Chimeric receptors and methods of use thereof

Abstract

Provided herein are chimeric proteins comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2). Also provided herein are cells, polynucleotides, vectors, compositions, and methods directed to the chimeric proteins comprising an antigen-binding domain specific for VSIG2.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 185,896, filed May 7, 2021, and U.S. Provisional Application No. 63 / 283,147, filed November 24, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] (Sequence Listing) This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

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

[0004] One challenge in developing CAR therapy for solid tumors is the lack of suitable targets. The ability to identify appropriate CAR targets is important to effectively target and treat tumors without damaging normal cells that express the same target antigen. Therefore, there is still a need for CAR-based solid tumor therapy that targets tumor cells without targeting normal cells or tissues. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pule et al., Nat. Med. (14):1264-1270 (2008) [Non-Patent Document 2] Maude et al., N Engl J Med. (371):1507-17 (2014) [Non-Patent Document 3] Brentjens et al., Sci Transl Med. (5):177ra38 (2013) Summary of the Invention

[0006] overview To meet the above-mentioned needs, the present disclosure relates to chimeric proteins comprising an antigen-binding domain specific for VSIG2 (VSIG2).

[0007] In one embodiment, there is provided a chimeric protein comprising an antigen binding domain specific for V-Set And Immunoglobulin Domain Containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) VH is A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4), and a VL A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14), or (b) VH is The heavy chain complementarity determining region 1 (CDR-H1) contained in the VH region amino acid sequence of SEQ ID NO: 1, the heavy chain complementarity determining region 2 (CDR-H2) contained in the VH region amino acid sequence of SEQ ID NO: 1, and the heavy chain complementarity determining region 3 (CDR-H3) contained in the VH region amino acid sequence of SEQ ID NO: 1, VL, comprising a light chain complementarity determining region 1 (CDR-L1) contained in the VL region amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) contained in the VL region amino acid sequence of SEQ ID NO: 9, and a light chain complementarity determining region 3 (CDR-L3) contained in the VL region amino acid sequence of SEQ ID NO: 9, or (c) VH is The heavy chain complementarity determining region 1 (CDR-H1) contained in the VH region amino acid sequence of SEQ ID NO: 1, the heavy chain complementarity determining region 2 (CDR-H2) contained in the VH region amino acid sequence of SEQ ID NO: 1, and the heavy chain complementarity determining region 3 (CDR-H3) contained in the VH region amino acid sequence of SEQ ID NO: 1, VL, light chain complementarity determining region 1 (CDR-L1) contained within the VL region amino acid sequence of SEQ ID NO: 10, light chain complementarity determining region 2 (CDR-L2) contained within the VL region amino acid sequence of SEQ ID NO: 10, and light chain complementarity determining region 3 (CDR-L3) contained within the VL region amino acid sequence of SEQ ID NO: 10; Optionally, provided herein are chimeric proteins in which the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat or Chothia numbering scheme.

[0008] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 9, or the VL region comprises the amino acid sequence of SEQ ID NO: 10.

[0009] In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv), optionally wherein the VH and VL of the scFv are separated by a peptide linker, optionally wherein the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain, and / or optionally wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-35 and 69-74.

[0010] In some embodiments, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0011] In some embodiments, the CAR is an inhibitory CAR that comprises one or more intracellular inhibitory domains that inhibit an immune response, wherein each of the one or more intracellular inhibitory domains comprises an enzyme inhibitory domain or an intracellular inhibitory co-signaling domain.

[0012] In another aspect, provided herein is an engineered polynucleotide encoding a chimeric protein of any one of the above aspects or embodiments.

[0013] In another aspect, provided herein is an expression vector comprising an engineered polynucleotide of any one of the above aspects or embodiments.

[0014] In another aspect, provided herein is an isolated cell comprising a chimeric protein of any one of the above aspects or embodiments, an engineered polynucleotide of the above aspects or embodiments, or an expression vector of the above aspects or embodiments.

[0015] In another aspect, provided herein is a population of engineered cells expressing a chimeric protein of any one of the above aspects, an engineered polynucleotide of any one of the above aspects or embodiments, or an expression vector of any one of the above aspects or embodiments.

[0016] In some embodiments, the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface, optionally each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0017] In some embodiments, the cell or population of cells is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of a cell or population of engineered cells of any one of the above aspects or embodiments, a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0019] In another aspect, provided herein is a method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective dose of a chimeric protein of any one of the above aspects or embodiments, an engineered polynucleotide of any one of the above aspects or embodiments, an expression vector of any one of the above aspects or embodiments, a cell or population of engineered cells of any one of the above aspects or embodiments, or a composition of any one of the above aspects or embodiments.

[0020] In another aspect, provided herein is a method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of a chimeric protein of any one of the above aspects or embodiments, an engineered polynucleotide of any one of the above aspects or embodiments, an expression vector of any one of the above aspects or embodiments, a cell or population of engineered cells of any one of the above aspects or embodiments, or a composition of any one of the above aspects or embodiments.

[0021] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 3 (CDR-H3) having an amino acid sequence of QGVRPFFDY (SEQ ID NO:4). In some aspects, the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some aspects, the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0022] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3), and wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO:1. In some embodiments, the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14). In some embodiments, the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0023] In some aspects, the present disclosure provides a chimeric protein comprising an antigen binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; The VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4). In some embodiments, the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some aspects, the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0024] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the VH comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4).

[0025] In some aspects, the disclosure provides a chimeric protein comprising an antigen binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14). In some embodiments, the VH comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4).

[0026] In some aspects, the disclosure provides a chimeric protein comprising an antigen binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heterologous molecule or moiety. The VL comprises a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of PFFDY (sequence number 4), and the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (sequence number 11) or RASENLYSYLA (sequence number 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (sequence number 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0027] In some aspects, the VH region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some aspects, the VH region comprises the amino acid sequence of SEQ ID NO:1.

[0028] In some embodiments, the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0029] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some aspects, the VH region comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 10.

[0030] In some aspects, the present disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region; 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 1.

[0031] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain competes with a reference antibody, or antigen-binding fragment thereof, for binding to VSIG2; The reference antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4), and the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO:11) or RASENLYSYLA (SEQ ID NO:12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO:13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO:14). In some embodiments, the VH region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0032] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain binds essentially the same VSIG2 epitope as a reference antibody or antigen-binding fragment thereof, the reference antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the VH having an amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 1 (CDR-H1) having an amino acid sequence of SDGGLY (SEQ ID NO:3), and the VL comprises a light chain complementarity determining region 1 (CDR-L1) having an amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having an amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having an amino acid sequence of QHHYVIPWT (SEQ ID NO: 14). In some embodiments, the VH region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0033] In some aspects, the disclosure provides a chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, wherein the antigen-binding domain binds to an epitope of human VSIG2 that is the same as a VSIG2 epitope bound by a reference antibody or antigen-binding fragment thereof, the reference antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region; The VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4), and the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO:11) or RASENLYSYLA (SEQ ID NO:12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO:13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO:14). In some embodiments, the VH region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the VL region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0034] In some embodiments, the antigen-binding domain of the chimeric protein comprises an F(ab) fragment, an F(ab') fragment, or a single chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the VH and VL of the scFv are separated by a peptide linker. In some embodiments, the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-35. In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-75.

[0035] In some aspects, the chimeric protein is an antibody-drug conjugate and the heterologous molecule or moiety comprises a therapeutic agent.

[0036] In some embodiments, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR is an activating CAR comprising one or more intracellular signaling domains that stimulate an immune response. In some embodiments, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some embodiments, the intracellular inhibitory domain comprises an enzyme inhibitory domain. In some embodiments, the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain. In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain. In some embodiments, the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-51.

[0037] In some aspects, the present disclosure provides a composition comprising a chimeric protein as described herein and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0038] In some aspects, the disclosure provides an engineered polynucleotide that encodes a chimeric protein as described herein.

[0039] In some aspects, the disclosure provides an expression vector comprising an engineered polynucleotide as described herein.

[0040] In some aspects, the disclosure provides a composition comprising an engineered polynucleotide as described herein, or an expression vector as described herein, and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0041] In some aspects, the disclosure provides a method of making an engineered cell comprising transducing an isolated cell with an engineered polynucleotide as described herein or an expression vector as described herein. In some aspects, the disclosure provides an engineered cell produced by the above method.

[0042] In some aspects, the disclosure provides an isolated cell comprising an engineered polynucleotide as described herein, an expression vector as described herein, or a composition comprising an engineered polynucleotide or expression vector as described herein and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0043] In some aspects, the disclosure provides a population of engineered cells that express an engineered polynucleotide as described herein, or an expression vector as described herein.

[0044] In some aspects, the present disclosure provides an isolated cell comprising the chimeric protein as described herein. In some aspects, the present disclosure provides a population of engineered cells expressing the chimeric protein as described herein. In some aspects, the chimeric protein is recombinantly expressed in the isolated cell or population of cells. In some aspects, the isolated 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. In some embodiments, the cell or cell population is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the cell or cell population is autologous. In some embodiments, the cell or cell population is allogeneic.

[0045] In some aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of a cell or a population of engineered cells as described herein and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0046] In some aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of a genetically modified cell expressing a chimeric protein as described herein and a pharma- ceutical acceptable carrier, a pharma-ceutical acceptable excipient, or a combination thereof. In some aspects, the pharmaceutical composition is for treating and / or preventing tumors.

[0047] In some aspects, the disclosure provides a method for treating a subject in need thereof, the method comprising administering a therapeutically effective dose of a composition or pharmaceutical composition comprising a chimeric protein, a polynucleotide, an expression vector as described herein, a cell or population of cells as described herein.

[0048] In some aspects, the disclosure provides a method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective dose of a composition or pharmaceutical composition comprising a chimeric protein, a polynucleotide, an expression vector, a cell or population of cells as described herein.

[0049] In some aspects, the disclosure provides a method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of a composition or pharmaceutical composition comprising a chimeric protein, a polynucleotide, an expression vector, a cell or population of cells as described herein.

[0050] In some aspects, the present disclosure provides a kit for treating and / or preventing a tumor comprising a chimeric protein as described herein. In some aspects, the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0051] In some embodiments, the present disclosure provides a kit for treating and / or preventing a tumor comprising a cell or a population of cells as described herein. In some embodiments, the kit further comprises written instructions for using the cells to treat and / or prevent a tumor in a subject.

[0052] In some aspects, the present disclosure provides a kit for treating and / or preventing a tumor comprising an isolated polynucleotide as described herein. In some aspects, the kit further comprises written instructions for using the polynucleotide to generate one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0053] In some embodiments, the present disclosure provides a kit for treating and / or preventing a tumor comprising a vector as described herein. In some embodiments, the kit further comprises written instructions for using the vector to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0054] In some embodiments, the present disclosure provides a kit for treating and / or preventing a tumor comprising a composition as described herein. In some embodiments, the kit further comprises written instructions for using the composition to treat and / or prevent a tumor in a subject. [Brief description of the drawings]

[0055] This 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.

[0056] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0057] [Figure 1] FIG. 1 shows the results of sequencing the light chain variable region of an anti-VSIG2 antibody (Ab) using the Chothia naming scheme. [Diagram 2] FIG. 2 shows the results of sequencing the heavy chain variable region of an anti-VSIG2 antibody (Ab) using the Chothia naming scheme. [Figure 3-1]FIG. 3 shows the expression of various anti-VSIG2 inhibitory CARs and anti-CEA activating CARs from CAR / iCAR-transduced NK cells. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4] FIG. 4 shows killing of CEA-positive target cells expressing VSIG2 or lacking VSIG2 expression by NK cells expressing an anti-VSIG2 inhibitory CAR and an anti-CEA activating CAR. [Diagram 5] FIG. 5 shows the killing of VSIG2-positive target cells by NK cells using an anti-VSIG2 activating CAR. [Figure 6] FIG. 6 shows the expression of various anti-VSIG2 activating CARs on transduced NK cells. [Figure 7A] Figure 7A shows killing of target cells Ls174t by NK cells expressing an anti-VSIG2 activating CAR. [Figure 7B] Figure 7B shows the killing of target cells DLD1 by NK cells expressing an anti-VSIG2 activating CAR. [Figure 7C] Figure 7C shows killing of target cells Ls174t by NK cells expressing an anti-VSIG2 activating CAR. [Figure 7D] Figure 7D shows the killing of target cells DLD1 by NK cells expressing an anti-VSIG2 activating CAR. [Figure 8] FIG. 8 shows the killing of FLT3-positive target cells by NK cells expressing FLT3-positive target cells and various anti-VSIG2 inhibitory CARs. [Figure 9] FIG. 9 shows quantification of TNFα production in FLT3-activated CAR / VSIG2-inhibitory CAR NK cell / target cell co-cultures. [Figure 10A] FIG. 10A shows the expression of various anti-VSIG2 inhibitory and anti-CEA activating CARs on transduced NK cells. [Figure 10B] FIG. 10B shows the expression of various anti-VSIG2 inhibitory and anti-CEA activating CARs on control NK cells. [Figure 11]FIG. 11 shows the killing of CEA-positive target cells by NK cells expressing CEA-activating CARs and various anti-VSIG2 inhibitory CARs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description 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 (SL Tan ed., Taylor & Francis, 2006); Fundamental Virology, 3 rd Edition, vol. I & II (BN Fields and DM Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); AL 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.).

[0059] definition Unless otherwise defined, all terms, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference to what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well known and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and conditions defined by the manufacturer, unless otherwise stated.

[0060] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Terms such as "including," "etc," and the like are intended to convey inclusion without limitation unless otherwise indicated.

[0061] As used herein, the term "comprising" also specifically includes embodiments "consisting of" and "consisting essentially of" the listed elements, unless otherwise indicated.

[0062] The term "about" refers to and includes the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" refers to the specified value ± one standard deviation of that value.

