chimeric receptor-expressing cell can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can be further coupled to an agent that induces cell killing, such as a toxin. In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express a target protein recognized by a cell depleting agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depleting agent is an anti-CD20 antibody. In such embodiments, the cell depleting agent is administered once it is desirable to reduce or eliminate the chimeric receptor-expressing cell. In some embodiments, the cell depleting agent is an anti-CD52 antibody.

[00368] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides, in which the components of a chimeric receptor of the present disclosure are partitioned on separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, when in the presence of a dimerization molecule, can couple the polypeptides to one another to form a functional chimeric receptor.

Chimeric Receptor-encoding Nucleic Acid Constructs

[00369] Certain aspects of the present disclosure relate to nucleic acids (e.g., isolated nucleic acids) encoding one or more chimeric receptors of the present disclosure. 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.

[00370] 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. , a solid tumor antigen), 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 an amino acid sequence selected from any of the nucleic acid sequences listed in Table 5. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[00371] 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. [00372] 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.

[00373] 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.

[00374] 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.

[00375] 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.

[00376] 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.

[00377] 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 (e.g., gammaretroviruses and lentiviruses), adenoviruses, adeno-associated viruses, and herpes viruses. In some embodiments, a vector of the present disclosure is a retroviral vector. Types of retroviral vectors include lentiviral vectors and gammaretroviral vectors. In some embodiments, a vector of the present disclosure is a lentiviral vector. Lentiviral vectors are derived from lentiviruses, such as human immunodeficiency virus (HIV), and can be suitable for long-term gene transfer as such vectors generally allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors can be advantageous over other retroviral vectors (e.g., murine leukemia viruses) for transducing certain cell types because lentiviral vectors can typically transduce non-proliferating cells. In some embodiments, a vector of the present disclosure is a gammaretroviral vector. Gammaretroviral vectors are derived from viruses of the genus Gammaretrovirus, which includes murine leukemia virus (MLV) and feline leukemia virus.

[00378] Viral particles produced using retroviral vectors (e.g., lentiviral and gammaretroviral vectors) are typically pseudotyped to include an envelope protein that is exogenous to the retrovirus. A common envelope protein used for pseudotyping is the Vesicular stomatitis virus (VSV) G glycoprotein. In some embodiments, the retroviral vector (e.g., lentiviral vector or gammaretrovirus vector) is pseudotyped using a baboon endogenous retrovirus (BaEV) envelope protein. The BaEV envelope protein may be a wild-type BaEV envelope, or may be a variant BaEV envelope, such as a chimeric BaEV having the cytoplasmic tail replaced with that of the MLV-A virus or a version truncated to remove the R peptide at the C-terminus (e.g. , Anais Girard-Gagnepain, et al. Blood 2014; 124 (8): 1221-1231. doi: https://doi.org/10.1182/blood-2014-02-558163).

[00379] 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.

[00380] 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.

[00381] 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 mammalian expression plasmids and has been shown to be effective in driving chimeric receptor expression from nucleic acids cloned into a lentiviral vector.

[00382] 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.

[00383] 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.

[00384] 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. An exemplary signal sequence is provided in Table 20. [00385] 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.

[00386] 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 a polypeptide 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.

[00387] In some embodiments, a vector comprising a nucleic acid sequence encoding a chimeric receptor of the present disclosure further comprises a second nucleic acid encoding a polypeptide that alters the activity of the chimeric receptor as described herein (e.g., modulates or inhibits).

[00388] In some embodiments, a second 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. , a solid tumor antigen), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the second 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 second spacer region. In some embodiments, the second antigen-binding domain is connected to the second transmembrane domain by the spacer region. In some embodiments, the spacer region comprises an amino acid sequence selected from any of the nucleic acid sequences listed in Table 5. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[00389] In embodiments where a chimeric receptor-expressing cell comprises two or more chimeric receptors, a single nucleic acid may encode the two or more chimeric receptors under a single regulatory control element (e.g., promoter) or under separate regulatory control elements for each chimeric receptor-encoding nucleotide sequence comprised in the nucleic acid. In some embodiments where a chimeric receptor-expressing cell comprises two or more chimeric receptors, each chimeric receptor may be encoded by 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.

[00390] 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 nucleic acid 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.

[00391] In some embodiments, a vector can include a dual expression vector system, such as a vector encoding multiple cassettes, where each cassette encodes a separate heterologous construct that can drive expression of a heterologous payload. For example, a dual expression vector system can include vectors where separate cassettes each include separate promoters each driving expression of separate heterologous payloads. Separate promoters can include one of the engineered promoters described herein. In some embodiments, each of the promoters in a dual expression vector include one of the engineered promoters described herein.

[00392] 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 can entrap 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.

[00393] 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.

[00394] 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.

[00395] In embodiments where a chimeric receptor of the present disclosure is transiently expressed in a cell, a chimeric receptor-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 chimeric receptor 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.

[00396] In some embodiments, the nucleic acid construct encodes a bicistronic chimeric antigen receptor, comprising a chimeric receptor and a chimeric inhibitory receptor. In some embodiments, the nucleic acid construct comprises a multicistronic chimeric antigen receptor, comprising two or more chimeric receptors and a chimeric inhibitory receptor. In some embodiments, multiple nucleic acid constructs are used, wherein one construct encodes the chimeric receptor and one construct encodes the chimeric inhibitory receptor.

[00397] In some embodiments, the chimeric receptor binds a CEA-family antigen and the chimeric inhibitory receptor binds a VSIG2 antigen. In some embodiments, the chimeric receptor binds a CEA-family antigen and the chimeric inhibitory receptor binds a CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[00398] In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEA antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM6 antigen.

[00399] In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a CAR with an antigen binding domain targeting VSIG2 and an antigen binding domain targeting any antigen provided in Table 1. In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a CAR with an antigen binding domain derived from an antibody as provided in Table 2 and an antigen binding domain derived from an antibody as provided in Table 3. In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a CAR with an antigen binding domain including an scFv as provided in Table 23.

[00400] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor, comprising a chimeric receptor and a chimeric inhibitory receptor. In some embodiments, the chimeric receptor binds a CEA family antigen and the chimeric inhibitory receptor binds a VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[00401] In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEA antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen and the chimeric receptor binds a CEACAM6 antigen.

[00402] In some embodiments, the encoded bivalent chimeric antigen receptor comprises a CAR with an antigen binding domain targeting a VSIG2 antigen and any antigen provided in Table 1. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a CAR with an antigen binding domain derived from an antibody as provided in Table 2 and antigen binding domain derived from an antibody as provided in Table 3. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a CAR with an antigen binding domain including an scFv as provided in Table 23.

Pharmaceutical Compositions and Administration

[00403] Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising one or more VSIG2-specific proteins (e.g., chimeric receptors) of the present disclosure or immunoresponsive cells of the present disclosure that express such one or more VSIG2-specific proteins. In some embodiments, compositions comprising VSIG2-specific proteins (e.g., chimeric receptors) or genetically modified immunoresponsive cells that express such VSIG2-specific proteins can be provided systemically or directly to a subject for the treatment of a proliferative disorder, such as a myeloid disorder. In certain embodiments, the composition is directly injected into an organ of interest (e.g., an organ affected by a disorder). Alternatively, the composition may be provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during, or after administration of the composition to increase production of T cells, NK cells, or CTL cells in vitro or in vivo.

[00404] Compositions comprising genetically modified cells of the present disclosure may be administered in any physiologically acceptable vehicle, for example intravascularly, although they may also be introduced into bone or other convenient sites where the genetically modified cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In some embodiments, at least IxlO⁵ cells may be administered, eventually reaching IxlO¹⁰ or more cells. Compositions comprising genetically modified cells of the present disclosure can comprise a purified population of cells. Methods for determining the percentage of genetically modified cells in a population of cells are well known in the art and include, without limitation, fluorescence activated cell sorting (FACS). In some embodiments, the purity of genetically modified cells in a population of cells may be about 50%, about 55%, about 60%, or about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more of the cells in the population of cells. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like. In some embodiments, factors can also be included, for example, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, G-CSF, MCSF, GM- CSF, gamma-interferon, and erythropoietin.

[00405] In certain embodiments, the compositions are pharmaceutical compositions comprising genetically modified cells, such as immunoresponsive cells or their progenitors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. In some embodiments, immunoresponsive cells of the present disclosure or their progeny may be derived from peripheral blood cells (e.g., in vivo, ex vivo, or in vitro derived) and may be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition containing a genetically modified cell of the present disclosure), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Formulations

[00406] Certain aspects of the present disclosure relate to formulations of compositions comprising VSIG2-specific proteins (e.g., chimeric receptors) of the present disclosure or genetically modified cells (e.g., immunoresponsive cells of the present disclosure) expressing such proteins. In some embodiments, compositions of the present disclosure comprising genetically modified cells may be provided as sterile liquid preparations, including without limitation isotonic aqueous solutions, suspensions, emulsions, dispersions, and viscous compositions, which may be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions may be more convenient to administer, especially by injection. In some embodiments, viscous compositions can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[00407] In some embodiments, sterile injectable solutions can be prepared by incorporating genetically modified cells of the present disclosure in a sufficient amount of the appropriate solvent with various amounts of any other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. In some embodiments, the compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing agents, pH buffering agents, and antimicrobials depending upon the route of administration and the preparation desired.