[0063] As used herein, the term "stimulating a cell-mediated immune response" or "stimulating an immune response" refers to the generation of a signal by one or more cell types or cell populations that results in an immune response. Immunostimulatory activity can include proinflammatory activity. In various embodiments, the immune response occurs after activation of immune cells (e.g., T cells or NK cells) or is simultaneously mediated through receptors, including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides can be present in the tumor microenvironment and can activate an immune response against tumor cells. In various embodiments, promoting, stimulating, or ligand-stimulating receptors of proinflammatory polypeptides and / or their ligands can enhance the immune response of immunoresponsive cells. Without being bound by any particular theory, receiving multiple stimulatory signals (e.g., costimulation) is important to support robust, long-lasting cell-mediated immune responses, such as T cell-mediated immune responses in which T cells are inhibited and may not respond to antigens in the absence of costimulatory signals (also referred to as "T cell anergy"). Although the various effects of costimulatory signals vary and are only partially understood, especially in combination with one another, costimulation generally results in increased gene expression to generate long-lived, proliferative, and apoptosis-resistant cells, such as T cells or NK cells, that respond potently to antigens, for example, in mediating complete and / or sustained elimination of target cells expressing the cognate antigen.

[0064] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain that includes a functional signaling domain (also referred to herein as an "intracellular signaling domain").

[0065] As used herein, the term "activated CAR" or "aCAR" refers to a CAR construct / structure that is capable of inducing signaling or protein expression changes in activated CAR-expressing cells that initiate, activate, stimulate, or increase an immune response upon binding to a cognate aCAR ligand.

[0066] As used herein, the term "inhibitory CAR" or "iCAR" refers to a CAR construct / structure capable of inducing a change in signal transduction or protein expression in an inhibitory CAR-expressing cell that inhibits, attenuates, reduces, diminishes, suppresses, or inhibits an immune response upon a cognate iCAR ligand, such as reducing activation of an immunoresponsive cell that is receiving or has received one or more stimulatory signals, including costimulatory signals.

[0067] As used herein, the term "intracellular signaling domain" refers to a functional portion of a protein that acts by transmitting information intracellularly to regulate cellular activity via a defined signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0068] 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 a given antigen or epitope, such as the polypeptide sequence or polypeptide complex portion of the chimeric proteins described herein that result in VSIG2 specific binding. The ABD (or antibody, antigen-binding fragment, and / or chimeric protein containing it) 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 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 molecule. Affinity, i.e., the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), with a lower KD value indicating a stronger interaction between the molecules. The KD values ​​of antibody constructs are measured by methods well known in the art, including, but not limited to, biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays (e.g., flow cytometry). Specific binding, as assessed by affinity, may refer to a binding molecule that has affinity between the ABD and its cognate antigen or epitope, and the KD value is greater than or equal to 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. Specific binding can also include recognition and binding of a biological molecule (e.g., a polypeptide) of interest, while not specifically recognizing and binding to other molecules in a sample, such as a biological sample that naturally contains the polypeptide of the present disclosure. In certain embodiments, specifically binding refers to the binding of an epitope or antigen or antigenic determinant of an ABD, antibody, or antigen-binding fragment in such a way that the binding can be displaced or competed with a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0069] The ABD may be an antibody. The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0070] The ABD may be an antigen-binding fragment of an antibody. As used herein, the term "antigen-binding fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, that is sufficient to confer recognition and specific binding to a target, such as an antigen or epitope. Examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fv, scFv, linear antibodies, single domain antibodies such as sdAb (VL or VH), camel V H These include, but are not limited to, multispecific antibodies formed from antigen-binding fragments, such as bivalent fragments comprising an H domain and two Fab fragments linked by disulfide bridges at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1 136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides, such as fibronectin type III (Fn3) (see, for example, U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0071] 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 with a single ABD is "monovalent". A binding molecule with multiple ABDs is said to be "multivalent". A multivalent binding molecule with two ABDs is "bivalent". A multivalent binding molecule with three ABDs is "trivalent". A multivalent binding molecule with four ABDs is "tetravalent". In various multivalent embodiments, all of the multiple 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 multiple ABDs have different recognition specificities. Such a binding molecule is multivalent and "multispecific". In multivalent embodiments where the ABDs collectively have two recognition specificities, the binding molecule is "bispecific". In multivalent embodiments where the ABDs collectively have three recognition specificities, the binding molecule is "trispecific". In multivalent embodiments in which the ABDs collectively have multiple 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 "dual paratopic."

[0072] In various multivalent embodiments, the multivalent binding molecule improves the avidity of the binding molecule to a specific target. As described herein, "avidity" refers to the overall strength of the interaction between two or more molecules, e.g., a multivalent binding molecule to a specific target, and avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs. Avidity can be measured by the same methods used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule to a specific target is measured by measuring the avidity of the binding molecule between two molecules, where the interaction is a specific binding interaction, and the avidity between the two molecules is greater than 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10In 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, and the affinity of one or more of the individual ABDs does not have a KD value that recognizes the specific binding to their own respective antigen or epitope. In certain embodiments, the avidity is the cumulative strength of the interaction caused by the affinity of multiple ABDs for separate antigens on a shared specific target or complex, such as separate antigens found on individual cells. In certain embodiments, the avidity is the cumulative strength of the interaction caused by the affinity of multiple ABDs for separate epitopes on a shared individual antigen.

[0073] 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 linked via a short flexible polypeptide linker and 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. As used herein, unless specified, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and an scFv can comprise a VL-linker-VH or a VH-linker-VL.

[0074] As used herein, "variable region" refers to the variable region resulting from a recombination event, for example, after V, J, and / or D segment recombination in immunoglobulin genes in B cells or T cell receptor (TCR) genes in T cells. 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 selected VH and a selected VL can combine together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0075] The term "complementarity determining region" or "CDR" as used herein refers to sequences within the antibody variable regions VH and VL that confer antigen specificity and binding affinity. For example, typically, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and there are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al, (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acid residues of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL 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 amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of 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) of a VL, e.g., a mammalian VL, e.g., a human VL. In various embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences.

[0076] As used herein, the term "framework region" or "FR" refers to the generally conserved sequences in antibody variable regions VH and VL that typically serve as scaffolds for interspersed CDRs, in the following configuration (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In various embodiments, the FRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In certain embodiments, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthetic sequences, including but not limited to rationally designed sequences.

[0077] 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 native conformations and which usually determine the class to which the antibody belongs.

[0078] 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 native conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0079] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0080] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response can involve either antibody production or activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen.

[0081] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life span, a reduction in tumor cell proliferation, a reduction in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure to prevent the development of tumors in the first place, such as prophylactic therapy or treatment.

[0082] As used herein, the term "autologous" refers to any material derived from the same subject that is subsequently reintroduced into the subject.

[0083] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the subject into which the material is introduced. Two or more subjects are said to be allogeneic to each other if 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 different to interact antigenically at certain genes, such as MHC alleles. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically identical to not interact antigenically at certain genes, such as MHC alleles.

[0084] The isolated nucleotide molecules of the present disclosure include any polynucleotide molecule or nucleic acid sequence that encodes a polypeptide or fragment thereof of the present disclosure. Such polynucleotide molecules do not need to be 100% homologous or identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A nucleic acid sequence that has "substantial identity" or "substantial homology" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded polynucleotide molecule. As used herein, "hybridization" refers to pairing to form a double-stranded molecule between a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof under various stringency conditions. For example, stringent salt concentrations can usually 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. In the absence of organic solvent, such as formamide, low stringency hybridization can be obtained, while in the presence of at least about 35% formamide or at least about 50% formamide, high stringency hybridization can be obtained.Stringent temperature conditions usually include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency can be achieved by combining these various conditions as needed.

[0085] "Substantially identical" or "substantially homologous" means that a polypeptide or polynucleotide molecule exhibits at least 50% homology or identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., 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 at the nucleic acid level to the sequence used for comparison. Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. An exemplary method for measuring the degree of identity may use the BLAST program, where a probability score of e-3 to e-100 indicates closely related sequences.

[0086] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the 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, or a biological property resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise indicated, 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 encoding a protein or RNA" can also include introns to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0087] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, allowing for intercellular recognition and / or interaction.

[0088] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result. In some embodiments, an "effective amount" or "therapeutically effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis of a disease or disorder of interest, e.g., a solid tumor.

[0089] 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 precursor or progeny thereof. Examples of immune effector cells include, but are not limited to, alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0090] As used herein, the term "immune effector response" or "immune effector function" refers to, for example, the function or response of an immunoresponsive cell that enhances or promotes the immune attack of a target cell. For example, immune effector function or response can refer to the property of T cells or NK cells that promotes the killing or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0091] As used herein, the term "flexible polypeptide linker" or "linker" refers to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and has the amino acid sequence (Gly-Gly-Gly-Gly-Ser). n or (Gly-Gly-Gly-Ser) n where n is a positive integer greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10. In some embodiments, the flexible polypeptide linker comprises a Gly 4 Ser [SEQ ID NO: 27] or (Gly 4 Ser) 3 [SEQ ID NO: 29]. In another embodiment, the linker is 2 Ser), (GlySer), or (Gly 3In some embodiments, the flexible polypeptide linker comprises multiple repeats of (Ser) [SEQ ID NO: 22]. In some embodiments, the flexible polypeptide linker comprises a Whitlow linker (e.g., GSTSGSGKPGSGEGSTKG [SEQ ID NO: 32]). In some embodiments, the flexible polypeptide linker comprises (EAAAK) 3 [SEQ ID NO: 33]. Also included within the scope of the present disclosure are linkers described, for example, in WO2012 / 138475.

[0092] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or alleviation of the progression, severity, and / or duration of a proliferative disorder (e.g., cancer), or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the present disclosure). In some embodiments, the reduction or amelioration refers to the improvement of at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to the inhibition of progression of the proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In some embodiments, the reduction or amelioration includes the reduction or stabilization of tumor size or cancer cell number.

[0093] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0094] Other aspects of the disclosure are described in the following sections and are within the scope of the claims.

[0095] Other Rules of Interpretation Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0096] VSIG2-specific chimeric proteins and antigen-binding domains The present disclosure provides chimeric proteins and polynucleotides encoding such chimeric proteins that bind to V-set and immunoglobulin domain-containing protein 2 (VSIG2). In some embodiments, the VSIG2-specific chimeric protein binds to human VSIG2 (e.g., Uniprot Q96IQ7, incorporated herein by reference for all purposes) or an epitopic fragment thereof. VSIG2 may be expressed on epithelial cells. VSIG2 may be expressed on cells that are generally considered healthy, such as healthy epithelial cells. Examples of VSIG2-specific antibodies include OTI2D8 (also known as "2D8" and referred to herein as Ab) and OTI5A10 (also known as "5A10").

[0097] The present disclosure provides chimeric proteins and polynucleotides encoding such chimeric proteins, comprising a VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table A.

[0098] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4). In some embodiments, the VH further comprises a heavy chain complementarity determining region 1 (CDR-H1) and a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequences of CDR-H1 and CDR-H2 contained in the VH region amino acid sequence of SEQ ID NO:1. In some embodiments, a VSIG2-specific antigen-binding domain further comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a VSIG2-specific antigen binding domain comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0099] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where the VH comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) with the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, a VSIG2-specific antigen-binding domain further comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), where the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, a VSIG2-specific antigen binding domain comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0100] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4). In some embodiments, the VSIG2-specific antigen-binding domain further comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a VSIG2-specific antigen binding domain comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0101] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a VSIG2-specific antigen-binding domain further comprises a heavy chain complementarity determining region (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3), where the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO:1. In some embodiments, a VSIG2-specific antigen binding domain comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4).

[0102] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14). In some embodiments, a VSIG2-specific antigen-binding domain further comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, a VSIG2-specific antigen binding domain comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4).

[0103] In some embodiments, a VSIG2-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4), and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO:11) or RASENLYSYLA (SEQ ID NO:12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO:13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO:14).

[0104] In some embodiments, a VSIG2-specific antigen-binding domain has a VH region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, a VSIG2-specific antigen-binding domain has a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0105] In some embodiments, a VSIG2-specific antigen-binding domain has a VL region comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a VSIG2-specific antigen-binding domain has a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a VSIG2-specific antigen-binding domain has (1) a VH region comprising the amino acid sequence of SEQ ID NO:1, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a VL region comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0106] In some embodiments, a VSIG2-specific antigen-binding domain has (1) a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a VL region comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0107] In some embodiments, a VSIG2-specific antigen-binding domain has (1) a VL region comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and (2) a VH region comprising the amino acid sequence of SEQ ID NO:1 or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, a VSIG2-specific antigen-binding domain has (1) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and (2) a VH region comprising the amino acid sequence of SEQ ID NO:1 or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0108] In some embodiments, the VSIG2-specific antigen-binding domain competes with a reference antibody or antigen-binding fragment having a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO:4), and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO:11) or RASENLYSYLA (SEQ ID NO:12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO:13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO:14).

[0109] In some embodiments, a VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a distinct human VSIG2 epitope) as a reference antibody, or antigen-binding fragment thereof, having a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and QGVRPFF (1) the VL comprises a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of DY (SEQ ID NO: 4); and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14). In some embodiments, the VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a distinct human VSIG2 epitope) as a reference antibody or antigen-binding fragment thereof having a VH comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a distinct human VSIG2 epitope) as a reference antibody or antigen-binding fragment thereof having a VL comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0110] The VSIG2-specific antigen-binding domain can be in any of the formats described herein, such as Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH, and multispecific formats. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab) format. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab') format.

[0111] In some embodiments, the VSIG2-specific antigen-binding domain is in a single chain variable fragment (scFv) format, including scFv formats with any of the peptide linkers described herein (see, e.g., Table 1). In some embodiments, the VSIG2-specific antigen-binding domain has the structure VH-L-VL or VL-L-VH, where L is a peptide linker.

[0112] Chimeric antigen receptors (CARs) Certain aspects of the present disclosure relate to a chimeric receptor having any one of the VSIG2-specific antigen binding domains described herein and capable of specifically binding to a VSIG2 protein, a VSIG2-derived antigen, or a VSIG2-derived epitope. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, a CAR is a chimeric protein that includes an antigen binding domain and a polypeptide molecule that is heterologous to the antigen binding domain, such as a peptide that is heterologous to the antibody from which the antigen binding domain can be derived. The polypeptide molecule that is heterologous to the antigen binding domain includes, but is not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or a combination thereof.