[00408] In some embodiments, compositions of the present disclosure may further include various additives that may enhance the stability and sterility of the compositions. Examples of such additives include, without limitation, antimicrobial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination may be prevented by the inclusions of any of various antibacterial and antifungal agents, including without limitation parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of an injectable pharmaceutical formulation of the present disclosure can be brought about by the use of suitable agents that delay absorption, such as aluminum monostearate and gelatin.

[00409] In some embodiments, compositions of the present disclosure can be isotonic, i.e., having the same osmotic pressure as blood and lacrimal fluid. In some embodiments, the desired isotonicity may be achieved using, for example, sodium chloride, dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.

[00410] In some embodiments, the components of the formulations of the present disclosure are selected to be chemically inert and to not affect the viability or efficacy of the genetically modified cells of the present disclosure.

[00411] One consideration concerning the therapeutic use of the genetically modified cells of the present disclosure is the quantity of cells needed to achieve optimal efficacy. In some embodiments, the quantity of cells to be administered will vary for the subject being treated. In certain embodiments, the quantity of genetically modified cells that are administered to a subject in need thereof may range from 1 x 10⁴ cells to 1 x IO¹⁰ cells. In some embodiments, the precise quantity of cells that would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art based on the present disclosure and the knowledge in the art.

Heterologous Moieties and Modifications

[00412] In a further series of embodiments, the VSIG2-specific chimeric proteins herein (e.g., an VSIG2-specific chimeric protein including an 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) include additional moieties and/or modifications.

Drug Conjugates

[00413] In various embodiments, the protein including the VSIG2-specific antigen-binding domain as described herein is conjugated to a therapeutic agent (i.e. drug) to form an antibodydrug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immune modulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In certain embodiments, the therapeutic agents are attached to the antigen-binding domain through a linker peptide, as discussed in more detail herein.

[00414] Methods of preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate drugs to the antigen-binding domains disclosed herein (e.g., 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) are described, e.g., in U.S. Pat. No. 8,624,003 (pot method), U.S. Pat. No. 8,163,888 (one-step), U.S. Pat. No. 5,208,020 (two-step method), U.S. Pat. No. 8,337,856, U.S. Pat. No. 5,773,001, U.S. Pat. No. 7,829,531, U.S. Pat. No. 5,208,020, U.S. Pat. No. 7,745,394, WO 2017/136623, WO 2017/015502, WO 2017/015496, WO 2017/015495, WO 2004/010957, WO 2005/077090, WO 2005/082023, WO 2006/065533, WO 2007/030642, WO 2007/103288, WO 2013/173337, WO 2015/057699, WO 2015/095755, WO 2015/123679, WO 2015/156786, WO 2017/165851, WO 2009/073445, WO 2010/068759, WO 2010/138719, WO 2012/171020, WO 2014/008375, WO 2014/093394, WO 2014/093640, WO 2014/160360, WO 2015/054659, WO 2015/195925, WO 2017/160754, Storz (MAbs. 2015 November-December; 7(6): 989-1009), Lambert et al. (Adv Ther, 2017 34: 1015). Diamantis et al. (British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007. 3: 321- 2), We et al. (Proc Natl Acad Sci USA, 2009. 106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7: 1052-67), Agarwal et al. (Proc Natl Acad Sci USA., 2013, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. (Drug Dev. and D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2014, 25: 1331-41), Liang et al. (J Am Chem Soc., 2014, 136: 10850-3), Drake et al. (Curr Opin Chem Biol., 2015, 28: 174-80), and York et al. (BMC Biotechnology, 2016, 16( 1) :23), each of which is hereby incorporated by reference in its entirety for all that it teaches.

Additional Binding Moieties

[00415] In various embodiments, the VSIG2-specific protein includes an 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 one or more additional binding moieties. In certain embodiments the binding moieties are antibody fragments or antibody formats including, but not limited to, full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, Diabodies, scDiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), herein incorporated by reference for all that it teaches.

[00416] In particular embodiments, the one or more additional binding moieties are attached to the C-terminus of one or more peptides of the VSIG2-specific antigen-binding domain, such as the VH and/or VL, Fab heavy and/or light-chain fragment, or scFv. In particular embodiments, the one or more additional binding moieties are attached to the N-terminus of one or more peptides of the VSIG2-specific antigen-binding domain, such as the VH and/or VL, Fab heavy and/or light-chain fragment, or scFv.

[00417] In certain embodiments, the one or more additional binding moieties are specific for a different antigen or epitope than VSIG2. In certain embodiments, the one or more additional binding moieties are specific for VSIG2.

[00418] In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding domains described herein (e.g., 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) using in vitro methods including, but not limited to, reactive chemistry (e.g., Click-chemistry) and affinity tagging systems. In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding domains described herein (e.g, 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) through Fc-mediated binding (e.g., Protein A/G). In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding domains described herein (e.g., 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) using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the antigen-binding domains described herein and the additional binding moieties on the same expression vector (e.g., plasmid).

Functional/Reactive Groups

[00419] In various embodiments, the antigen-binding domains described herein (e.g., 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) have modifications that comprise functional groups or chemically reactive groups that can be used in downstream processes, such as linking to additional moieties (e.g., drug conjugates and additional binding moieties) and downstream purification processes.

[00420] In certain embodiments, the modifications are chemically reactive groups including, but not limited to, reactive thiols (e.g., maleimide based reactive groups), reactive amines (e.g., N-hydroxy succinimide based reactive groups), “click chemistry” groups (e.g., reactive alkyne groups), and aldehydes bearing formylglycine (FGly). In certain embodiments, the modifications are functional groups including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems etc.). In certain embodiments, the functional groups or chemically reactive groups have a cleavable peptide sequence. In particular embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In particular embodiments, protease cleavage is carried out by intracellular proteases. In particular embodiments, protease cleavage is carried out by extracellular or membrane associated proteases. ADC therapies adopting protease cleavage are described in more detail in Choi et al. (Theranostics, 2012; 2(2): 156-178.), the entirety of which is hereby incorporated by reference for all it teaches. Methods of Treatment

[00421] Certain aspects of the present disclosure relate to methods of using the VSIG2- specific proteins (e.g., chimeric receptors) and genetically modified cells of the present disclosure (e.g., immunoresponsive cells) that express such proteins to treat subjects in need thereof. In some embodiments, the methods of the present disclosure are useful for treating cancer in a subject, such as colorectal cancer (CRC). Other aspects of the present disclosure relate to use of VSIG2-specific chimeric receptors and genetically modified cells of the present disclosure (e.g., immunoresponsive cells) that express such chimeric receptors in methods for treating a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. In some embodiments, the methods of the present disclosure may comprise administering genetically modified cells of the present disclosure in an amount effective to achieve the desired effect, including without limitation palliation of an existing condition, prevention of a condition, treatment an existing condition, management of an existing condition, or prevention of recurrence or relapse of a condition. In some embodiments, the effective amount can be provided in one or a series of administrations of the genetically modified cells of the present disclosure (e.g., immunoresponsive cells). In some embodiments, an effective amount can be provided in a bolus or by continuous perfusion.

[00422] As disclosed herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the immunoresponsive cells administered.

[00423] For adoptive immunotherapy using antigen-specific cells (e.g., immunoresponsive cells such as T cells), cell doses in the range of about 1 x 10⁶ to 1 xlO¹⁰ cells (e.g., about 1 x 10⁹ cells) are typically infused. Upon administration of the cells into the subject and subsequent differentiation, immunoresponsive cells are induced that are specifically directed against the specific antigen. In some embodiments, induction of immunoresponsive cells can include, without limitation, inactivation of antigen-specific cells such as by deletion or anergy. Inactivation is particularly useful to establish or reestablish tolerance such as in autoimmune disorders. The genetically modified cells can be administered by any method known in the art including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal and directly to the thymus.

[00424] In some embodiments, methods of use encompass methods of inhibiting an immune response. Inhibiting an immune response can refer to preventing, attenuating, or inhibiting a cell-mediated immune response, such as, for example, induced by a chimeric receptor expressed on the surface of an immunomodulatory cell. In embodiments, the methods include preventing, attenuating, or inhibiting activation of an activating chimeric receptor expressed on the surface of an immunomodulatory cell.

[00425] In some embodiments, a chimeric inhibitory receptor of the present disclosure is used to prevent, attenuate, inhibit, or suppress an immune response initiated by a tumor targeting chimeric receptor (e.g., an activating CAR). For example, an immunomodulatory cell expresses an inhibitory chimeric antigen that recognizes an antigen target 1 (e.g., a non-tumor antigen) and a tumor-targeting chimeric receptor that recognizes a different antigen target 2 (e.g. , a tumor target). In this example, when the immunomodulatory cell contacts a target cell, the inhibitory and tumor targeting chimeric receptors may or may not bind to their cognate antigen. In a scenario of this example, where the target cell is a non-tumor cell that expresses both antigen target 1 and antigen target 2, both the inhibitory chimeric receptor and the tumor-targeting receptor can be activated. In such cases, the activation of the inhibitory chimeric receptor results in the prevention, attenuation, or inhibition of the tumor targeting chimeric receptor signaling and the immunomodulatory cell is not activated. Similarly, in exemplary instances where the target cell is a non-tumor cell that expresses only antigen target 1, only the inhibitory chimeric receptor can be activated. In contrast, in exemplary instances where the target cell is a tumor cell that expresses only antigen target 2, the inhibitory chimeric receptor cannot be activated while the tumor-targeting chimeric receptor can be activated, resulting in signal transduction that results in activation of the immunomodulatory cell.

[00426] Inhibition of an immune response initiated by a tumor targeting chimeric receptor can be an inhibition or reduction in the activation of the tumor targeting chimeric receptor, an inhibition or reduction in the signal transduction of a tumor targeting chimeric receptor, or an inhibition or reduction in the activation of the immunomodulatory cell. The inhibitory chimeric receptor can inhibit activation of the tumor targeting chimeric receptor, signal transduction by the tumor targeting chimeric receptor, or activation of the immunomodulatory cell by the tumor targeting chimeric receptor by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to the activation of the tumor targeting chimeric receptor, signal transduction, or activation of the immunomodulatory cell as compared to an immunomodulatory cell lacking an inhibitory chimeric receptor. In some embodiments, inhibition refers to a decrease or reduction of the activity of a tumor targeting chimeric receptor before or after it has been activated.

[00427] The immune response can be cytokine or chemokine production and secretion from an activated immunomodulatory cell. The immune response can be a cell-mediated immune response to a target cell.

[00428] In some embodiments, the chimeric inhibitory receptor is capable of suppressing cytokine production from an activated immunomodulatory cell. In some embodiments, the chimeric inhibitory receptor is capable of suppressing a cell-mediated immune response to a target cell, wherein the immune response is induced by activation of the immunomodulatory cell.

Therapeutic Treatment

[00429] In some embodiments, the methods of the present disclosure increase an immune response in a subject in need thereof. In some embodiments, the methods of the present disclosure include methods for treating and/or preventing a myeloid disorder in a subject. In some embodiments, the subject is a human. In some embodiments, suitable human subjects for therapy may comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those who bear a clinically measurable tumor. A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., based on percentage of leukemic cells, by palpation, CAT scan, sonogram, mammogram or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population). In some embodiments, a pharmaceutical composition of the present disclosure is administered to these subjects to elicit an anti-tumor response, with the objective of palliating their condition. In some embodiments, reduction in tumor mass occurs as a result of administration of the pharmaceutical composition, but any clinical improvement will constitute a benefit. In some embodiments, clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor. In some embodiments, a second group of suitable human subjects are "adjuvant group" subjects. These subjects are individuals who have had a history of a myeloid disorder, but have been responsive to another mode of therapy. The prior therapy may have included, without limitation, surgical resection, radiotherapy, and/or traditional chemotherapy. As a result, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. In some embodiments, this group can be further subdivided into high-risk and low-risk individuals. The subdivision can be made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different myeloid disorder. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

[00430] In any and all aspects of increasing an immune response as described herein, any increase or decrease or alteration of an aspect of characteristic(s) or function(s) is as compared to a cell not contacted with an immunoresponsive cell as described herein.

[00431] Increasing an immune response can be both enhancing an immune response or inducing an immune response. For instance, increasing an immune response encompasses both the start or initiation of an immune response, or ramping up or amplifying an on-going or existing immune response. In some embodiments, the treatment induces an immune response. In some embodiments, the induced immune response is an adaptive immune response. In some embodiments, the induced immune response is an innate immune response. In some embodiments, the treatment enhances an immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the treatment increases an immune response. In some embodiments, the increased immune response is an adaptive immune response. In some embodiments, the increased immune response is an innate immune response. [00432] In some embodiments, a further group of subjects are those having a genetic predisposition to a myeloid disorder, but that have not yet evidenced clinical signs of the myeloid disorder. For example, women testing positive for a genetic mutation associated with CRC, but still of childbearing age, may benefit from receiving one or more of the cells of the present disclosure (e.g., immunoresponsive cells) in treatment prophylactically to prevent the occurrence of CRC until it is suitable to perform preventive surgery. In some embodiments, the subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects. In some embodiments, the subjects may have a history of the condition, for which they have already been treated, in which case the therapeutic objective may typically include a decrease or delay in the risk of recurrence.

Combination Therapies

[00433] In some embodiments, genetically modified cells of the present disclosure (e.g., immunoresponsive ells) expressing one or more proteins including an antigen-binding domain (e.g., scFv) of the present disclosure, such as a chimeric receptor of the present disclosure, may be used in combination with other known agents and therapies. In some embodiments, a combination therapy of the present disclosure comprises a genetically modified cells of the present disclosure that can be administered in combination with one or more additional therapeutic agents. In some embodiments, the genetically modified cell and the one or more additional therapeutic agents can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the genetically modified can be administered first, and the one or more additional agents can be administered second, or the order of administration can be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[00434] In some embodiments, a genetically modified cell of the present disclosure may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunoablative agents (e.g., CAMPATH or anti-CD3 antibodies), cytoxin, fludarabme, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, and peptide vaccines.

[00435] In some embodiments, a genetically modified cell of the present disclosure may be used in combination with a lymphodepleting agent. Suitable lymphodepleting agents reduce or decrease lymphocytes, e.g., B cell lymphocytes and/or T cell lymphocytes, prior to immunotherapy. Examples of suitable lymphodepleting agents include, without limitation, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[00436] In some embodiments, a genetically modified cell of the present disclosure may be used in combination with a chemotherapeutic agent. Suitable chemotherapeutic agents include, without limitation, an anthracycline (e.g., doxorubicin), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), an antimetabolite (e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, such as fludarabine), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide).

[00437] Examples of general chemotherapeutic agents suitable for use in combination therapies include, without limitation, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC- Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxy citidine), hydroxyurea (Hydrea®), Idarubicin (Idaniycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[00438] Examples of suitable alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®. Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Rev immune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamme (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Aitretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®).

[00439] Examples of suitable mTOR inhibitors include, without limitation, temsirolimus, ridaforolimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RADOO1), rapamycin (AY22989, Sirolmius®), and XL765.

[00440] Examples of suitable immunomodulators include, without limitation, afutuzumab, pegfdgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047), and IRX-2.

[00441] Examples of suitable anthracy clines include, without limitation, doxorubicin

(Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomyem, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PES®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacet Iravidomycin.

[00442] Examples of suitable vinca alkaloids include, without limitation, vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbme®).

[00443] Examples of suitable proteosome inhibitors include, without limitation, bortezomib (Velcade®); carfilzomib; marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and ONX-0912.

[00444] In some embodiments, a genetically modified cell of the present disclosure is administered in combination with a CD20 inhibitor, e.g., an anti-CD20 antibody, or fragment thereof. Exemplary anti-CD20 antibodies include, without limitation, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU- 015 (Trubion Pharmaceuticals), ocaratuzumab, and Prol31921.

[00445] In some embodiments, a genetically modified cell of the present disclosure is administered in combination with an oncolytic virus. In some embodiments, oncolytic viruses are capable of selectively replicating in and triggering the death of or slowing the growth of a cancer cell. In some cases, oncolytic viruses have no effect or a minimal effect on non-cancer cells. Suitable oncolytic viruses include, without limitation, an oncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)). In some embodiments, the oncolytic virus is a recombinant oncolytic virus.

[00446] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with a protein tyrosine phosphatase inhibitor, e.g., a SHP-I inhibitor or a SHP-2 inhibitor. In one embodiment, a genetically modified cell of the present disclosure can be used in combination with a kinase inhibitor. Examples of suitable kinase inhibitors include, without limitation, CDK4 inhibitors, CDK4/6 inhibitors, BTK inhibitors, phosphatidylinositol 3 -kinase (PI3K) inhibitors, mTOR inhibitors, MNK inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors.