[0113] In some embodiments, CARs are engineered receptors that transfer or confer specificity of interest (e.g., VSIG2) to immune effector cells. In certain embodiments, CARs can be used to transfer antibody specificity to immune responsive cells such as T cells or NK cells. In some embodiments, the CARs of the present disclosure comprise an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0114] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, and also commonly referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of an immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of an immunoresponsive cell. In some embodiments, activation of the immunoresponsive cell results in killing of the target cell. In some embodiments, activation of the immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of the immunoresponsive cell can be a combination of the above responses.

[0115] The CAR of the present disclosure can be a first, second, or third generation CAR. A "first generation" CAR contains a single intracellular signaling domain, generally derived from a T cell receptor chain. A "first generation" CAR generally has an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the main transmitter of signals from the endogenous TCR. A "first generation" CAR provides de novo antigen recognition and transduces CD4 T cells via the CD3ζ chain signaling domain in a single fusion molecule, independent of antigen presentation via HLA. + and CD8 +"Second generation" CARs add a second intracellular signaling domain from one of a variety of costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide an additional signal to the T cell. "Second generation" CARs provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second generation" CARs can enhance the antitumor activity of immune responsive cells such as T cells. "Third generation" CARs have multiple intracellular costimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0116] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, the one or more chimeric inhibitory receptors bind to an antigen expressed on a non-tumor cell derived from a tissue selected from brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0117] In some embodiments, a chimeric inhibitory receptor (e.g., a VSIG2-specific chimeric inhibitory receptor) may be used with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, for example, as a non-logical gate to control, regulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. For example, if a healthy cell expresses both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by an inhibitory chimeric receptor, the immunoresponsive cell expressing the tumor antigen may bind to the healthy cell. In such a case, the inhibitory chimeric antigen also binds to its cognate ligand on the healthy cell, and the inhibitory function of the inhibitory chimeric receptor reduces, reduces, prevents, or inhibits the activation of the immunoresponsive cell via the tumor-targeting chimeric receptor ("non-logical gating"). In some embodiments, the inhibitory chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, the immunoresponsive cells may contain one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors that target antigens that are not expressed or generally not considered to be expressed on tumors (e.g., VSIG2). A combination of tumor-targeting chimeric receptors and inhibitory chimeric receptors on the same immunoresponsive cells can be used to reduce extratumoral toxicity on the target.

[0118] In some embodiments, the extracellular antigen binding domain of a CAR of the disclosure comprises about 2×10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 M or less, approximately 2×10 -9 M or less, or about 1 x 10 -9 The dissociation constant (K d ) binds to one or more antigens (e.g., VSIG2). In some embodiments,d is about 2 x 10 -7 M ~ approx. 1×10 -9 The range is M.

[0119] Binding of the extracellular antigen-binding domain of the CAR of the present disclosure can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeling reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFvs can be radioactively labeled and used in RIA assays. Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific for the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., with a radioactive or fluorescent marker).

[0120] In some embodiments, a CAR of the present disclosure comprises an extracellular antigen binding domain that binds to VSIG2 (e.g., a VSIG2 protein, a VSIG2-derived antigen, or a 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 comprises an F(ab) 2 It is a fragment.

[0121] Extracellular antigen-binding domain The extracellular antigen binding domain of the CAR of the present disclosure specifically binds to VSIG2 (e.g., a VSIG2 protein, a VSIG2-derived antigen, or a VSIG2-derived epitope). In certain embodiments, the extracellular antigen binding domain binds to VSIG2 expressed on epithelial cells. In certain embodiments, the extracellular antigen binding domain binds to VSIG2 expressed on cells that are generally considered healthy, such as healthy epithelial cells. In some embodiments, the VSIG2 is human VSIG2.

[0122] Antigen binding domains of the present disclosure can include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single domain antibodies (sdAbs), such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), as well as any domain associated with an alternative scaffold known in the art to function as an antigen binding domain, such as recombinant fibronectin domains, T cell receptors (TCR), recombinant affinity-improved TCRs, or fragments thereof, e.g., single chain TCRs. In some cases, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used.

[0123] 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 chimeric antibody. In some embodiments, the extracellular antigen-binding domain comprises an antigen-binding fragment of an antibody.

[0124] In some embodiments, the extracellular antigen-binding domain comprises an F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises an F(ab') fragment.

[0125] 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 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).

[0126] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 74.

[0127] In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, a polynucleotide encoding an scFv of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 84. 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 1. In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked to each other, each scFv can be linked to the next scFv to which a peptide is linked. In some embodiments, each of the one or more scFvs is separated by a peptide linker.

[0128] Table 1 Peptide linkers TIFF2024516713000002.tif95128

[0129] In some embodiments, the present disclosure provides a first CAR and a second CAR. The antigen-binding domain of the first CAR and the antigen-binding domain of the second CAR can be suitable antigen-binding domains 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 antibodies, F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFv), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first CAR and / or the second CAR comprises two single-chain variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first CAR can be specific for VSIG2, and the antigen-binding domain of the second CAR can be specific for a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on tumor cells, such as colorectal cancer cells).

[0130] In some embodiments, the extracellular antigen-binding domain comprises a single domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0131] 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 antigen binding domains, or ten or more antigen binding domains. In some embodiments, each of the two or more antigen binding domains binds to the same antigen. In some embodiments, each of the two or more antigen binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen binding domains binds to a different antigen.

[0132] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains can be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, an sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-improved recombinant TCR, and a single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0133] In certain embodiments, the bispecific CAR or tanCAR comprises an antigen-binding domain that comprises 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 located upstream of its VL (VL 1 ) upstream of its VH (VH 1 ), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH 2 ) upstream of that VL(VL 2 ) so that the overall bispecific antibody molecule has the arrangement VH 1 -VL 1 -VL 2 -VH 2 In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VH (VH 1 ) upstream of that VL(VL 1 ) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL 2 ) upstream of its VH (VH 2 ) and the overall bispecific antibody molecule is arranged in the arrangement VL 1 VH 1 -VH 2 -VL 2 In some embodiments, a linker is provided between two antibodies or antibody fragments (e.g., scFv), for example, when the construct has a VH 1 -VL1 -VL 2 -VH 2 If placed as VL 1 and V.L. 2 Between or between the construct VL 1 -VH 1 -VH 2 -VL 2 If placed as VH 1 and V.H. 2 The linker may be any of the linkers described herein, e.g., (Gly 4 -Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. In general, the linker between the two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is placed between the VL and VH of the first scFv. In some embodiments, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR comprises a VL, a VH, and may further comprise one or more linkers in an arrangement as described herein.

[0134] In some embodiments, the chimeric receptor of the present disclosure comprises a bivalent CAR. In some embodiments, the bivalent CAR is a VSIG2 bivalent CAR. In some embodiments, the bivalent VSIG2 CAR comprises one or more of the anti-VSIG2 sequences shown in Table A. In some embodiments, the ABDs of the bivalent VSIG2 CAR each comprise the same ABD.

[0135] In some embodiments, the chimeric receptor comprises a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises a VSIG2 CAR. In some embodiments, the bicistronic VSIG2 CAR comprises one or more of the anti-VSIG2 sequences shown in Table A.

[0136] Transmembrane domain In some embodiments, the transmembrane domain of the CAR of the present disclosure (e.g., the VSIG2-specific CAR described herein) comprises a hydrophobic alpha helix that spans at least a portion of the cell membrane. It has been shown that different transmembrane domains can result in different receptor stabilities. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR of the present disclosure can comprise a transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0137] In some embodiments, the transmembrane domain is from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. Examples of CD8 transmembrane domains include IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 36), IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 37), and IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 38). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 36). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 37). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 38).

[0138] In some embodiments, the transmembrane domain is from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide may be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide may be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2.In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Exemplary BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0139] In some embodiments, the transmembrane domain is selected from the group consisting of NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009 , NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a fragment thereof. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can include one conservative amino acid substitution, no more than two conservative amino acid substitutions, or no more than three conservative amino acid substitutions.In some embodiments, the polypeptide 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 (NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_00333.2, NP_00333.3, NP_00333.4, NP_00333.5, NP_00333.6, NP_00333.7, NP_00333.8, NP_00333.9, NP_00333.1, NP_00333.1, NP_00333.2, NP_00333.3, NP_00333.4, NP_00333.5, NP_00333.6, NP_00333.7, NP_00333.8, NP_00333.9, NP_00333.9, NP_00333.1 ... 1668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0140] Further examples of suitable polypeptides from which a transmembrane domain may be derived include T cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD10 3, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (antennary), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and the transmembrane domains of the alpha, beta, or zeta chains of NG2C.

[0141] In some embodiments, the transmembrane domain is derived from the same protein as the intracellular domain of the CAR.

[0142] Spacer Region In some embodiments, the CAR of the present disclosure (e.g., the VSIG2-specific CAR described herein) can also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region can be a hinge derived from a human protein. For example, the hinge can be a human Ig (immunoglobulin) hinge, including but not limited to an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region can include an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some aspects, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may comprise any of the amino acid sequences listed in Table 2, 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 2.

[0143] (Table 2) Spacer amino acid sequences TIFF2024516713000003.tif94157

[0144] In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 39. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 40. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 47. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 48.

[0145] In some embodiments, the spacer region comprises the sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 49). In some embodiments, the spacer region comprises the sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 50). In some embodiments, the spacer region comprises the sequence FVPVFLPAKPTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 51).

[0146] In some embodiments, a polynucleotide encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table 3, 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 3.

[0147] Table 3: Spacer nucleic acid sequences TIFF2024516713000004.tif163157

[0148] In some embodiments, the CAR of the present disclosure may further comprise a short oligopeptide or polypeptide linker that is 2 to 10 amino acid residues in length and can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine-serine duplex. In some embodiments, the linker comprises the amino acid sequence of GGCKJSGGCKJS (SEQ ID NO: 62).

[0149] Intracellular signaling domains In some embodiments, a CAR of the present disclosure (e.g., a VSIG2-specific CAR described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of a CAR may comprise an intracellular signaling domain.

[0150] Examples of suitable intracellular signaling domains that may be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act coordinately to regulate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0151] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of T cells, and thus secondary and / or costimulatory signals are typically also required for full activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). In addition, T cell signaling and function (e.g., activation signaling cascades) can be negatively controlled by inhibitory receptors present in T cells via intracellular inhibitory co-signaling domains.

[0152] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can include an inhibitory intracellular signaling domain. 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., transmembrane domains) via a peptide linker (see, e.g., Table 1) or a spacer or hinge sequence (see, e.g., Table 2). 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 via a peptide linker (see, e.g., Table 1) or a spacer or hinge sequence (see, e.g., Table 2). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, one or more intracellular inhibitory co-signaling domains of the chimeric protein include one or more ITIM-containing proteins, or fragments thereof. ITIMs are conserved amino acid sequences found in the cytoplasmic tail of many inhibitory immunoreceptors. In some embodiments, the one or more ITIM-containing proteins, or fragments thereof, are selected from PD-1, CTLA4, TIGIT, BTLA, LAIR1, KIR2DL1, KIR3DL1, LIR1, SIGLEC2, and SIRPalpha (SIRPa). In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, GITR, and PD-L1. The inhibitory intracellular signaling domain can further comprise an enzyme inhibitory domain. In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain.In some embodiments, the enzyme catalytic domain is derived from an enzyme including, but not limited to, CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, or RasGAP. Examples of enzymatic regulation of signal transduction are described in detail by Pavel Otahal et al. (Biochim Biophys Acta. 2011 Feb;1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 Jun; 14(6): 669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep; 137(1): 1-19), each of which is incorporated herein by reference for all purposes.

[0153] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can include a primary signaling domain that controls the primary activation of the TCR complex, either in a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can include signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in the CAR of the present disclosure include, but are not limited to, those of CD3-zeta, FcR gamma, FcR beta beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0154] In some embodiments, a CAR of the present disclosure (e.g., a VSIG2-specific CAR described herein) comprises an intracellular signaling domain, e.g., a primary signaling domain of a 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 of NCBI reference number NP_932170 or NP_001106864.2. In some embodiments, a CD3-zeta polypeptide may include one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, a polypeptide can have an amino acid sequence that is a contiguous portion of NCBI reference number 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.

[0155] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the 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, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0156] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can comprise a CD3-zeta signaling domain by itself, or it can be combined with any other desired intracellular signaling domain useful in the context of the CAR of the present disclosure.For example, the intracellular signaling domain of the CAR can comprise a portion of the CD3-zeta chain and a costimulatory signaling domain.A costimulatory signaling domain can refer to a portion of the CAR that comprises 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 ligand that can be required for the efficient response of lymphocytes to antigens.Examples of suitable costimulatory molecules include CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, MHC class I molecules, TNF receptor proteins, immunoglobulins, and the like. globulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4 , VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGA X, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, C These include, but are not limited to, D96 (antennary), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and the like.

[0157] In some embodiments, the intracellular signaling sequences within the cytoplasmic portion of the CAR of the present disclosure can be linked together in a random or specific order. In some embodiments, a short oligopeptide or polypeptide linker, for example, 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form a bond between the intracellular signaling sequences. In one embodiment, a glycine-serine duplex can be used as a suitable linker. In one embodiment, a single amino acid, for example, alanine or glycine, can be used as a suitable linker.

[0158] 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, ten 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 1). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0159] In some embodiments, a cell of the disclosure expresses a CAR that includes an antigen binding domain that binds to VSIG2, a transmembrane domain of the disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0160] In some embodiments, a cell of the disclosure expresses an iCAR that includes an antigen binding domain that binds VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A), a transmembrane domain of the disclosure, and one or more intracellular inhibitory co-signaling domains. In some embodiments, a cell of the disclosure expresses (1) a CAR that includes an antigen binding domain that binds VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A), a transmembrane domain of the disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0161] Natural killer cell receptor (NKR) CAR In some embodiments, a CAR of the disclosure (e.g., a VSIG2-specific CAR described herein) comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR components include KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS The NKR-CAR may be a transmembrane domain, hinge domain, or cytoplasmic domain from any suitable natural killer cell receptor, including, but not limited to, killer cell immunoglobulin-like receptors (KIRs) such as DPI; natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0162] Further chimeric receptor targets Certain embodiments of the present disclosure relate to chimeric receptors that bind to an antigen of interest in addition to VSIG2 and polynucleotides encoding such chimeric receptors. Certain embodiments of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, genetically modified to express one or more of such chimeric receptors that bind to an antigen of interest in addition to VSIG2, and methods of using such receptors and cells to treat and / or prevent malignant tumors, such as solid tumors, and other conditions 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 to treat such malignant cells.