[00447] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and at the tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the efficacy of chimeric receptorexpressing cell therapy. Without being bound by theory, it is believed that administration of a MDSC modulator enhances the efficacy of a genetically modified cell of the present disclosure. Examples of suitable modulators of MDSCs include, without limitation, MCS110 and BLZ945.

[00448] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with an agent that inhibits or reduces the activity of immunosuppressive plasma cells. Immunosuppressive plasma cells have been shown to impede T cell-dependent immunogenic chemotherapy, such as oxaliplatin (Shalapour et al., Nature 2015, 521 :94- 101). In one embodiment, immunosuppressive plasma cells can express one or more of IgA, interleukin (IL)- 10, and PD-L1.

[00449] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with an interleukin- 15 (IL-15) polypeptide, an interleukin- 15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide. In some embodiments, a genetically modified cell of the present disclosure is further modified to express an interleukin- 15 (IL- 15) polypeptide, an interleukin- 15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL- 15 Ra polypeptide.

[00450] In some embodiments, a subject having cancer (e.g., CRC) is administered a genetically modified cell of the present disclosure in combination with an agent, e.g., cytotoxic or chemotherapy agent, a biologic therapy (e.g., antibody, e.g., monoclonal antibody, or cellular therapy), or an inhibitor (e.g., kinase inhibitor). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is a liposomal formulation comprising cytarabine and daunorubicin at a 5: 1 molar ratio. In some embodiments, the subject is administered a chimeric receptorexpressing cell described herein in combination with a hypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g., azacytidine or decitabine. In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a biologic therapy, e.g., an antibody or cellular therapy, e.g., 225Ac-lintuzumab (Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma/Bristol Myers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination a FLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo), crenoianib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), or ASP2215 (Astelias). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with an isocitrate dehydrogenase (IDH) inhibitor, e.g., AG-221 (Celgene/ Agios) or AG- 120 (Agios/Celgene). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a cell cycle regulator, e.g., inhibitor of polo-like kinase 1 (Plkl), e.g., volasertib (Boehringer Ingelheim); or an inhibitor of cyclin-dependent kinase 9 (Cdk9), e.g., alvocidib (Tolero Pharmaceuticals/Sanofi Aventis). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a B cell receptor signaling network inhibitor, e.g., an inhibitor of B-cell lymphoma 2 (Bel- 2), e.g., venetoclax (Abbvie/Roche); or an inhibitor of Button's tyrosine kinase (Btk), e.g., ibrutinib (Pharmacy clics/Johnson & Johnson Janssen Pharmaceutical). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with an inhibitor of Ml aminopeptidase; an inhibitor of histone deacetylase (HDAC), e.g., pracinostat (MEI Pharma); a multi-kinase inhibitor, e.g., rigosertib (Onconova Therapeutics/Baxter/SymBio); or a peptidic CXCR4 inverse agonist, e.g., BL-8040 (BioLineRx).

[00451] In some embodiments, a subject can be administered an agent which enhances the activity or fitness of a genetically modified cell of the present disclosure. For example, the agent may inhibit a molecule that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule. In some embodiments, inhibitory molecules, such as Programmed Death 1 (PD-1) can decrease the ability of the genetically modified cell to mount an immune effector response. Examples of suitable inhibitory molecules include, without limitation, PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LIR-1 (LILRB1), CD 160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta. Inhibition of a molecule that modulates or regulates, e.g., inhibits, T cell function, e.g., by inhibition at the DNA, RNA or protein level, can optimize the performance of genetically modified cells of the present disclosure. In some embodiments, an agent, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the genetically modified cell. In one embodiment, the inhibitor is an shRNA. In some embodiments, a genetically modified cell of the present disclosure may be further modified to express an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the genetically modified cell.

[00452] In one embodiment, the agent that modulates or regulates, e.g., inhibits, T-cell function is inhibited within a genetically modified cell of the present disclosure. In such embodiments, a dsRNA molecule that inhibits expression of a molecule that modulates or regulates, e.g., inhibits, T-cell function is linked to the nucleic acid that encodes a component, e.g., all of the components, of a chimeric receptor of the present disclosure. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is operably linked to a promoter, e.g., a HI- or a U6-derived promoter such that the dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is expressed, e.g., is expressed within a the genetically modified cell. In one embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is present on the same vector, e.g., a lentiviral vector, that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the chimeric receptor. In such an embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T- cell function is located on the vector, e.g., the lentiviral vector, 5'- or 3'- to the nucleic acid that encodes a component, e.g., all of the components, of the chimeric receptor. The nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function can be transcribed in the same or different direction as the nucleic acid that encodes a component, e.g., all of the components, of the chimeric receptor. In one embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T- cell function is present on a vector other than the vector that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the chimeric receptor. In one embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function it transiently expressed within the genetically modified cell. In one embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is stably integrated into the genome of a genetically modified cell of the present disclosure.

[00453] In one embodiment, an agent that modulates or regulates, e.g., inhibits, T-cell function can be an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-1 , PD-L1, PD-L2 or CTLA4. In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3. [00454] In some embodiments, the agent which enhances the activity of the genetically modified cell is a CEACAM inhibitor (e.g., CEA CAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. In one embodiment, the agent which enhances activity of a genetically modified cell of the present disclosure is miR-17-92. In some embodiments, the agent which enhances the activity of the genetically modified cell is CD40L. In some embodiments, the agent which enhances the activity of the genetically modified cell is GM-CSF. In some embodiments, a genetically modified cell of the present disclosure is further modified to express an antibody or antibody fragment that binds to an inhibitory molecule of the present disclosure.

[00455] In one embodiment, the agent which enhances activity of a genetically modified cell of the present disclosure is a cytokine. Cytokines have important functions related to immunoresponsive cell expansion, differentiation, survival, and homeostats. Cytokines that can be administered to the subject receiving a genetically modified cell of the present disclosure include, without limitation, IL-2, IL-4, IL-7, IL-9, IIL-12, L-15, IL-18, and IL-21, or a combination thereof. The cytokine can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The cytokine can be administered for more than one day, e.g., the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine is administered once a day for 7 days. In some embodiments, a genetically modified cell of the present disclosure is further modified to express one or more cytokines, such as IL-2, IL-4, IL-7, IL-9, IL-12, L-15, IL-18, and IL-21.

[00456] In some embodiments, the cytokine can be administered simultaneously or concurrently with the genetically modified cells, e.g., administered on the same day. The cytokine may be prepared in the same pharmaceutical composition as the genetically modified cells, or may be prepared in a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after administration of the genetically modified cells, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In some embodiments where the cytokine is administered in a dosing regimen that occurs over more than one day, the first day of the cytokine dosing regimen can be on the same day as administration with the genetically modified cells, or the first day of the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In one embodiment, on the first day, the genetically modified cells are administered to the subject, and on the second day, a cytokine is administered once a day for the next 7 days. In some embodiments, the cytokine is administered for a period of time after administration of the genetically modified cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of the genetically modified cells. In one embodiment, the cytokine is administered after assessment of the subject's response to the genetically modified cells.

Kits

[00457] Certain aspects of the present disclosure relate to kits for the treatment and/or prevention of a cancer (e.g., CRC) or other diseases (e.g., immune-related or autoimmune disorders). In certain embodiments, the kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more proteins including an antigen-binding domain (e.g., scFv) of the present disclosure, such as a chimeric receptor of the present disclosure, isolated nucleic acids of the present disclosure, vectors of the present disclosure, and/or cells of the present disclosure (e.g., immunoresponsive cells). In some embodiments, the kit comprises a sterile container. In some embodiments, such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[00458] In some embodiments, therapeutic or prophylactic composition is provided together with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., CRC). In some embodiments, the instructions may include information about the use of the composition for the treatment and/or prevention of the disorder. In some embodiments, the instructions include, without limitation, a description of the therapeutic or prophylactic composition, a dosage schedule, an administration schedule for treatment or prevention of the disorder or a symptom thereof, precautions, warnings, indications, counter-indications, over-dosage information, adverse reactions, animal pharmacology, clinical studies, and/or references. In some embodiments, the instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

ENUMERATED EMBODIMENTS

Embodiment 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, 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: 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 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.

Embodiment 2 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: 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 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.

Embodiment 3 The antibody or antigen binding fragment thereof of any one of embodiments 1-2, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 88-107.

Embodiment 4 The antibody or antigen binding fragment thereof of any one of embodiments 1-3, wherein the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

Embodiment 5 The antibody or antigen binding fragment thereof of any one of embodiments 1-4, 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; 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 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.

Embodiment 6 The antibody or antigen binding fragment thereof of any one of embodiments 1-4, wherein 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.

Embodiment 7 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: a. 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 b. 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.

Embodiment 8 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.

Embodiment 9 The antibody or antigen binding fragment thereof of claim 8, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88- 107.

Embodiment 10 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 VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-107.

Embodiment 11 The antibody or antigen binding fragment thereof of embodiment 10, wherein the VL has an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

Embodiment 12 The antibody or antigen binding fragment thereof of any one of embodiments 1-11, wherein the antibody or antigen binding fragment thereof is an antigen binding fragment.

Embodiment 13 The antibody or antigen binding fragment thereof of any one of embodiments 1-12, wherein the antigen binding fragment comprises a F(ab) fragment, a F(ab’) fragment, or a single chain variable fragment (scFv).

Embodiment 14 The antibody or antigen binding fragment thereof of any one of embodiments 1-13, wherein the antigen binding fragment comprises a single chain variable fragment (scFv).

Embodiment 15 The antibody or antigen binding fragment thereof of embodiment 14, wherein the scFv comprises a VH and a VL separated by a peptide linker.

Embodiment 16 The antibody or antigen binding fragment thereof of embodiment 15, 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. Embodiment 17 The antibody or antigen binding fragment thereof embodiment 15 or embodiment 16, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 21-37.

Embodiment 18 The antibody or antigen binding fragment thereof of any one of embodiment 14-17, wherein the scFv comprises an amino acid sequence selected from the group consisting of: SEQ ID Nos: 108-132.

Embodiment 19 A chimeric protein comprising an antibody or antigen binding fragment thereof of any one of embodiments 1-18 and a heterologous molecule or moiety.

Embodiment 20 The chimeric protein of embodiment 19, wherein the chimeric protein is an antibody-drug conjugate, and wherein the heterologous molecule or moiety comprises a therapeutic agent.

Embodiment 21 The chimeric protein of embodiment 19, 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.

Embodiment 22 The chimeric protein of embodiment 21, wherein the CAR comprises a transmembrane domain.

Embodiment 23 The chimeric protein of embodiment 21 or 22, wherein the CAR comprises one or more intracellular signaling domains.

Embodiment 24 The chimeric protein of any one of embodiments 21-23, wherein the CAR is an activating CAR comprising one or more intracellular signaling domains that stimulate an immune response.

Embodiment 25 The chimeric protein of any one of embodiments 21-23, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response.

Embodiment 26 The chimeric protein of embodiment 25, 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.

Embodiment 27 The chimeric protein of embodiment 25, wherein the intracellular inhibitory domain comprises the amino acid sequence VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPN NHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPR K (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

VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPN NHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPR K (SEQ ID NO: 139).

Embodiment 28 The chimeric protein of embodiment 25, wherein the intracellular inhibitory domain comprises an enzymatic inhibitory domain.

Embodiment 29 The chimeric protein of any one of embodiments 25-28, wherein the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain.

Embodiment 30 The chimeric protein of any one of embodiments 21-29, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain.

Embodiment 31 The chimeric protein of embodiment 30, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52.

Embodiment 32 A composition comprising the antibody or antigen binding fragment thereof of any one of embodiments 1-18 or the chimeric protein of any one of embodiments 19-31 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 33 An engineered nucleic acid encoding the antibody or antigen binding fragment of any one of embodiments 1-18, the chimeric protein of any one of embodiments 19-31, or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 34 An expression vector comprising the engineered nucleic acid of embodiment 33 or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 35 A composition comprising the engineered nucleic acid of embodiment 33, the expression vector of embodiment 34, or the engineered expression system of any one of embodiments 69 - 114, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 36 A method of making an engineered cell, comprising transducing an isolated cell with the engineered nucleic acid of embodiment 33, the expression vector of embodiment 34, or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 37 An isolated cell comprising the engineered nucleic acid of embodiment 33, the expression vector of embodiment 34, the composition of embodiment 35, or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 38 A population of engineered cells expressing the engineered nucleic acid of embodiment 33, the expression vector of embodiment 34, or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 39 An isolated cell comprising the antigen binding fragment of any one of embodiments 1-18, the chimeric protein of any one of embodiments 19-31, or the engineered expression system of any one of embodiments 69 - 114.

Embodiment 40 A population of engineered cells expressing the antigen binding fragment of any one of embodiments 1-18 or the chimeric protein of any one of embodiments 19- 31.

Embodiment 41 The cell or population of cells of any one of embodiments 37-40, wherein the chimeric protein is recombinantly expressed.

Embodiment 42 The cell or population of cells of any one of embodiments 37-41, wherein the chimeric protein is expressed from a vector or a selected locus from the genome of the cell.

Embodiment 43 The cell or population of cells of any one of embodiments 37-42, wherein the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface.

Embodiment 44 The cell or population of cells of embodiment 43, 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.

Embodiment 45 The cell or population of cells of any one of embodiments 37-44, 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. Embodiment 46 The cell or population of cells of any one of embodiments 37-45, wherein the cell is autologous.

Embodiment 47 The cell or population of cells of any one of embodiments 37-45, wherein the cell is allogeneic.

Embodiment 48 A pharmaceutical composition comprising an effective amount of the cell or population of engineered cells of any one of embodiments 37-47 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 49 A pharmaceutical composition comprising an effective amount of genetically modified cells expressing the antigen binding fragment of any one of embodiments 1-18 or the chimeric protein of any one of embodiments 19-31 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 50 The pharmaceutical composition of embodiment 48 or embodiment 49, which is for treating and/or preventing a tumor.

Embodiment 51 A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of the composition of embodiment 32 or embodiment 35, or any of the cells of any one of embodiments 37-47, or the pharmaceutical composition of embodiment 48 or embodiment 49.

Embodiment 52 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 the composition of embodiment 32 or embodiment 35, or any of the cells of any one of embodiments 37-47, or the composition of embodiment 48 or embodiment 49.

Embodiment 53 The method of embodiment 52, comprising administering to the subject the isolated cell or the population of cells of any one of embodiments 37-47, wherein the isolated cell or population of cells express the chimeric protein comprising the activating CAR of embodiment any one of the above embodiments.

Embodiment 54 A method of inhibiting a cell-mediated immune response to a normal cell in a subject, the method comprising administering to a subject a therapeutically effective dose of the composition of embodiment 32 or embodiment 35, or any of the cells of any one of embodiments 37-47, or the composition of embodiment 48 or embodiment 49.

Embodiment 55 The method of embodiment 54, comprising administering to the subject any of the cell of any one of embodiments 37-47, wherein the isolated cell or population of cells express the chimeric protein comprising the inhibitory CAR of embodiment 25. Embodiment 56 The method of embodiment 55, 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.

Embodiment 57 The method of any one of embodiments 54-56, 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., nontumor) epithelial cell.

Embodiment 58 A method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of the composition of embodiment 32 or embodiment 35, or any of the cells of any one of embodiments 37-47, or the composition of embodiment 48 or embodiment 49.

Embodiment 59 A kit for treating and/or preventing a tumor, comprising the chimeric protein of any one of embodiments 19-31.

Embodiment 60 The kit of embodiment 59, 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.

Embodiment 61 A kit for treating and/or preventing a tumor, comprising the cell or population of cells of any one of embodiments 37-47.

Embodiment 62 The kit of embodiment 61, wherein the kit further comprises written instructions for using the cell for treating and/or preventing a tumor in a subject.

Embodiment 63 A kit for treating and/or preventing a tumor, comprising the engineered nucleic acid of embodiment 41.

Embodiment 64 The kit of embodiment 63, 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.

Embodiment 65 A kit for treating and/or preventing a tumor, comprising the vector of embodiment 34.

Embodiment 66 The kit of embodiment 65, 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.

Embodiment 67 A kit for treating and/or preventing a tumor, comprising the composition of embodiment 32, 35, 48, or 49. Embodiment 68 The kit of embodiment 67, wherein the kit further comprises written instructions for using the composition for treating and/or preventing a tumor in a subject.

Embodiment 69 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; and iii. 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 embodiments 1 - 18 or the chimeric protein of any one of embodiments 19 - 31.

Embodiment 70 The engineered expression system of embodiment 69, wherein the first CAR comprises a first spacer between the first extracellular antigen-binding domain and the first transmembrane domain.

Embodiment 71 The engineered expression system of embodiment 69, wherein the first spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52.

Embodiment 72 The engineered expression system of embodiment 69 or 70, wherein the first spacer comprises the amino acid sequence of SEQ ID NO: 50.

Embodiment 73 The engineered expression system of any one of embodiments 69-72, wherein the second CAR comprises a second spacer between the second extracellular antigen-binding domain and the second transmembrane domain.

Embodiment 74 The engineered expression system of embodiment 73, wherein the second spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52.

Embodiment 75 The engineered expression system of embodiment 73 or 74, wherein the second spacer comprises the amino acid sequence of SEQ ID NO: 50.

Embodiment 76 The engineered expression system of any one of embodiments 69-75, 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 CD3 epsilon-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.