[0163] In some embodiments, the first chimeric receptor comprises an antigen binding domain that binds VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A) and the second chimeric receptor comprises an additional antigen binding domain that binds a second antigen, such as a tumor associated antigen (e.g., a colorectal cancer associated antigen). In some embodiments, the cell can express a first chimeric receptor specific for VSIG2 (e.g., a CAR comprising a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A) and a second chimeric receptor specific for a second antigen, such as a tumor associated antigen (e.g., a colorectal cancer associated antigen). In some embodiments, the cell can express a first inhibitory chimeric receptor specific for VSIG2 (e.g., an inhibitory CAR comprising a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A) and a second chimeric receptor specific for a second antigen, such as a tumor associated antigen (e.g., a colorectal cancer associated antigen). For example, a cell (e.g., an immunoresponsive cell) can be engineered to co-express, or be capable of co-expressing, an iCAR comprising an antigen binding domain that binds VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A) and an aCAR that targets a tumor-associated antigen (e.g., a colorectal cancer-associated antigen). In addition to VSIG2, suitable antibodies that bind antigens 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 as a tumor-associated antigen (e.g., a colorectal cancer-associated antigen). In some embodiments, a commercially available antibody that binds to a second antigen, such as a tumor-associated antigen (e.g., a colorectal cancer-associated antigen), may be used. The CDRs of commercially available antibodies are readily accessible by one of skill in the art using conventional sequencing techniques. Furthermore, one of skill in the art can construct polynucleotides encoding scFvs and chimeric receptors (e.g., CARs and TCRs) based on the CDRs of such commercially available antibodies.

[0164] T cell receptor (TCR) Certain aspects of the present disclosure relate to chimeric receptors that specifically bind to a second antigen, such as a tumor-associated antigen (e.g., a colorectal cancer-associated antigen), where the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). The TCR of the present disclosure is a disulfide-linked heterodimeric protein that includes two variable chains expressed as part of a complex with an invariant CD3 chain molecule. The TCR is found on the surface of T cells and is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR of the present disclosure includes an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR includes a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0165] Each chain of the TCR consists of two extracellular domains, the variable (V) region and the constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable regions bind to the peptide / MHC complex. Each of the variable regions has three complementarity determining regions (CDRs).

[0166] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. Upon complexing of the TCR complex with its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0167] In some embodiments, the 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 the naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from the 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 a naturally occurring TCR modified by at least one amino acid residue. In some embodiments, the TCR is a naturally occurring TCR modified 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.

[0168] Chimeric TCR In some embodiments, the TCR of the present disclosure comprises one or more antigen binding domains that can be grafted onto one or more constant domains of a TCR chain, e.g., a TCR alpha chain or a TCR beta chain, to create a chimeric TCR that specifically binds to a second antigen of interest, such as a tumor-associated antigen (e.g., a colorectal cancer-associated antigen). Without wishing to be bound by theory, it is believed that the chimeric TCR can signal through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., scFv) can be grafted onto at least a portion of the constant domains, e.g., the extracellular constant domain, the transmembrane domain, and the cytoplasmic domain, of a TCR chain, such as a TCR alpha chain and / or a TCR beta chain. As another example, the CDRs of an antibody or antibody fragment can be grafted onto a TCR alpha chain and / or a beta chain to create a chimeric TCR that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., a colorectal cancer-associated antigen). Such chimeric TCRs can be produced by methods well known in the art (see, 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).

[0169] Immunoresponsive cells Certain aspects of the present disclosure relate to cells (such as immunoresponsive cells) genetically engineered to contain one or more chimeric receptors of the present disclosure or one or more polynucleotides encoding such chimeric receptors, and methods of using such cells to treat solid tumors (e.g., colorectal cancer).

[0170] 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 is a bone marrow cell, a blood cell, a skin cell, a bone cell, a muscle cell, a neuronal cell, an adipocyte, a liver cell, or a cardiac cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, but are not limited to, 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 (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell derived from or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. The immune cell of the present disclosure can be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or an iPSC). Exemplary immune cells include, but are not limited to, 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 can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary neuronal cells include, but are not limited to, 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.

[0171] In some embodiments, the cell is an immunoresponsive cell. The immunoresponsive cells of the present disclosure can be isolated or differentiated from the stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary immunoresponsive cells of the present disclosure include, but are not limited to, cells of lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, provides antibody production, control of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, and the like. Examples of immunoresponsive cells of lymphoid lineage include, but are not limited to, T cells, natural killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those derived from or that can be differentiated into lymphoid cells). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present disclosure include T helper cells, cytotoxic T cells, memory T cells (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., T EM Cells and T EMRA The T cells can be any type of T cell, including but not limited to T cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as chimeric TCRs or CARs.

[0172] 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 to exert their cytotoxic effect on target cells.

[0173] In some embodiments, the immunoresponsive cells of the present disclosure are T cells. The T cells of the present disclosure may be derived in vitro from autologous, allogeneic, or engineered progenitor or stem cells.

[0174] In some embodiments, the immunoresponsive cell of the present disclosure is a universal T cell with defective TCR-αβ. Methods for developing universal T cells are described in the art, for example, Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0175] In some embodiments, the immunoresponsive cells of the present disclosure are isolated immunoresponsive cells comprising one or more chimeric receptors of the present disclosure, hi some embodiments, the immunoresponsive cells comprise 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.

[0176] In some embodiments, the immunoresponsive cell is a T cell. In some embodiments, the immunoresponsive cell is a natural killer (NK) cell.

[0177] In some embodiments, the immunoresponsive cells express or are capable of expressing an immunoreceptor. An immunoreceptor can generally induce signal transduction or changes in protein expression in an immunoreceptor-expressing cell, which modulates an immune response upon binding to a cognate ligand (e.g., modulates, activates, initiates, stimulates, increases, prevents, attenuates, inhibits, reduces, decreases, inhibits, or suppresses an immune response). For example, when the CD3 chains present on a TCR / CAR cluster in response to ligand binding, a signal transduction cascade occurs via an immunoreceptor tyrosine-based activation motif (ITAM). Specifically, in certain embodiments, binding of an endogenous TCR, an exogenous TCR, a chimeric TCR, or a CAR (specifically an activated CAR) to its respective antigen results in the formation of an immune synapse, which includes the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. Such clustering of membrane-bound signaling molecules results in phosphorylation of ITAM motifs contained within the CD3 chains, which initiates the T cell activation pathway and ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors can induce global gene expression in T cells to initiate T cell-mediated immune responses such as cytokine production and / or T cell-mediated killing by increasing IL-2 production for proliferation and expression of master regulator T cell proteins.

[0178] Cells expressing multiple chimeric receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the disclosure comprises two or more chimeric receptors of the disclosure. In some embodiments, the cell comprises two or more chimeric receptors, where one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, where 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, where 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, where at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0179] In some embodiments, each of the two or more chimeric receptors comprises a different antigen binding domain, e.g., antigen binding domains that bind the same antigen or different antigens. In some embodiments, each antigen bound by the two or more chimeric receptors is expressed in the same cell, such as an epithelial cell type (e.g., the same epithelial cell type).

[0180] In embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more distinct chimeric receptors, the antigen-binding domains of each of the different chimeric receptors can be designed so that the antigen-binding domains do not interact with each other. For example, a cell (e.g., an immunoresponsive cell) of the present disclosure expressing a first chimeric receptor (e.g., a VSIG2-specific chimeric receptor) and a second chimeric receptor can include a first chimeric receptor that includes 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 can include an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can include a VHH.

[0181] Without wishing to be bound by theory, it is believed that in cells with multiple chimeric membrane-embedded receptors, each of which contains an antigen-binding domain, interactions between the antigen-binding domains of each receptor may be undesirable as such interactions may inhibit the ability of one or more of the antigen-binding domains to bind their cognate antigen. Thus, in embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more chimeric receptors, the chimeric receptors contain 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 a second chimeric receptor comprises a single VH domain, e.g., a camel, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0182] In some embodiments, when present on the surface of a cell, the binding of the antigen binding domain of the first chimeric receptor to its cognate antigen (e.g., a VSIG2-specific chimeric receptor that binds to VSIG2) is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, the 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 the 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 each other less than when both are scFv antigen binding domains. In some embodiments, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other less than when both are scFv antigen binding domains.

[0183] Chimeric inhibitory receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the disclosure comprises one or more chimeric inhibitory receptors of the disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen binding domain that binds to an antigen that is typically expressed on normal cells (e.g., cells typically considered to be healthy) but not expressed on tumor cells, such as colorectal cancer cells. In some embodiments, the inhibitory chimeric receptor comprises an antigen binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table A).

[0184] In some embodiments, the one or more chimeric inhibitory receptors bind to an antigen expressed on a non-tumor cell derived from a tissue selected from brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0185] In some embodiments, a chimeric inhibitory receptor (e.g., a VSIG2-specific chimeric inhibitory receptor) may be used with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, e.g., as a non-logic gate to control, regulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. In some embodiments, an inhibitory chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure.

[0186] Costimulatory Ligands In some embodiments, the cells (e.g., immunoresponsive cells) of the present disclosure can further include one or more recombinant or exogenous costimulatory ligands. For example, the cells can be further transduced with one or more costimulatory ligands such that the cells co-express or are induced to co-express one or more chimeric receptors of the present disclosure (e.g., VSIG2-specific CARs described herein) and one or more costimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between one or more chimeric receptors and one or more costimulatory ligands may provide a non-antigen-specific signal that is important for the full activation of the cells. Examples of suitable costimulatory ligands include, but are not limited to, 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 response. Its main role is the regulation of immune cells. Members of the TNF superfamily share several common characteristics. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) that contain a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas Ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell activating factor (B AFF) / Bly s / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and contain immunoglobulin domains (folds).Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, and PD-L1 / (B7-H1), a ligand for PD-1. In certain embodiments, the one or more costimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0187] Chemokine Receptors In some embodiments, the cells (e.g., immunoresponsive cells) of the present disclosure comprise one or more chimeric receptors (e.g., VSIG2-specific CARs described herein) and may further comprise one or more chemokine receptors. For example, transgenic expression of chemokine receptors CCR2b or CXCR2 in cells, such as T cells, enhances trafficking to CCL2- 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 the chemokine receptors expressed on the chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by the tumor and improve targeting of the cells to the tumor, which may promote invasion of the cells into the tumor and enhance the anti-tumor effect of the cells. The chemokine receptor of the present disclosure may include naturally occurring chemokine receptors, recombinant chemokine receptors, or chemokine-binding fragments thereof.Suitable chemokine receptors that may be expressed on the cells of the present disclosure include, but are not limited to, CXC chemokine receptors such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7, CC chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11, CX3C chemokine receptors such as CX3CR1, XC chemokine receptors such as XCR1, and chemokine-binding fragments thereof.In some embodiments, the chemokine receptors expressed on the cells are selected based on the chemokines secreted by tumors.

[0188] Chimeric receptor regulation Some embodiments of the present disclosure relate to controlling one or more chimeric receptor activities of the chimeric receptor expressing cells of the present disclosure (e.g., VSIG2-specific CARs described herein). There are several ways to control chimeric receptor activity. In some embodiments, a controllable chimeric receptor that can control one or more chimeric receptor activities may be desirable to optimize the safety and / or efficacy of 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 chimeric receptor therapy. In some embodiments, cells expressing a chimeric receptor of the present disclosure can also express inducible caspase-9 (iCaspase-9), which upon administration of a dimerizing agent such as rimiduside (IUPAC name: [(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-[[2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-oxoethoxy]phenyl]propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate), induces activation of caspase-9, resulting in cellular apoptosis. In some embodiments, iCaspase-9 contains a binding domain that contains a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, resulting in inducible and selective depletion of chimeric receptor-expressing cells.

[0189] Alternatively, in some embodiments, the chimeric receptor of the present disclosure may be controlled by utilizing small molecules or antibodies that inactivate or inhibit chimeric receptor activity. For example, an antibody may delete chimeric receptor-expressing cells by inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the chimeric receptor-expressing cells of the present disclosure may further express an antigen that is recognized by a molecule that can induce cell death by ADCC or complement-induced cell death. For example, the chimeric receptor-expressing cells of the present disclosure may further express a receptor that can be targeted by an antibody or antibody fragment. Examples of suitable receptors that can be targeted by an antibody or antibody fragment include EpCAM, VEGFR, integrins (e.g., ανβ3, α4, αΙ3 / 4β3, α4β7, α5β1, ανβ3, αν), 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-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, C D5, CD11, CD11a / 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.

[0190] In some embodiments, the chimeric receptor-expressing cells of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capability but retains an epitope recognized by a molecule capable of inducing ADCC (e.g., WO2011 / 056894).

[0191] In some embodiments, the chimeric receptor expressing cells of the present disclosure further comprise a highly expressed small marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor expressing cells that binds with an anti-CD20 antibody (e.g., rituximab) and results in selective depletion of the chimeric receptor expressing cells by ADCC. Other methods for depleting the chimeric receptor expressing cells of the present disclosure include, but are not limited to, administration of a monoclonal anti-CD52 antibody that selectively binds and targets the chimeric receptor expressing cells for destruction by inducing ADCC. In some embodiments, the chimeric receptor expressing cells can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can trigger effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can further be bound to an agent that induces cell death, such as a toxin. In some embodiments, the chimeric receptor expressing cells of the present disclosure can further express a target protein that is recognized by the cell depletion agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depletion agent is an anti-CD20 antibody. In such embodiments, the cell depletion agent is administered when it is desired to reduce or eliminate chimeric receptor expressing cells. In some embodiments, the cell depletion agent is an anti-CD52 antibody.