Embodiment 77 The engineered expression system of any one of embodiments 69-76, wherein the first CAR comprises a CD28 intracellular signaling domain and a CD3zeta- chain intracellular signaling domain.

Embodiment 78 The engineered expression system of any one of embodiments 69-77, 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, 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.

Embodiment 79 The engineered expression system of any one of embodiments 69-78, wherein the first CAR comprises a CD28 transmembrane domain.

Embodiment 80 The engineered expression system of any one of embodiments 69-79, wherein the first and second nucleic acid sequences are comprised within a single expression vector.

Embodiment 81 The engineered expression system of any one of embodiments 69-80-, 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.

Embodiment 82 The engineered expression system of any one of embodiments 69-81, wherein the first antigen-binding domain binds CEACAM5. Embodiment 83 The engineered expression system of any one of embodiments 69-82, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 84 The engineered expression system of any one of embodiments 69-83, 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 hMN 14 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.

Embodiment 85 The engineered expression system of any one of embodiments 69-84, wherein the VH comprises the amino acid sequence of an hMN14 VH, and the VL comprises the amino acid sequence of an hMN14 VL.

Embodiment 86 The engineered expression system of any one of embodiments 69-82, wherein 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.

Embodiment 87 The engineered expression system of any one of embodiments 69-86, 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.

Embodiment 88 The CAR or engineered expression system of embodiment 86 or 87, 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.

Embodiment 89 The engineered expression system of any one of embodiments 69-88, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 90 The engineered expression system of any one of embodiments 69-89, 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.

Embodiment 91 The engineered expression system of any one of embodiments 69-90, wherein the VH comprises the amino acid sequence of an A5B7 VH, and the VL comprises the amino acid sequence of an A5B7 VL.

Embodiment 92 The engineered expression system of any one of embodiments 69-91, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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. Embodiment 93 The CAR or engineered expression system of any one of embodiments 69-92, 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 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.

Embodiment 94 The engineered expression system of any one of embodiments 69-93, wherein the VH comprises the amino acid sequence of an MFE23 VH, and the VL comprises the amino acid sequence of an MFE23 VH.

Embodiment 95 The engineered expression system of any one of embodiments 69-94, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 96 The engineered expression system of any one of embodiments 69-95, 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,.

Embodiment 97 The engineered expression system of any one of embodiments 69-96, wherein the VH comprises the amino acid sequence of an hMFE23 VH, and the VL comprises the amino acid sequence of an hMFE23 VL.

Embodiment 98 The engineered expression system of any one of embodiments 69-97, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 99 The engineered expression system of any one of embodiments 69-98, 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.

Embodiment 100 The engineered expression system of any one of embodiments 69-99, wherein the VH comprises the amino acid sequence of an FM4 VH, and the VL comprises the amino acid sequence of an FM4 VL.

Embodiment 101 The engineered expression system of any one of embodiments 69-100, wherein 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.

Embodiment 102 The engineered expression system of any one of embodiments 69-101, 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.

Embodiment 103 The engineered expression system of any one of embodiments 69-102, 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. Embodiment 104 The engineered expression system of any one of embodiments 69-103, wherein 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.

Embodiment 105 The engineered expression system of any one of embodiments 69-104, 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.

Embodiment 106 The engineered expression system of any one of embodiments 69-105, 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.

Embodiment 107 The engineered expression system of any one of embodiments 69-106, wherein the first antigen-binding domain binds CECAM1.

Embodiment 108 The engineered expression system of any one of embodiments 69-107, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 109 The engineered expression system of any one of embodiments 69-108, 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.

Embodiment 110 The engineered expression system of any one of embodiments 69-109, wherein the VH comprises the amino acid sequence of an MRG1 VH, and the VL comprises the amino acid sequence of an MRG1 VL.

Embodiment 111 The engineered expression system of any one of embodiments 69-110, wherein the first antigen-binding domain binds CEACAM6.

Embodiment 112 The engineered expression system of any one of embodiments 69-111, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), a. 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; b. 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.

Embodiment 113 The engineered expression system of any one of embodiments 69-112, 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.

Embodiment 114 The engineered expression system of any one of embodiments 69-113, 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.

Embodiment 115 The engineered expression system of any one of embodiments 69-114, further comprising: a. A fourth nucleotide sequence encoding a first cytokine; and b. A fifth nucleotide sequence encoding a second cytokine.

Embodiment 116 The engineered expression system of any one of embodiments 69-115, wherein at least one of the first and the second cytokines is a controlled release cytokine.

Embodiment 117 The engineered expression system of any one of embodiments 69-116, wherein 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 ADAM 10 and/or ADAM 17, 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-l, 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.

Embodiment 118 The engineered expression system of any one of embodiments 69-117, wherein the first cytokine is IL 15, optionally wherein the IL 15 comprises the amino acid sequence of IL 15 set forth in Table 10.

Embodiment 119 The engineered expression system of any one of embodiments 69-118, wherein the IL15 is controlled-release IL15 (crIL15).

Embodiment 120 The engineered expression system of any one of embodiments 69-119, 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).

Embodiment 121 The engineered expression system of any one of embodiments 69-120, wherein the first or second cytokine comprises an amino acid sequence set forth in Table 10.

Embodiment 122 The engineered expression system of any one of embodiments 69-121, wherein 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. EXAMPLES [00459] The following are examples of methods and compositions of the present disclosure. It is understood that various other embodiments may be practiced, given the general description provided herein.

[00460] Below are examples of specific embodiments for carrying out the claimed subject matter of the present disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Example 1: Construction of Anti-VSIG2 Antigen-Binding Domains Variants [00461] A series of anti-VSIG2 antigen-binding domain variants (e.g., for scFvs), were constructed using murine derived anti-VSIG2 VH and VL sequences as shown in Table 21. Framework regions surrounding the CDRs (as designated by the Kabat annotation and numbering scheme) were replaced with human germline antibody sequences IGHV3-21 for the heavy chain and IGKV1-5 for the light chain, with further individual residues in the human framework regions back-mutated to their murine counterparts. Heavy chain J segment IGHJ4*02 (WGQGTLVTVSS; SEQ ID NO: 10) light chain J segments IGKJl*01 (FGQGTKVEIK; SEQ ID NO: 11) were also used for framework region 4 (FR4) following the respective CDR3 sequence.

[00462] Additional anti-VSIG2 VH and VL variants were generated. Specifically, variants of the heavy chain and light chain CDR3 domains were generated, while heavy chain CDR1 (NSGMS; SEQ ID NO: 1), heavy chain CDR2 (SISDGGLYTHYPDSVKG; SEQ ID NO:3), light chain CDR1 (RASENIYSYLA; SEQ ID NO: 6), and light chain CDR2 (NAETLPE; SEQ ID NO:7) were kept constant, with CDR sequences defined by Kabat. The sequences of the anti- VSIG2 VH and VL variants are shown in Table 21. Pairings of VH and VL variants and their respective light chain and heavy chain CDR3s are shown in Table 22. The anti-VSIG2 VH and VL variants were constructed into scFvs shown in Table 23.

Table 21. Anti-VSIG2 VH and VL Variant Sequences

Table 22. Anti-VSIG2 VH and VL Variant Pairing and CDR3 Sequences

Table 23. Anti-VSIG2 Variant scFv Sequences

Example 2: Assessment of Anti-VSIG2 Antigen-Binding Domains Variants in iCAR Mediated Protection in NK Cells

[00463] To assesses anti-VSIG2 antigen-binding domain variants, the scFvs described in Example 1 and shown in Table 23 were assessed in an iCAR format for their ability to protect against NK-mediated killing.

Methods

[00464] NK cells were isolated from peripheral blood and cultured and expanded for 11 days with irradiated K562 feeder cells engineered to express membrane-bound IL-21 and IL- 15 in NK MACS® media with 5% human AB serum. During expansion and subsequent NK cell culture, NK cell media was supplemented with 500 U/mL IL-2 and 10 ng/mL IL-15 every 3-4 days. At day 11, NK cells were transduced with gamma-retrovirus carrying gene payloads encoding an CEA-targeting aCAR and/or a VSIG2 -targeting iCAR proteins, e.g., iCARs including scFvs with anti-VSIG2 antigen-binding domain variants. Constructs with the anti- VSIG2 antigen-binding domain variants were in the following format (linearly linked in N- terminal to C-terminal orientation):

[00465] CD8ss - aCEA hMN14 LH - myc NGAA linker - CD8 hinge - CD28 TM - CD28 ICD - CD3z - GSG P2A - CD8SS - anti-VSIG Variant scFv - V5 NGAA linker - CD8 hinge - SIRPa TM - SIRPa ICD

[00466] The anti-VSIG2 scFv sequences assessed are shown in Table 23 with additional CAR construct sequences shown in Table 24.

Table 24 - aCAR/iCAR Sequences [00467] An additional construct including an aVSIG2.13 scFv was generated replacing the SIRPa TM and ICD with a LIR1 TM and ICD, respectively. Additionally, a construct was generated that included a LIR1 TM and ICD with the anti-VSIG scFv replaced with a Hemtargeting scFv (H3B1 clone, described in Rudnick SI, Lou J, Shaller CC, Tang Y, Klein-Szanto AJ, Weiner LM, Marks JD, Adams GP. Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors. Cancer Res. 2011 Mar 15;71(6):2250-9. doi: 10.1158/0008-5472.CAN-10-2277. PMID: 21406401; PMCID: PMC3077882, which is hereby incorporated by reference).

[00468] After 3 days post-transduction, NK cells media was removed and fresh media with 500U/mL IL-2 and 10 ng/mL IL- 15 was added. After an additional 3 days, media was replaced again with fresh media and cytokines, and expression of aCAR and iCAR was measured by antibody staining. Myc tags on aCARs and V5 tags on iCARs were stained with anti-myc Alex Fluor 488 (Cell Signaling Technology) at 1:50 dilution and anti-V5-Alexa Fluor 647 (Invitrogen) at 1:300 dilution, respectively. Cells were washed with phosphate-buffered saline supplemented with 5% fetal (FBS) bovine serum and resuspended in the viability dye SytoxBlue (Invitrogen) at 1: 1000 dilution.

[00469] After an additional 2 days, cytotoxicity assays were performed using the colorectal adenocarcinoma cell line DLD-1 engineered to express CEACAM5, Her2, GFP (DLD-1 CEA+Her2+GFP+) and DLD-1 engineered to express CEACAM5, VSIG2, mCherry (DLD-1 CEA+VSIG2+mCherry+) as targets and RPMI supplemented with 10% FBS were used as the assay media. All cytotoxicity assays were analyzed using the Sartorius Incucyte imaging instrument. These target cells were harvested by trypsinization, washed, and counted. The two types of target cells were diluted to le6 cells/mL and then mixed 1: 1. Then, 2e4 target cells were added in a 100 uL volume to wells of 96 well flat bottom plates. The targets were then incubated overnight (16-18 hours) prior to NK cell addition. To prepare NK cells forthe assay, they were harvested, washed, and counted. Then, le4 NK cells were added in alOOuL volume in triplicates to the mixed target plates.

[00470] These plates were then moved to the Incucyte® for whole-well imaging every 4 hours. The target cell reduction (also called killing) was quantified as 100% x (1 - No. Targets / No. Targets (targets alone)). Suppression was quantified as 100% x (1 - killing of VSIG2+ cells / killing of all VSIG2- cells) and A%VSIG2+ was quantified as the change in the following quantity relative to untransduced NK cells: 100% x (No. VSIG2+ / No. all target cells).

Results

[00471] NK cells transduced with the iCARs including scFvs with anti-VSIG2 antigenbinding domain variants were assessed for their protective effect of suppressing NK killing of VSIG2-expressing target cells. Specifically, as shown in FIG. 1, the ability of the anti-VSIG2 iCAR to protect against killing of CEA+/VSIG2+ target cells (left column) in comparison to killing of CEA+ target cells that did not express VSIG2 (right column) was assessed.

[00472] As shown in FIG. 1, the iCARs with the anti-VSIG2 antigen-binding domain variants all demonstrated protection, as assessed by percent suppression of killing, ranging from 17.4% to 96.2% suppression. All anti-VSIG2 antigen-binding domain variants shown demonstrated at least the same level of protection as the parental anti-VSIG2 antigen-binding domain (aVSIG2-SIRPa), with the majority showing noticeable improvements in suppressing killing. Protection was also provided when replacing the SIRPa inhibitory intracellular domain with LIR1 (aVSIG2.13-LIRl). Replacing the anti-VSIG2 scFv with an anti-Her2 scFv also switched the protection to CEA+/Her2+ target cells, indicating the protective effect was mediated by iCAR scFv’s target specificity.

[00473] A summary of the results for the VSIG2 iCAR protection assay is shown in FIG. 2. As the summary shows, the optimized anti-VSIG2 antigen-binding domain variant a VSIG2.21 demonstrated significantly improved protection. Notably, while the parental aVSIG2-SIRPa (non-optimized; column 3) reduced the increased aCAR-mediated killing (no NOT gate; column 2) back to basal NK-mediated killing levels (column 1), in contrast the aVSIG2 variant (optimized; column 4) further protected against even basal NK-mediated killing almost to complete protection against any NK-mediated killing in a VSIG2-specific manner.

Example 3: Assessment of Variant Anti-VSIG2 Antigen-Binding Domain in iCAR Mediated Protection with Various Inhibitory Domains

[00474] The NK-mediated protection assay described in Example 2 was used to assess an anti-VSIG2 variant’s ability to provide iCAR-mediated protection with various inhibitory intracellular domains (ICDs).

[00475] Constructs with the various inhibitory ICDs were in the following format (linearly linked in N-terminal to C-terminal orientation):

[00476] CD8ss - aCEA hMN14 LH - myc NGAA linker - CD8 hinge - CD28 TM - CD28 ICD - CD3z - GSG P2A - CD8SS - aVSIG2. 13 - V5 NGAA linker - CD8 hinge - ICD TM - ICD Domain

[00477] Sequences for the various inhibitory ICDs assessed are shown in Table 25 with additional CAR construct sequences shown in Table 24. The constructs for NKG2A and CD72 used a LIR1 transmembrane domain in place of the native NKG2A and CD72 transmembrane sequence. A construct that included a SIRPa TM and ICD with the anti-VSIG scFv replaced with a Her2 -targeting scFv was also assessed. Table 25 - Inhibitory Intracellular Domain Sequences

[00245] Expression of the iCARs with the various inhibitory ICDs was assessed. As shown in FIG. 3, expression was generally equivalent across all constructs.

[00246] Anti-VSIG2 iCAR-mediated protection with the various inhibitory ICDs was assessed. As shown in FIG. 4, a range of protection was shown with the majority of constructs demonstrating a protective effect indicating the anti-VSIG2 variant’s ability to provide iCAR- mediated protection across a variety of inhibitory ICDs. Example 4: Assessment of Anti-VSIG2 Antigen-Binding Domains Variants in iCAR Mediated Protection in T Cells

Methods

[00478] T cells were isolated from peripheral blood and cultured and expanded using CD3/CD28 DynaBeads (Invitrogen) for 3 days. Throughout culturing and transduction, T cells were maintained in T cell media (CTS OpTmizer with serum replacement, Life Technologies) with 200 U/mL IL-2 at a concentration of 0.5-le6 cells/mL. T cells were were then removed from beads and transduced with gamma-retrovirus carrying gene payloads encoding an CEA- targeting aCAR and/or a VSIG2 -targeting iCAR protein including the anti-VSIG2 antigenbinding domain variant aVSIG2.21. Constructs with the anti-VSIG2 antigen-binding domain variants were in the following format (linearly linked in N-terminal to C-terminal orientation): [00479] CD8ss - aCEA hMN14 LH - myc NGAA linker - CD8 hinge - CD28 TM - CD28 ICD - CD3z - GSG P2A - CD8SS -aVSIG2.21 scFv - V5 NGAA linker - CD8 hinge - SIRPa TM - SIRPa ICD

[00480] The anti-VSIG2.21 scFv sequence assessed is shown in Table 23 with additional CAR construct sequences shown in Table 24, above.

[00481] After 3 days post-transduction, T cell media was removed and fresh media with 200U/mL IL-2. Expression of aCAR and iCAR was measured by antibody staining. Myc tags on aCARs and V5 tags on iCARs were stained with anti-myc Alex Fluor 488 (Cell Signaling Technology) at 1:50 dilution and anti -V5 -Alexa Fluor 647 (Invitrogen) at 1:300 dilution, respectively. Cells were washed with phosphate-buffered saline supplemented with 5% fetal (FBS) bovine serum and resuspended in the viability dye SytoxBlue (Invitrogen) at 1: 1000 dilution.

[00482] After an additional 4 days of culturing as described above, cytotoxicity assays were performed using the colorectal adenocarcinoma cell line DLD-1 engineered to express CEACAM5, Her2, GFP (DLD-1 CEA+Her2+GFP+) and DLD-1 engineered to express CEACAM5, VSIG2, mCherry (DLD-1 CEA+VSIG2+mCherry+) as targets and RPMI supplemented with 10% FBS were used as the assay media. All cytotoxicity assays were analyzed using the Sartorius Incucyte imaging instrument. These target cells were harvested by trypsinization, washed, and counted. Then, 2e4 target cells of each type were added in a 100 uL volume to separate wells of 96 well flat bottom plates. The targets were then incubated overnight (16-18 hours) priorto T cell addition. Then, T cells were harvested, washed, and counted, and 5e3 T cells were added in a volume of 100 uL to each well. All conditions were set up in triplicate. [00483] These plates were then moved to the Incucyte® for whole-well imaging every 4 hours. The target cell reduction (also called killing) was quantified as 100% x (1 - No. Targets / No. Targets (targets alone)). Suppression was quantified as 100% x (1 - killing of VSIG2+ cells / killing of all VSIG2- cells) and A%VSIG2+ was quantified as the change in the following quantity relative to untransduced T cells: 100% x (No. VSIG2+ / No. all target cells).