[0192] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides in which the components of the chimeric receptor of the present disclosure are distributed on separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, in the presence of a dimerization molecule, can bind the polypeptides to each other to form a functional chimeric receptor.

[0193] Polynucleotide constructs encoding chimeric receptors Certain aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) encoding one or more chimeric receptors of the present disclosure (e.g., VSIG2-specific CARs described herein). In some embodiments, the polynucleotide is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the polynucleotide is a DNA construct.

[0194] In some embodiments, the polynucleotides of the present disclosure encode a chimeric receptor comprising one or more antigen binding domains, each domain binding to a target antigen (e.g., VSIG2), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the polynucleotide encodes a chimeric receptor comprising an antigen binding domain, a transmembrane domain, a primary signaling domain (e.g., a CD3-zeta domain), and one or more costimulatory signaling domains. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a spacer region. In some embodiments, the antigen binding domain is connected to the transmembrane domain by a spacer region. In some embodiments, the spacer region comprises a nucleic acid sequence selected from any of the nucleic acid sequences listed in Table 3. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0195] The polynucleotides of the present disclosure can be obtained using any suitable recombinant method known in the art, including, but not limited to, screening libraries from cells expressing the gene of interest, inducing the gene of interest from a vector known to contain the gene, or isolating the gene of interest directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0196] In some embodiments, the polynucleotides of the present disclosure are contained within a vector. In some embodiments, the polynucleotides of the present disclosure are expressed in cells via transposons, CRISPR / Cas9 systems, TALENs, or zinc finger nucleases.

[0197] In some embodiments, expression of a polynucleotide encoding a chimeric receptor of the present disclosure can be achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. Suitable vectors are capable of replicating and integrating in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling expression of the desired nucleic acid.

[0198] In some embodiments, the expression constructs of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols (e.g., US5399346, US5580859, and US5589466). In some embodiments, the vectors of the present disclosure are gene therapy vectors.

[0199] The polynucleotide of the present disclosure can be cloned into several types of vectors.For example, the polynucleotide can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, or cosmids.In some embodiments, the vector can be an expression vector, a replication vector, a probe generation vector, or a sequencing vector.

[0200] In some embodiments, the plasmid vector comprises a transposon / transposase system for integrating the polynucleotide of the present disclosure into a host cell genome. Methods for expressing proteins in immune cells using transposon and transposase plasmid systems are generally described in Chicaybam L, Hum Gene Ther. 2019 Apr; 30 (4): 511-522. doi: 10.1089 / hum.2018.218; and Ptackova P, Cytotherapy. 2018 Apr; 20 (4): 507-520. doi: 10.1016 / j.jcyt.2017.10.001, each of which is incorporated herein by reference in its entirety. In some embodiments, the transposon system is Sleeping Beauty transposon / transposase or piggyBac transposon / transposase.

[0201] In some embodiments, the expression vector of the present disclosure can be provided to cells in the form of a viral vector. Suitable viral vector systems are well known in the art. For example, viral vectors can be derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, the vector of the present disclosure is a lentiviral vector. Lentiviral vectors are suitable for long-term gene transfer, because such vectors allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors are also advantageous over vectors derived from oncoretroviruses (e.g., murine leukemia viruses) in that lentiviral vectors can transduce non-proliferating cells. In some embodiments, the vector of the present disclosure is an adenoviral vector (A5 / 35). In some embodiments, the vector of the present disclosure comprises a functional origin of replication in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and US6326193). Numerous virus-based systems have been developed for gene transfer into mammalian cells. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to mammalian cells either in vivo or ex vivo. Several retroviral systems are known in the art.

[0202] In some embodiments, vectors of the present disclosure contain additional promoter elements, such as enhancers, that control the frequency of transcription initiation. Enhancers are typically located in the region 30 bp to 110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements can be flexible, such that promoter function is maintained when elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decrease. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Exemplary promoters can include, but are not limited to, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EF1a promoter, the ubiquitin C promoter, and the phosphoglycerokinase (PGK) promoter.

[0203] In some embodiments, the promoter that can express the polynucleotide of the present disclosure in mammalian cells, such as the immunoresponsive cells of the present disclosure, is EF1a promoter.Natural EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes.EF1a promoter is widely used in mammalian expression plasmids, and has been shown to be effective in promoting chimeric receptor expression from polynucleotides cloned into lentiviral vectors.

[0204] In some embodiments, the promoter capable of expressing the polynucleotide of the present disclosure in mammalian cells, such as the immunoresponsive cells of the present disclosure, is a constitutive promoter. For example, a suitable constitutive promoter is the spleen focus forming virus (SFFV) promoter. Another example of a suitable constitutive promoter is the immediate early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter that can drive high-level expression of any polynucleotide sequence operably linked to the promoter. Other suitable constitutive promoters include, but are not limited to, the ubiquitin C (UbiC) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the elongation factor 1a promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0205] In some embodiments, the promoter that can express the polynucleotide of the present disclosure in mammalian cells, such as the immunoresponsive cells of the present disclosure, is an inducible promoter.The use of an inducible promoter can provide a molecular switch that can induce or suppress the expression of the polynucleotide of the present disclosure when the promoter is operably linked with the polynucleotide.Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0206] In some embodiments, vectors of the present disclosure can further include a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator, elements that allow for episomal replication, and / or elements that allow for selection.

[0207] In some embodiments, the vectors of the present disclosure may further comprise a selectable marker gene and / or a reporter gene to facilitate identification and selection of chimeric receptor expressing cells from a population of cells transduced with the vector. In some embodiments, the selectable marker may be encoded by a polynucleotide that is separated from the vector and used in the co-transfection procedure. Either the selectable marker or the reporter gene may be flanked by appropriate control sequences to allow expression in the host cell. Examples of selectable markers include, but are not limited to, antibiotic resistance genes, such as neo.

[0208] In some embodiments, reporter genes can be used to identify transduced cells and to evaluate the functionality of control sequences. As disclosed herein, reporter genes are genes that encode a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression results in an easily detectable property, such as an enzymatic activity. Expression of the reporter gene can be assayed at a suitable time after the polynucleotide is introduced into the recipient cell. Examples of reporter genes include, but are not limited to, genes encoding luciferase, genes encoding beta-galactosidase, genes encoding chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, and genes encoding green fluorescent protein. Suitable expression systems are well known in the art and can be prepared using known techniques or obtained commercially. In some embodiments, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0209] In some embodiments, a vector comprising a polynucleotide sequence encoding a chimeric receptor of the present disclosure further comprises a second polynucleotide encoding a polypeptide that increases the activity of the chimeric receptor.

[0210] In embodiments in which the chimeric receptor-expressing cell comprises two or more chimeric receptors, a single polynucleotide may encode two or more chimeric receptors under a single regulatory control element (e.g., promoter) or under separate regulatory control elements for the nucleotide sequence encoding each chimeric receptor contained in the polynucleotide. In some embodiments in which the chimeric receptor-expressing cell comprises two or more chimeric receptors, each chimeric receptor may be encoded by a separate polynucleotide. In some embodiments, each separate polynucleotide comprises its own regulatory element (e.g., promoter). In some embodiments, a single polynucleotide encodes two or more chimeric receptors, and the nucleotide sequences encoding the chimeric receptors 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 autocleavage sites or substrates for intracellular proteases. Suitable peptide cleavage sites may include, but are not limited to, T2A peptide cleavage sites, P2A peptide cleavage sites, E2A peptide cleavage sites, and F2A peptide cleavage sites. In some embodiments, the two or more chimeric receptors comprise a T2A peptide cleavage site. In some embodiments, two or more chimeric receptors comprise an E2A peptide cleavage site.In some embodiments, two or more chimeric receptors comprise a T2A and an E2A peptide cleavage site.

[0211] Methods for introducing and expressing genes into cells are well known in the art. For example, in some embodiments, expression vectors can be transferred into host cells by physical, chemical, or biological means. Examples of physical means for introducing polynucleotides into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Examples of chemical means for introducing polynucleotides into host cells include, but are not limited to, colloidal dispersion systems, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of biological means for introducing polynucleotides into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0212] In some embodiments, liposomes may be used as a non-viral delivery system for introducing the disclosed polynucleotides or vectors into host cells in vitro, ex vivo, or in vivo. In some embodiments, the polynucleotide may be associated with a lipid, for example, by being encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, by being attached to the liposome via a linking molecule that is associated with both the liposome and the polynucleotide, by being entrapped in the liposome, by being complexed with the liposome, by being dispersed in a solution containing lipids, by being mixed with lipids, by being combined with lipids, by being contained as a suspension in lipids, by being contained or complexed in micelles, or by being otherwise associated with lipids. As disclosed herein, lipid-associated polynucleotide or vector compositions are not limited to any particular structure in solution. In some embodiments, such compositions may exist as micelles or in bilayer structures with a "collapsed" structure. Such compositions may also be interspersed in the solution to form aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that can be naturally occurring or synthetic. In some embodiments, lipids can include lipid droplets that naturally occur in the cytoplasm, or a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Suitable lipids may be obtained from commercial sources, and include, but are not limited to, dimyristyl phosphatidylcholine ("DMPC"), dicetyl phosphate ("DCP"), cholesterol, and dimyristyl phosphatidylglycerol ("DMPG"). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as a solvent because it evaporates more easily than methanol. As used herein, "liposomes" can encompass a variety of single and multi-layer lipid vesicles formed by the production of enclosed lipid bilayers or aggregates.In some embodiments, liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. In some embodiments, multilamellar liposomes can 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, the lipid components may undergo self-rearrangement before the formation of a closed structure, and can trap water and dissolved solutes between the lipid bilayers. In some embodiments, the lipids can assume a micellar structure or exist only as heterogeneous aggregates of lipid molecules.

[0213] In some embodiments, the polynucleotide 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 the polynucleotide or vector of the present disclosure in the host cell can be confirmed by any suitable assay known in the art, including, but not limited to, Southern blot assay, Northern blot assay, RT-PCR, PCR, ELISA assay, and Western blot assay.

[0214] In some embodiments, the polynucleotide or vector of the present disclosure is stably transduced into the immunoresponsive cell of the present disclosure. In some embodiments, the cell exhibiting stable expression of the polynucleotide or vector expresses 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.

[0215] In embodiments in which the chimeric receptor of the present disclosure is expressed transiently in a cell, a polynucleotide or vector encoding the chimeric receptor 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.

[0216] In some embodiments, the polynucleotide construct encodes a bicistronic chimeric antigen receptor. In some embodiments, the encoded bicistronic chimeric antigen receptor comprises a VSIG2 CAR (e.g., a VSIG2 inhibitory CAR) and a CAR specific for a second antigen (e.g., a tumor-targeting chimeric receptor).

[0217] In some embodiments, the polynucleotide construct encodes a bivalent chimeric antigen receptor. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a VSIG2 antigen binding domain and a second antigen binding domain.

[0218] Pharmaceutical Compositions and Administration Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising one or more chimeric receptors of the present disclosure or immunoresponsive cells of the present disclosure expressing such one or more chimeric receptors. In some embodiments, compositions comprising chimeric receptors or genetically modified immunoresponsive cells expressing such chimeric receptors can be provided systemically or directly to a subject for the treatment of a proliferative disorder, such as a bone marrow disorder. In certain embodiments, the composition is directly injected into an organ of interest (e.g., an organ affected by the disorder). Alternatively, the composition can be provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). Proliferative and differentiation agents can be provided before, during, or after administration of the composition to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

[0219] Compositions containing the genetically modified cells of the present disclosure can be administered in any physiologically acceptable vehicle, for example, intravascularly, but they can also be introduced into bone or other convenient sites where the genetically modified cells can find a suitable site for regeneration and differentiation (e.g., the thymus). In some embodiments, at least 1×10 5 cells may be administered, ultimately resulting in a total of 1 x 10 10 100% or more of cells. The composition comprising the genetically modified cells of the present disclosure can comprise a purified cell population. Methods for determining the percentage of genetically modified cells in a cell population are well known in the art and include, but are not limited to, fluorescence activated cell sorting (FACS). In some embodiments, the purity of genetically modified cells in a population of cells can 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. The dosage can be easily adjusted by one skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc. In some embodiments, factors can also include, 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.

[0220] In certain embodiments, the composition is a pharmaceutical composition comprising genetically modified cells, such as immunoresponsive cells or their precursor cells, and a pharma- ceutically acceptable carrier. Administration can be autologous or xenogeneic. For example, immunoresponsive cells or precursors can be obtained from one subject and administered to the same subject or to a different compatible subject. In some embodiments, the immunoresponsive cells or their progeny of the present disclosure can be derived from peripheral blood cells (e.g., derived in vivo, ex vivo, or in vitro) and can be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the therapeutic composition of the present disclosure (e.g., pharmaceutical composition comprising the genetically modified cells of the present disclosure), it is generally formulated in a unit dose injectable form (solution, suspension, emulsion).

[0221] formulation Certain aspects of the present disclosure relate to formulations of compositions comprising the chimeric receptors of the present disclosure, or genetically modified cells expressing such chimeric receptors (e.g., immunoresponsive cells of the present disclosure). In some embodiments, the compositions of the present disclosure comprising genetically modified cells may be provided as sterile liquid preparations, including, but not limited to, isotonic aqueous solutions, suspensions, emulsions, dispersions, and viscous compositions that 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 conveniently administered, particularly by injection. In some embodiments, viscous compositions may be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0222] In some embodiments, a sterile injectable solution can be prepared by incorporating the genetically modified cells of the present disclosure in a sufficient amount of a suitable solvent with various amounts of any other components as desired. Such compositions can be mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, dextrose, etc. In some embodiments, the compositions can also be lyophilized. The compositions can include auxiliary substances such as wetting agents, dispersants, pH buffers, and antibacterial agents, depending on the route of administration and the desired preparation.

[0223] In some embodiments, the composition of the present disclosure may further comprise various additives that can enhance the stability and sterility of the composition. Examples of such additives include, but are not limited to, antibacterial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination can be prevented by including any of various antibacterial and antifungal agents, including, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable preparation of the present disclosure can be achieved by using a suitable agent that delays absorption, such as aluminum monostearate and gelatin.

[0224] In some embodiments, the compositions of the present disclosure can be isotonic, i.e., have the same osmotic pressure as blood and tears. In some embodiments, the desired isotonicity can be achieved using, for example, sodium chloride, dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.

[0225] In some embodiments, the components of the formulations of the present disclosure are selected to be chemically inert and not affect the viability or efficacy of the genetically modified cells of the present disclosure.

[0226] One consideration regarding therapeutic use of the genetically modified cells of the present disclosure is the amount of cells necessary to achieve optimal efficacy. In some embodiments, the amount of cells administered varies depending on the subject being treated. In certain embodiments, the amount of genetically modified cells administered to a subject in need thereof is greater than 1×10 4 cells ~ 1×10 10 The amount of cells that is considered an effective dose may range from 100 to 1000 cells. In some embodiments, the exact amount of cells that is considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage amount can be easily ascertained by those skilled in the art based on this disclosure and technical knowledge.

[0227] Heterologous Moieties and Modifications In a further set of embodiments, the VSIG2-specific proteins herein (e.g., VSIG2-specific chimeric proteins comprising an antigen-binding domain having one or more of the amino acid sequences listed in Table A) comprise additional moieties and / or modifications.

[0228] Drug Conjugates In various embodiments, a VSIG2-specific chimeric protein comprises an antigen-binding domain having one or more of the amino acid sequences listed in Table A that is conjugated to a therapeutic agent (i.e., a drug) to form an antibody-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In certain embodiments, the therapeutic agent is attached to the antigen-binding domain via a linker peptide, as discussed in more detail herein.

[0229] Methods for preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate a drug to an antigen-binding domain disclosed herein (e.g., having one or more of the amino acid sequences listed in Table A) are described, for example, in U.S. Pat. No. 8,624,003 (the Pott method), U.S. Pat. No. 8,163,888 (one-step method), U.S. Pat. No. 5,208,020 (two-step method), U.S. Pat. No. 8,337,626 (a method for preparing antibody-drug conjugates), and U.S. Pat. ,856, U.S. Patent No. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, U.S. Patent 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 / 157286, WO 2017 / 165851, WO 2009 / 073445 No. 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 incorporated herein by reference in its entirety for all of its teachings.

[0230] Further binding moieties In various embodiments, the VSIG2-specific chimeric protein comprises an antigen-binding domain having one or more of the amino acid sequences listed in Table A and one or more additional binding moieties. In certain embodiments, the binding moiety is an antibody fragment or antibody format, including, but not limited to, a full-length antibody, a Fab fragment, an Fv, an scFv, a tandem scFv, a diabody, a sc diabody, a DART, a tandAb, a minibody, a camelid VHH, and other antibody fragments or formats known to those of skill 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), the entire teachings of which are incorporated herein by reference.

[0231] In certain embodiments, the one or more additional binding moieties are attached to the C-terminus of one or more peptides of a VSIG2-specific antigen-binding domain, such as a VH and / or VL, a Fab heavy and / or light chain fragment, or an scFv. In certain embodiments, the one or more additional binding moieties are attached to the N-terminus of one or more peptides of a VSIG2-specific antigen-binding domain, such as a VH and / or VL, a Fab heavy and / or light chain fragment, or an scFv.

[0232] 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.

[0233] In certain embodiments, one or more additional binding moieties are attached to an antigen binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) using in vitro methods including, but not limited to, reactive chemistry (e.g., click chemistry) and affinity tagging systems. In certain embodiments, one or more additional binding moieties are attached to an antigen binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) via Fc-mediated attachment (e.g., protein A / G). In certain embodiments, one or more additional binding moieties are attached to an antigen binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) using recombinant DNA techniques, such as encoding the nucleotide sequence of a fusion product between an antigen binding domain described herein and an additional binding moiety in the same expression vector (e.g., plasmid).

[0234] Functional / Reactive Groups In various embodiments, the antigen binding domains described herein (e.g., having one or more of the amino acid sequences listed in Table A) have functional or chemically reactive groups that can be used in downstream processes, such as attaching additional moieties (e.g., drug conjugates and additional binding moieties), as well as downstream purification processes.

[0235] In certain embodiments, the modification is a chemically reactive group, including, but not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkyne groups), and aldehydes resulting in formylglycine (FGly). In certain embodiments, the modification is a functional group, including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and streptosystems, etc.). In certain embodiments, the functional group or chemically reactive group has a cleavable peptide sequence. In certain embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In certain embodiments, the protease cleavage is performed by an intracellular protease. In certain embodiments, the protease cleavage is performed by an extracellular or membrane-bound protease. ADC therapy using protease cleavage is described in detail by Choi et al. (Theranostics, 2012; 2(2): 156-178.), the entire teachings of which are incorporated herein by reference.

[0236] Treatment method Certain aspects of the present disclosure relate to methods of using the chimeric receptors of the present disclosure and genetically modified cells (e.g., immunoresponsive cells) expressing such chimeric receptors to treat a subject in need of such a chimeric receptor. In some embodiments, the methods of the present disclosure are useful for treating cancer in a subject, such as a solid tumor. In some embodiments, the solid tumor is selected from colorectal cancer, pancreatic cancer, lung cancer, and / or gastric cancer. In some embodiments, the solid tumor is colorectal cancer. In some embodiments, the colorectal cancer is colorectal carcinoma. In some embodiments, the solid tumor is lung cancer. In some embodiments, the lung cancer is lung adenocarcinoma. Other aspects of the present disclosure relate to the use of the chimeric receptors of the present disclosure and genetically modified cells (e.g., immunoresponsive cells) expressing such chimeric receptors in methods for treating pathogen infections or other infectious diseases in a subject, such as an immunocompromised human subject. In some embodiments, the methods of the present disclosure may include administering the genetically modified cells of the present disclosure in an amount effective to achieve a desired effect, including, but not limited to, alleviation of an existing condition, prevention of a condition, treatment of an existing condition, management of an existing condition, or prevention of recurrence or recurrence of a condition. In some embodiments, the effective amount may be provided in a single or series of administrations of the genetically modified cells (e.g., immunoresponsive cells) of the present disclosure. In some embodiments, the effective amount may be provided by bolus or continuous perfusion.

[0237] As disclosed herein, an "effective amount" or a "therapeutically effective amount" is an amount sufficient to affect beneficial or desired clinical results during 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 sufficient to palliate, improve, stabilize, reverse, or delay the progression of a disease, or reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person of ordinary skill in the art. In determining the appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the 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 being administered.

[0238] For adoptive immunotherapy using antigen-specific cells (e.g., immune-responsive cells such as T cells or NK cells), approximately 1 × 10 5 ~1×10 10 cells / kg (e.g., approximately 1 x 10 9 Cell doses ranging from 100 to 1000 cells are usually injected. Upon administration of the cells to a subject and subsequent differentiation, immunoresponsive cells are induced to be specifically directed against a particular antigen. In some embodiments, induction of immunoresponsive cells can include, but is not limited to, inactivation of antigen-specific cells, such as by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance, such as in autoimmune disorders. Genetically modified cells can be administered by any method known in the art, including, but not limited to, intravenously, subcutaneously, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

[0239] Treatment 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 bone marrow disorders in a subject. In some embodiments, the subject is a human. In some embodiments, human subjects suitable for treatment may include two treatment groups that may be distinguished by clinical criteria. Subjects with "progressive disease" or "high tumor burden" are subjects with clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., based on percentage of leukemic cells by palpation, CAT scan, ultrasound, mammogram, or X-ray; positive biochemical or histopathological markers are insufficient to identify this population by themselves). In some embodiments, the pharmaceutical compositions of the present disclosure are administered to these subjects to induce an anti-tumor response with the aim of alleviating their condition. In some embodiments, a reduction in tumor burden occurs as a result of administration of the pharmaceutical composition, but any clinical improvement would constitute a benefit. In some embodiments, clinical improvement includes a reduced risk or reduced rate of progression in pathological outcomes of the tumor. In some embodiments, a second group of suitable human subjects are "adjuvant group" subjects. These subjects are individuals who have a history of bone marrow disorders but have responded to another treatment. Previous therapy may include, but is not limited to, surgical resection, radiation therapy, and / or conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk for disease progression near the original tumor site or by metastasis. In some embodiments, this group can be further subdivided into high-risk and low-risk individuals. Subdivision can be based on characteristics observed before or after initial treatment. These characteristics are known in the clinical arts and are suitably defined for different bone marrow diseases. Typical characteristics of the high-risk subgroup are those in which the tumor has invaded adjacent tissues or shows lymph node involvement.

[0240] In any and all aspects of the increased immune response described herein, any increase or decrease or change in an aspect of a characteristic or function is compared to a cell that has not been contacted with an immunoresponsive cell as described herein.

[0241] Increasing an immune response can be both an enhancement of an immune response or an induction of an immune response. For example, increasing an immune response encompasses both initiating or initiating an immune response or increasing or amplifying an ongoing 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 enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response.

[0242] In some embodiments, a further group of subjects are those who have a genetic predisposition to malignant disease but have not yet demonstrated clinical signs of malignant disease. For example, women who have detected positive for gene mutations associated with malignant disease but are still of childbearing age may benefit from receiving one or more cells (e.g., immunoresponsive cells) of the present disclosure in a prophylactic treatment to prevent the occurrence of malignant disease until suitable for chemotherapy, radiation-based therapy, and / or prophylactic surgery. In some embodiments, subjects may have a progressive form of disease, in which case treatment goals may include alleviating or reversing disease progression and / or improving side effects. In some embodiments, subjects may have a history of a condition in which they have already been treated, in which case treatment goals may typically include reducing or delaying the risk of recurrence.

[0243] Combination therapy In some embodiments, the genetically modified cells (e.g., immunoresponsive cells) of the present disclosure expressing one or more chimeric receptors of the present disclosure may be used in combination with other known drugs and therapies. In some embodiments, the combination therapy of the present disclosure includes the genetically modified cells of the present disclosure, which may be administered in combination with one or more additional therapeutic agents. In some embodiments, the genetically modified cells and the one or more additional therapeutic agents may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the genetic modifier may be administered first and the one or more additional agents may be administered second, or the order of administration may be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[0244] In some embodiments, the genetically modified cells of the present disclosure may be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunosuppressants (e.g., CAMPATH or anti-CD3 antibodies), cytotoxin, fludarabme, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, and peptide vaccines.

[0245] In some embodiments, the genetically modified cells of the present disclosure can be used in combination with lymphodepleting agents.Suitable lymphodepleting agents reduce or reduce lymphocytes, for example, B-cell lymphocytes and / or T-cell lymphocytes, before immunotherapy.Examples of suitable lymphodepleting agents include, but are not limited to, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[0246] In some embodiments, the genetically modified cells of the present disclosure may be used in combination with a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, anthracyclines (e.g., doxorubicin), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclarubicin A, gliotoxin, bortezomib), immunoderivatives such as thalidomide or thalidomide derivatives (e.g., lenalidomide), and the like.

[0247] Examples of common chemotherapeutic agents suitable for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Bleomycin®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chloramphenicol (Citrosil), and rivaroxaban (Ricin®). Bucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosal®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyte®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubicin®), daunorubicin citrate liposome Infusion (Daunozom®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Bepcid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adolsil®, Efudex®), flutamide (Eurexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idahivir®), Syn (registered trademark), ifosfamide (IFEX (registered trademark), irinotecan (Camptosar (registered trademark)), L-asparaginase (ELSPAR (registered trademark)), leucovorin calcium, melphalan (Alkeran (registered trademark)), 6-mercaptopurine (Purintor (registered trademark)), methotrexate (Forex (registered trademark)), mitoxantrone (Novantron (registered trademark)), Mylotarg, paclitaxel (Taxol (registered trademark), Phoenix (Yttrium 90 / MX-DTPA), pentostatin,These include, but are not limited to, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Bumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velbon®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0248] Examples of suitable alkylating agents include 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 illustrative examples of alkylating agents include oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin D, Cosmegen®), melphalan (L-PAM, Also known as L-sarcolysin, and phenylalanine masterbate, Alkeran®), altretamine (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®), amphetamine (also known as hexamethylmelamine (HMM), Hexalen®), rifabutin (Rifabutin ... These include, but are not limited to, aitretamine (also known as hexamethylmelamine (HMM), Hexalen®), ifosfamide (Ifex®), Prednumustine, procarbazine (Matulane®), mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, Mustargen®), streptozocin (Zanosar®), thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine HC1 (Treanda®).

[0249] Examples of suitable mTOR inhibitors include, but are not limited to, temsirolimus, ridaforolimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RAD001), rapamycin (AY22989, Sirolimus®), and XL765.

[0250] Examples of suitable immunomodulatory agents include, but are not limited to, afutuzumab, pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047), and IRX-2.

[0251] Examples of suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin® and Rubex®), bleomycin (lenoxane®), daunorubicin (dauorubicin hydrochloride, daunomyem, and rubidomycin hydrochloride, Cerubidine®), daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®), mitoxantrone (DHAD, Novantrone®), epirubicin (Ellence™), idarubicin (idamycin®, idamycin PES®), mitomycin C (Mutamycin®), geldanamycin, herbimycin, rabidomycin, and desacetramycin.

[0252] Examples of suitable vinca alkaloids include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Erdisine®), vinblastine (also known as vinblastine sulfate, vincalucoblastine, and VLB, Alkaban-AQ® and Velban®), and vinorelbine (Navelbme®).

[0253] Examples of suitable proteosome inhibitors include, but are not limited to, bortezomib (Velcade®), carfilzomib, marizomib (NPI-0052), ixazomib citrate (MLN-9708), delanzomib (CEP-18770), and ONX-0912.

[0254] In some embodiments, the genetically modified cells of the present disclosure are administered in combination with a CD20 inhibitor, such as an anti-CD20 antibody, or a fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Prol31921.