Results

[00484] Anti-VSIG2 iCAR-mediated protection in T cells expressing the aCEA-aCAR and aVISG2.21-iCAR was assessed and compared to T cells expression the aCAR alone with no iCAR. As shown in FIG. 5, both T cell types demonstrated comparable killing of VSIG2- cells, which was expected since the lack of VSIG2 expression on the target cells did not stimulate the iCAR. In response to VSIG2+ targets, however, T cells expressing the iCAR killed the target cells at less than half the rate of T cells without the iCAR, confirming that the aVSIG2.21 iCAR is functional in T cells. Additionally, untransduced T cells were evaluated, which we confirmed had no killing function (data not shown), so the killing shown in FIG. 5 is aCAR-mediated.

Example 5: In Vitro Assessment of NOT Gate Systems using a CEA aCAR and a VSIG2 iCAR and Cytokine Payloads

Methods

Retrovirus Production

[00485] DNA was transfected into GP2-293 (y-retrovirus) producer cells following manufacturer recommendations. Viral supernatant was collected, clarified by centrifugation, treated by MgC12 and Benzonase, and concentrated using Lenti-X concentrator.

NK Cell Engineering

[00486] Primary NK cells were isolated from PBMCs from healthy donors and frozen in liquid nitrogen. For individual experiments, single vials of frozen NK cells were thawed and stimulated with irradiated feeder cells (engineered K562 cells). NK cells were expanded in 6- well G-Rex plates in NK media (NK MACS media with 5% human AB serum with 100 U/mL IL2). For virus transduction preparation, 12-well plates were coated with recombinant human fibronectin fragment (RetroNectin) according to manufacturer protocols. NK cells and retrovirus were added to coated plates and centrifuge at 1000g for 2 hours at 32°C. After transduction, the NK cells were transferred to 12-well G-Rex for expansion. After 7-14 days, expression was checked by flow cytometry and cells were harvested for use in assays. Flow Cytometry

[00487] For aCAR-Myc, iCAR-V5 detection and IL- 15 detection, Myc-tag antibody (Cell Signaling Technology), V5-tag antibody (ThermoFisher), anti-human IL-15 antibody (BioLegend) and Sytox Blue (Life Technologies) were used to stain the transduced NK cells. For staining preparation, transduced NK cells were washed twice with FACS buffer, then stained with antibodies and viability dye for 1 hour in 4°C. After incubation, cells were washed and resuspended in an appropriate volume of FACS buffer for flow cytometry (Beckman CytoFLEX).

Cytotoxicity Assay

[00488] For cytotoxicity assay, the VSIG2+ and VSIG2- target cells (engineered DLD1 cells) were mixed at 1 : 1 ratio to represent the “healthy” cells and “tumor” modal cells . V SIG2+ DLD 1 cells have mCherry fluorescent protein and VSIG2- DLD1 cells have GFP fluorescent protein for the flow cytometry readout. Non-transduced NK cells and CAR NK cells were mixed with target cells for co-culture (2 to 6 days). For cytotoxicity measurement, Zombie UV staining dye (BioLegend) was used to distinguish the live and dead target cell populations. Anti -human CD56 antibody (BioLegend) was used to exclude NK cells in the gating procedures.

Luminex Assay

[00489] The secreted cytokines, IL- 15 and IL-21 were quantified using Luminex assays (multiplexed ELISA assays). 5 x 10⁵ NK cells were plated in 96-well plate in a total volume of 200 pL of medium. After 24 hours, plates were centrifuged and 100 pL supernatant were collected. The secreted cytokines were measured using customized MILLIPLEX multiplex assay kit and Luminex MAGPIX System according to manufacturer protocols.

Results

[00490] NK cells were transduced with the following constructs in Table 26 to assess the expression of the CAR (double positive aCAR/iCAR populations) and membrane associated IL 15 (mIL15) as indicated in the Methods above to assess the payload expression in different arrangements of the payloads.

[00491] All three constructs showed increased CAR+ populations compared to IL15. Different arrangements of the payload show differing ratios of CAR+ versus IL 15+ cell populations (FIG. 5A). Table 26: Exemplary Constructs

[00492] Additional constructs were also assessed in the same manner with the following constructs in Table 27 to determine how the arrangement of the cytokine (IL 15 and IL21) affect payload expression. Comparing SB010383 and SB010572, for IL-21 first order and IL-15 first order, the CAR expression and mIL-15 expression are comparable (FIG. 5B).

Table 27: Additional Exemplary Constructs

[00493] Cells transduced with constructs SB10383, SB010573, and SB10385 were assessed for the ability to kill target cells. In FIGs. 6A - 6C, the expression profile is shown for CAR (double positive iCAR/aCAR) and mIL15 (FIG. 6A), soluble IL 15 (FIG. 6B), and soluble IL21 (FIG. 6C). SB 10573 showed the highest level of CAR expression of the three tested constructs, the different order of payloads affects the level of IL15 or IL21 expressed.

[00494] The same engineered cells were used in an in vitro cytotoxicity assay using a mixed VSIG2+ "Healthy" cells and VSIG2- "Tumor" cells (mixed at a ratio of 1.5: 1) to demonstrate the functionality of the NOT gate to protect VSIG2 -positive cells. Compared to the “Targets Only” and “No NV” controls, each of the cell populations transduced with the different constructs showed NOT gate protection (FIG. 6D), where SB10573 demonstrated approximately 95% enrichment of VSIG2 positive cells.

Example 6: In Vivo Assessment of NOT Gate Systems using a CEA aCAR and a VSIG2 iCAR and Cytokine Payloads

Methods

CAR NK Cell Characterization

[00495] For CAR expression and mIL-15 expression assessment, V5-tag antibody

(ThermoFisher), anti-human IL- 15 antibody (BioLegend) and Sytox Blue (Life Technologies) were used to stain the transduced NK cells. Vector copy number (VCN) was determined by qPCR against woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) within the construct.

CAR NK Cell Preparation

[00496] To generate CAR NK cells for in vivo study, after transduction, the NK cells were cultured for 2 days in cell culture plates, and then transferred to IL G-Rex (Wilson Wolf, P/N RU81100) for expansion. After 12-day expansion, expression was checked by flow cytometry and cells were harvested for copy number assay and in vivo injection.

In Vivo Study

[00497] NSG mice were use for in vivo model (Jackson Laboratories, 6-8 weeks, female). Based on previous kinetic study, the DLD1 CEA+ VSIG2+ mCherry+ “healthy” modal cells and DLD1 CEA+ VSIG2- GFP+ “tumor” modal cells were mixed (1.5: 1 ratio) for SQ injection. CARNK cells (or PBS control) were pre-mixed together for the same day injection. Body weight and tumor size were monitored every week. The mice were taken down and tumor was digested to single-cell suspension using Tumor Dissociation Kit (Miltenyi Biotec). Zombie UV staining dye (BioLegend) and anti-human CD56 antibody (BioLegend) were used in cell staining to exclude the dead cells and NK cells. Based on the fluorescent protein reporter, the cytotoxicity to “healthy” modal cells and “tumor” modal cells were evaluated (Q 1 vs Q3 in flow cytometry plot).

Results

[00498] NKs were transduced with SB12515 (FIG. 7A, Table 27) viruses with MOI 5.05. CARNK cells were expanded 15 days in G-Rex 100M plate for in vivo study. The CARNK cells showed 45.2% CAR+ expression (FIG. 7B, 4.27% mIL-15+ expression (FIG. 7C). The average copy number (WPRE) is 2.0.

[00499] In vivo characterization of the engineered NK cells was carried out as described in the methods and in FIG. 7D. The presence of healthy and tumor cells were assessed by flow cytometry, where reduction of events in Q3 indicated killing of tumor cells and increase in events in QI indicated protection of “healthy cells.

[00500] FIG. 7E shows that the engineered CAR NK cell has increased protection of “healthy” cells (VSIG2+) compared to the no treatment group. Table 28: Additional Exemplary Constructs

INCORPORATION BY REFERENCE

[00501] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

EQUIVALENTS

[00502] While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the present disclosure(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

CLAIMS What 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; 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 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.

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 is an 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 sequence

VRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTE 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; and iii. 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, 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, 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 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, 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 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, 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 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, 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; and b. 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 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- 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 the engineered 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.