[0255] In some embodiments, the genetically modified cells of the present disclosure are administered in combination with an oncolytic virus. In some embodiments, the oncolytic virus can selectively replicate in cancer cells and induce the death or slow the growth of cancer cells. In some cases, the oncolytic virus has no effect or minimal effect on non-cancer cells. Suitable oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, 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.

[0256] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, for example, an SHP-1 inhibitor or an SHP-2 inhibitor.In one embodiment, the genetically modified cells of the present disclosure can be used in combination with a kinase inhibitor.Examples of suitable kinase inhibitors include, but are not limited to, CDK4 inhibitors, CDK4 / 6 inhibitors, BTK inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, mTOR inhibitors, MNK inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors.

[0257] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with a regulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the effectiveness of chimeric receptor-expressing cell therapy. Without being bound by theory, it is believed that administration of MDSC regulators enhances the effectiveness of the genetically modified cells of the present disclosure. Examples of suitable regulators of MDSCs include, but are not limited to, MCS110 and BLZ945.

[0258] In some embodiments, the genetically modified cells of the present disclosure are 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 inhibit T cell-dependent immunogenic chemotherapy, such as oxaliplatin (Shalapour et al., Nature 2015, 521:94-101). In one embodiment, the immunosuppressive plasma cells can express one or more of IgA, interleukin (IL)-10, and PD-L1.

[0259] In some embodiments, the genetically modified cells of the disclosure are administered to a subject in combination with an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide. In some embodiments, the genetically modified cells of the disclosure are further modified to express an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide.

[0260] In some embodiments, a subject with a malignant tumor (e.g., a solid tumor) is administered the genetically modified cells of the present disclosure in combination with an agent, e.g., a cytotoxic or chemotherapeutic agent, a biological therapy (e.g., an antibody, e.g., a monoclonal antibody, or a cell therapy), or an inhibitor (e.g., a kinase inhibitor). In some embodiments, the subject is administered the genetically modified cells 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 containing cytarabine and daunorubicin in a 5:1 molar ratio. In some embodiments, the subject is administered the chimeric receptor-expressing cells described herein in combination with a hypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g., azacitidine or decitabine. In some embodiments, the subject is administered the genetically modified cells of the disclosure in combination with a biological therapy, e.g., an antibody or cell 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 the genetically modified cells of the disclosure in combination with a FLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo), crenolanib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), ASP2215 (Astelias).In some embodiments, the subject is administered the genetically modified cells 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 the genetically modified cells of the present disclosure in combination with an inhibitor of a cell cycle regulator, e.g., an 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 the genetically modified cells of the disclosure in combination with a B cell receptor signaling network inhibitor, e.g., an inhibitor of B cell lymphoma 2 (Bcl-2), e.g., venetoclax (Abbvie / Roche), or an inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib (Pharmacyclics / Johnson & Johnson Janssen Pharmaceutical). In some embodiments, the subject is administered the genetically modified cells of the disclosure in combination with an inhibitor of M1 aminopeptidase, an inhibitor of histone deacetylase (HDAC), e.g., pracinostat (MEI Pharma), a multikinase inhibitor, e.g., rigosertib (Onconova Therapeutics / Baxter / SymBio), or a peptidic CXCR4 inverse agonist, e.g., BL-8040 (BioLineRx).

[0261] In some embodiments, a subject may be administered an agent that enhances the activity or compatibility of the genetically modified cells of the present disclosure. For example, the agent may inhibit a molecule that regulates or controls the function of immune cells (e.g., T cells or NK cells). In some embodiments, the molecule that regulates or controls immune cell function is an inhibitory molecule. In some embodiments, an inhibitory molecule, such as programmed death 1 (PD-1), can reduce the ability of the genetically modified cells to initiate an immune effector response. Examples of suitable inhibitory molecules include, but are not limited to, PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 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 molecules that modulate or control, e.g., inhibit, immune cell function, e.g., by inhibition at the DNA, RNA, or protein level, can optimize the performance of the genetically modified cells of the present disclosure. In some embodiments, agents such as inhibitory polynucleotides, such as inhibitory polynucleotides, such as inhibitory polynucleotides, such as dsRNA, such as siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN) can be used to inhibit the expression of inhibitory molecules in genetically modified cells.In one embodiment, the inhibitor is shRNA.In some embodiments, the genetically modified cells of the present disclosure can be further modified to express an inhibitory polynucleotide, e.g., an inhibitory polynucleotide, e.g., an inhibitory polynucleotide, 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), and can be used to inhibit expression of an inhibitory molecule in the genetically modified cell.

[0262] In one embodiment, the agent that modulates or controls, e.g., inhibits, immune cell function is inhibited in the genetically modified cell of the present disclosure. In such an embodiment, the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, e.g., inhibits, immune cell function is linked to the polynucleotide that encodes the components, e.g., all components, of the chimeric receptor of the present disclosure. In one embodiment, the polynucleotide molecule that encodes the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, immune cell function is operably linked to a promoter, e.g., a promoter derived from HI or U6, so that the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, immune cell function is expressed, e.g., in the genetically modified cell. In one embodiment, the polynucleotide molecule that encodes the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, e.g., inhibits, immune cell function is present on the same vector, e.g., a lentiviral vector, that includes the polynucleotide molecule that encodes the components, e.g., all components, of the chimeric receptor. In such an embodiment, the polynucleotide molecule encoding the dsRNA molecule that inhibits the expression of a molecule that modulates or controls, e.g., inhibits, immune cell function is located on a vector, e.g., a lentiviral vector, 5'- or 3'- of the polynucleotide encoding the components, e.g., all components, of the chimeric receptor. The polynucleotide molecule encoding the dsRNA molecule that inhibits the expression of a molecule that modulates or controls, e.g., inhibits, immune cell function can be transcribed in the same or different direction as the polynucleotide encoding the components, e.g., all components, of the chimeric receptor. In one embodiment, the polynucleotide molecule encoding the dsRNA molecule that inhibits the expression of a molecule that modulates or controls, e.g., inhibits, immune cell function is present on a vector other than the vector containing the polynucleotide molecule encoding the components, e.g., all components, of the chimeric receptor. In one embodiment, the polynucleotide molecule encoding the dsRNA molecule that inhibits the expression of a molecule that modulates or controls, e.g., inhibits, immune cell function is transiently expressed in the genetically modified cell.In one embodiment, a polynucleotide molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates, e.g., inhibits, immune cell function is stably integrated into the genome of a genetically modified cell of the present disclosure.

[0263] In one embodiment, the agent that regulates or controls, for example inhibits, immune 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.

[0264] In some embodiments, the agent that enhances the activity of the genetically modified cells is a CEACAM inhibitor (e.g., a CEACAM-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 that enhances the activity of the genetically modified cells of the present disclosure is miR-17-92. In some embodiments, the agent that enhances the activity of the genetically modified cells is CD40L. In some embodiments, the agent that enhances the activity of the genetically modified cells is GM-CSF. In some embodiments, the genetically modified cells of the present disclosure are further modified to express an antibody or antibody fragment that binds to an inhibitory molecule of the present disclosure.

[0265] In one embodiment, the agent that enhances the activity of the genetically modified cells of the present disclosure is a cytokine. Cytokines have important functions related to immune responsive cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to a subject receiving the genetically modified cells of the present disclosure include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21, or combinations thereof. Cytokines can be administered once a day or more than once a day, for example, twice a day, three times a day, or four times a day. Cytokines can be administered for more than one day, for example, cytokines are administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, cytokines are administered once a day for 7 days. In some embodiments, the genetically modified cells of the present disclosure are further modified to express one or more cytokines, such as IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21.

[0266] In some embodiments, the cytokine can be administered simultaneously or contemporaneously with the genetically modified cells, for example, on the same day. The cytokine can be prepared in the same pharmaceutical composition as the genetically modified cells, or in a separate pharmaceutical composition. The cytokine can be administered immediately after administration of the genetically modified cells, for example, 1, 2, 3, 4, 5, 6, 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 one day, the first day of the cytokine dosing regimen can be the same day as administration with the genetically modified cells, or the first day of the cytokine dosing regimen can be 1, 2, 3, 4, 5, 6, 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, the cytokine is administered once a day for the next 7 days. In some embodiments, the cytokine is administered for a period of time following administration of the genetically modified cells, for example, 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 a year or more following administration of the genetically modified cells. In one embodiment, the cytokine is administered following evaluation of the subject's response to the genetically modified cells.

[0267] kit Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of cancer (e.g., solid tumors). In certain embodiments, the kits include a therapeutic or prophylactic composition comprising an effective amount of one or more chimeric receptors of the present disclosure, isolated nucleic acids of the present disclosure, vectors of the present disclosure, and / or cells (e.g., immunoresponsive cells) of the present disclosure. In some embodiments, the kits include a sterile container. In some embodiments, such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a drug.

[0268] In some embodiments, the therapeutic or prophylactic composition is provided with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., a solid tumor). In some embodiments, the instructions may include information regarding the use of the composition for the treatment and / or prevention of a disorder. In some embodiments, the instructions include, but are not limited to, a description of the therapeutic or prophylactic composition, a dosing schedule, a dosing schedule for the treatment or prevention of a disease or its symptoms, precautions, warnings, indications, contraindications, overdosing information, adverse reactions, animal pharmacology, clinical trials, and / or references. In some embodiments, the instructions may be printed directly on the container (if present), or may be printed as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0269] Enumerated Embodiments

[0270] 1. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; A chimeric protein, wherein the VH comprises a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (sequence number 4).

[0271] 2. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3); A chimeric protein, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO:1.

[0272] 3. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A chimeric protein comprising a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (sequence number 4).

[0273] 4. A chimeric protein described in any one of embodiments 1 to 3, wherein the VL comprises light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0274] 5. VL is A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENIYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and 5. The chimeric protein according to any one of embodiments 1 to 4, comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14).

[0275] 6. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VL comprises light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3); A chimeric protein, wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0276] 7. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VL, A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A chimeric protein comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0277] 8. The chimeric protein of embodiment 6 or embodiment 7, wherein the VH comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained in the VH region amino acid sequence of SEQ ID NO:1.

[0278] 9. VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and 9. The chimeric protein according to any one of embodiments 6 to 8, comprising a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4).

[0279] 10. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4), VL, A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A chimeric protein comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0280] 11. A chimeric protein described in any one of embodiments 1 to 10, wherein the VH region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0281] 12. A chimeric protein described in any one of embodiments 1 to 11, wherein the VH region comprises the amino acid sequence of SEQ ID NO:1.

[0282] 13. The chimeric protein of any one of embodiments 1 to 12, wherein the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9.

[0283] 14. A chimeric protein described in any one of embodiments 1 to 12, wherein the VL region comprises the amino acid sequence of SEQ ID NO:9.

[0284] 15. A chimeric protein described in any one of embodiments 1 to 12, wherein the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:10.

[0285] 16. A chimeric protein described in any one of embodiments 1 to 12, wherein the VL region comprises the amino acid sequence of SEQ ID NO: 10.

[0286] 17. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, The antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; The VH is a chimeric protein comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0287] 18. The chimeric protein of embodiment 17, wherein the VH region comprises the amino acid sequence of SEQ ID NO:1.

[0288] 19. The chimera of embodiment 17 or embodiment 18, wherein the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0289] 20. The chimera of embodiment 17 or embodiment 18, wherein the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9.

[0290] 21. The chimeric protein of embodiment 17 or embodiment 18, wherein the VL region comprises the amino acid sequence of SEQ ID NO:9.

[0291] 22. The chimera of embodiment 17 or embodiment 18, wherein the VL region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10.

[0292] 23. The chimeric protein of embodiment 17 or embodiment 18, wherein the VL region comprises the amino acid sequence of SEQ ID NO: 10.

[0293] 24. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises an antibody or an antigen-binding fragment; the antibody or antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region; A chimeric protein, wherein the VL comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0294] 25. The chimeric protein of embodiment 24, wherein the VL region comprises the amino acid sequence of SEQ ID NO:9.

[0295] 26. The chimeric protein of embodiment 24, wherein the VL region comprises the amino acid sequence of SEQ ID NO: 10.

[0296] 27. A chimeric protein described in any one of embodiments 24 to 26, wherein the VH region 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%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0297] 28. A chimeric protein described in any one of embodiments 24 to 26, wherein the VH region comprises the amino acid sequence of SEQ ID NO:1.

[0298] 29. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain competes with a reference antibody or antigen-binding fragment thereof for binding to VSIG2; the reference antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4), VL, A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A chimeric protein comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0299] 30. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, The antigen-binding domain is essentially binds to the same VSIG2 epitope as a reference antibody or antigen-binding fragment thereof, wherein the reference antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4), VL, A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or the amino acid sequence of RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A chimeric protein comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0300] 31. A chimeric protein comprising an antigen-binding domain specific for V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain binds to the same epitope of human VSIG2 as the VSIG2 epitope bound by a reference antibody or antigen-binding fragment thereof; the reference antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region; VH, A heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO: 2), A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO: 3), and A heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of QGVRPFFDY (SEQ ID NO: 4), VL, A light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12), A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and A chimeric protein comprising a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (sequence number 14).

[0301] 32. A chimeric protein described in any one of embodiments 29 to 31, wherein the VH region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:1.

[0302] 33. A chimeric protein described in any one of embodiments 29 to 32, wherein the VL region of the reference antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0303] 34. The chimeric protein of any one of embodiments 1 to 33, wherein the antigen-binding domain comprises an F(ab) fragment, an F(ab') fragment, or a single-chain variable fragment (scFv).

[0304] 35. The chimeric protein of embodiment 34, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0305] 36. A chimeric protein described in any one of embodiments 1 to 35, wherein the VH and VL are separated by a peptide linker.

[0306] 37. The chimeric protein of embodiment 36, wherein the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

[0307] 38. The chimeric protein of embodiment 36 or embodiment 37, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 35.

[0308] 39. The chimeric protein of embodiment 35 or embodiment 36, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 74.

[0309] 40. A chimeric protein described in any one of embodiments 1 to 39, wherein the chimeric protein is an antibody-drug conjugate and the heterologous molecule or moiety comprises a therapeutic agent.

[0310] 41. The chimeric protein of any one of embodiments 1 to 39, which is a chimeric antigen receptor (CAR), and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0311] 42. The chimeric protein of embodiment 41, wherein the CAR comprises a transmembrane domain.

[0312] 43. The chimeric protein of embodiment 41 or embodiment 42, wherein the CAR comprises one or more intracellular signaling domains.

[0313] 44. A chimeric protein described in any one of embodiments 41 to 43, wherein the CAR is an activated CAR comprising one or more intracellular signaling domains that stimulate an immune response.

[0314] 45. A chimeric protein described in any one of embodiments 41 to 43, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response.

[0315] 46. ​​The chimeric protein of embodiment 45, wherein the intracellular inhibitory domain comprises an enzyme inhibitory domain.

[0316] 47. The chimeric protein of embodiment 45, wherein the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain.

[0317] 48. A chimeric protein described in any one of embodiments 41 to 47, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain.

[0318] 49. The chimeric protein of embodiment 48, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 51.

[0319] 50. A composition comprising a chimeric protein described in any one of embodiments 1 to 49 and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0320] 51. An engineered polynucleotide encoding a chimeric protein described in any one of embodiments 1 to 49.

[0321] 52. An expression vector comprising the engineered polynucleotide of embodiment 51.

[0322] 53. A composition comprising an engineered polynucleotide according to embodiment 51 or an expression vector according to embodiment 52, and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0323] 54. A method for producing an engineered cell, comprising transducing an isolated cell with an engineered polynucleotide described in embodiment 51 or an expression vector described in embodiment 52.

[0324] 55. An engineered cell produced by the method of embodiment 54.

[0325] 56. An isolated cell comprising the engineered polynucleotide of embodiment 51, the expression vector of embodiment 52, or the composition of embodiment 53.

[0326] 57. A population of engineered cells expressing the engineered polynucleotide of embodiment 51 or the expression vector of embodiment 52.

[0327] 58. An isolated cell comprising a chimeric protein described in any one of embodiments 1 to 49.

[0328] 59. A population of engineered cells expressing a chimeric protein described in any one of embodiments 1 to 49.

[0329] 60. A cell or population of cells described in any one of embodiments 56 to 59, wherein the chimeric protein is recombinantly expressed.

[0330] 61. The cell or population of cells described in any one of embodiments 56 to 60, wherein the chimeric protein is expressed from a selected locus derived from the vector or the genome of the cell.

[0331] 62. A cell or population of cells described in any one of embodiments 56 to 61, wherein the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface.

[0332] 63. The cell or population of cells described in embodiment 62, wherein the one or more tumor-targeting chimeric receptors are chimeric antigen receptors (CARs) or engineered T cell receptors.

[0333] 64. The cell or population of cells according to any one of embodiments 56-63, wherein the cell or population of cells is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0334] 65. A cell or population of cells described in any one of embodiments 56 to 64, wherein the cells are autologous.

[0335] 66. A cell or population of cells described in any one of embodiments 56 to 64, wherein the cells are homogenous.

[0336] 67. A pharmaceutical composition comprising an effective amount of a cell or a population of engineered cells described in any one of embodiments 56 to 66, and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0337] 68. A pharmaceutical composition comprising an effective amount of a genetically modified cell expressing a chimeric protein described in any one of embodiments 1 to 49 and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

[0338] 69. A pharmaceutical composition described in embodiment 67 or embodiment 68, for treating and / or preventing tumors.

[0339] 70. A method of treating a subject in need thereof, comprising administering a therapeutically effective dose of the composition described in embodiment 53, or any of the cells described in any one of embodiments 55 to 66, or the composition described in any one of embodiments 67 to 69.

[0340] 71. A method for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a tumor-bearing subject a therapeutically effective dose of the composition described in embodiment 53, or any of the cells described in any one of embodiments 55 to 66, or the composition described in any one of embodiments 67 to 69.

[0341] 72. A method for treating a subject having a tumor, comprising administering a therapeutically effective dose of any of the cells described in embodiment 53, or any one of embodiments 55 to 66, or the composition described in any one of embodiments 67 to 69.

[0342] 73. A kit for treating and / or preventing tumors, comprising a chimeric protein described in any one of embodiments 1 to 49.

[0343] 74. The kit described in embodiment 73, further comprising written instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0344] 75. A kit for treating and / or preventing tumors, comprising a cell or a population of cells described in any one of embodiments 55 to 66.

[0345] 76. The kit of embodiment 75, further comprising written instructions for using the cells to treat and / or prevent a tumor in a subject.

[0346] 77. A kit for treating and / or preventing tumors, comprising an engineered polynucleotide described in embodiment 51.

[0347] 78. The kit described in embodiment 77, further comprising written instructions for using the polynucleotide to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0348] 79. A kit for treating and / or preventing a tumor, comprising the vector described in embodiment 52.

[0349] 80. The kit described in embodiment 79, further comprising written instructions for using the vector to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0350] 81. A kit for treating and / or preventing a tumor, comprising a composition described in embodiment 53, or any one of embodiments 67 to 69.

[0351] 82. The kit of embodiment 81, further comprising written instructions for using the composition to treat and / or prevent a tumor in a subject. EXAMPLES

[0352] The following are examples of methods and compositions of the present disclosure. It will be understood that various other embodiments may be practiced in light of the general description provided herein.

[0353] Below are examples of specific embodiments for carrying out the claimed subject matter of the present disclosure. The examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts are made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should of course be allowed for.

[0354] Example 1: Anti-VSIG2 Antibody Sequencing method Antibody sequencing Mouse anti-human VSIG2 monoclonal antibody clones ("Abs") were sequenced. Briefly, samples containing each of the immunoglobulin chains were digested with various enzymes and then analyzed by LC-MS / MS. Peptides were characterized from the LC-MS / MS data using de novo peptide sequencing, and then the antibody sequences were constructed.

[0355] The Ab anti-VSIG2 antibody was peptide sequenced. LC-MS / MS data of multiple enzymatic digests were mapped to the constructed antibody sequence. In the heavy and light chains, 100% of the amino acid residues were covered by at least five peptide scans with significant supporting fragment ions (data not shown).

[0356] The results of sequencing the light and heavy chain variable regions are shown in Figure 1 and Figure 2, respectively. The framework and complementarity determining regions (CDRs) are annotated according to the Chothia annotation and numbering scheme. The sequences are shown in Table A. Given that leucine (L) and isoleucine (I) have the same residue mass, additional analytical techniques were used to identify these residues. As shown in Figure 1, the isoleucine residue at position 21 of CDR-L1 was determined to be isoleucine, but was not confirmed with 100% confidence.

[0357] Table A: Anti-VSIG2 antibody (Ab) sequences TIFF2024516713000005.tif223159

[0358] Example 2: Production and evaluation of anti-VSIG2 activating CARs The CAR construct was cloned into a lentivirus vector. The lentivirus was produced using the Lenti-X 293T system. The antigen specificity and domain organization of the CAR construct examined are shown in Table B below, and the scFv amino acid sequence and nucleotide sequence are shown in Table C and Table D, respectively.

[0359] (Table B) Activation of CAR constructs (activation) TIFF2024516713000006.tif102157TIFF2024516713000007.tif186157

[0360] Table C: CAR scFv amino acid sequences TIFF2024516713000008.tif149152

[0361] Table D: CAR scFv nucleotide sequences TIFF2024516713000009.tif232152TIFF2024516713000010.tif183152TIFF2024516713000011.tif208152TIFF2024516713000012.tif70152

[0362] Activated CAR NK cell assay Primary NK cells are isolated from human donor PBMCs and frozen. NK cells are transduced with a CAR lentivirus containing a selected activating CAR (aCAR) as described in Table C.

[0363] Killing assays are performed to evaluate the killing of VSIG-2 expressing cells, including HT29 cells, a human colorectal adenocarcinoma cell line that endogenously expresses VSIG-2, Ls174t cells that exogenously express VSIG2, and DLD1 cells that exogenously express VSIG2. The killing assays demonstrate that NK cells expressing anti-VSIG2 aCAR have the ability to kill VSIG2 expressing cells.

[0364] Example 3: Generation of anti-VSIG2 inhibitory CARs The anti-VSIG2 inhibitory CAR (iCAR) constructs were each cloned into a lentiviral vector. The antigen specificity and domain organization of the CAR constructs are listed in Table E below. The amino acid sequence of each anti-VSIG2 scFv of the iCAR constructs is listed in Table C. The nucleotide sequence of each anti-VSIG2 scFv is listed in Table F below.

[0365] Table E. Inhibitory CAR constructs TIFF2024516713000013.tif44128

[0366] To test the inhibitory activity of the NOT-gated anti-VSIG2 iCAR, individual anti-VSIG2 iCAR and anti-CEA aCAR constructs are packaged into lentiviral particles and used to transduce primary NK cells. The amount of virus is set by p24 titer (750,000 pg per transduction). Because the iCAR constructs contained a puroR cassette, puromycin is added to NK cell cultures 4–7 days after transduction, at which point expression is assessed by flow cytometry, and NK cells are transferred to microwell plates for killing assays. NK cells are cultured (1) with tumor cells expressing only the aCAR antigen CEA, (2) with tumor cells expressing both the aCAR antigen CEA and the iCAR antigen VSIG2, or (3) with both tumor cell types mixed. After 16–18 h, cultures are analyzed by flow cytometry, and the remaining live target cells of each type are counted. aCAR-mediated killing (base difference) of a given NK cell type is quantified by first calculating the total killing (reduction of targets compared to the target-only condition) and then subtracting the total killing by control (iCAR-only) NK cells. iCAR-mediated protection is quantified as the change in aCAR-mediated killing between targets with and without iCAR antigen. Supernatants from killing assays are analyzed for TNFα secretion and aCAR and iCAR performance metrics are calculated as well as killing. For expression analysis, iCAR and aCAR are stained for their respective epitope tags. Results show the inhibitory activity of the NOT-gated anti-VSIG2 iCAR.

[0367] Example 4: Protection of safety antigen-positive cells based on NOT gate In this example, the use of NOT-gate in combination with a CEA-activated CAR was evaluated using cells expressing a safety antigen (VSIG2).

[0368] First, a cell line expressing a safety antigen (VSIG2) was generated. SEM cells were transduced with a lentivirus expressing the VSIG2 protein and a resistance selection marker (blasticidin). Cells were selected for antibiotic resistance and expression of the desired protein was assessed via flow cytometry. SEM cells were also transduced with a lentivirus encoding a membrane-bound form of CEACAM5-EGFP. Cells were sorted for EGFP positivity and co-expression of CEACAM5 and VSIG2 was measured by flow cytometry.

[0369] Next, NK cells expressing CEA-activated CARs and anti-HER2 inhibitory CARs were generated. Various anti-VSIG2 inhibitory CARs with various inhibitory domains (those with multiple intracellular domains are listed in order of proximity to the transmembrane domain: LIR1-LIR1, KIR3DL1, KIR3DL1-KIR3DL1, LIR1-KIR3DL1, KIR3DL1-LIR1, KIR2DL1, LAIR1, SIGLEC2, and SIRPa). For each with only a single intracellular domain, a transmembrane domain from the same protein was used. For those with two intracellular domains, a transmembrane domain from the same protein as the first intracellular domain was used. In addition, each CAR contained a CD8 hinge and a V5 epitope tag between the scFv and the hinge. A description of the constructs is provided in Table F.

[0370] Table F. Description of anti-VSIG2 inhibitory CAR domains and constructs TIFF2024516713000014.tif158128

[0371] Components of an anti-VSIG2 inhibitory CAR are provided in Table G.

[0372] Table G - Components and sequences of anti-VSIG2 inhibitory CARs TIFF2024516713000015.tif207154

[0373] Primary NK cells from donors were first expanded and then transduced with a retrovirus encoding aCAR. Expression of aCAR was determined via flow cytometry using the MYC tag. Expression for the VSIG2 safety antigen system is shown in Figure 3.

[0374] NK cells expressing CEA-aCAR and various anti-VSIG2 iCARs were co-cultured with target cells expressing either CEACAM5 alone or CEACAM5 / safety antigen (VSIG2). Appropriate controls such as aCAR alone, iCAR alone, and non-targeting iCAR were used.

[0375] Flow cytometry was used to measure the percent reduction of target cells after overnight co-culture with NOT-gated CAR NK cells, as shown in Figure 4. As seen in Figure 4, various VSIG2 inhibitory CARs containing various inhibitory domains show a significant reduction in CAR-mediated cell killing in target cells expressing safety antigens (VSIG2, blue bars) but not in target cells expressing only CEACAM5 (red bars). This result indicates that expression of VSIG2 inhibitory CARs reduces activation of CAR-mediated signaling in a safety antigen-dependent manner.

[0376] Example 5: Characterization of VSIG2-binding agents and their use as safety antigens In this example, CARs containing the VSIG2-binding agents described in the examples above were analyzed for their activity as activating CARs (aCARs) and inhibitory CARs (iCARs).

[0377] An overview of the VSIG2 CARs used in this example is provided below in Table H. The corresponding sequences are provided in Example 3 above.

[0378] Table H - Summary of VSIG2 CAR constructs evaluated TIFF2024516713000016.tif126159

[0379] The VSIG2 scFv sequence was used to construct CD28z aCARs using different linkers and orientations to confirm expression, binding, and killing of VSIG2-positive target cells by NK cells. NK cells were transduced with retroviruses expressing different VSIG2-aCARs (as shown in Table H above). Different VSIG2 binders were designed based on the mouse sequence of the monoclonal VSIG2 antibody and constructed as activating CARs with CD28 ICD and CD3z signaling domains. Using the methodology described in Example 4 above, transduced NK cells were co-cultured with SEM target cells engineered to overexpress human VSIG2 at an effector cell:target cell (E:T) ratio of 1:8. Target cell death was quantified compared to wild-type (no VSIG2 expression) target cells. The results are shown in Figure 5.

[0380] On day 6 after transduction, the expression of VSIG2 aCAR with different binders was determined (Figure 6). It was observed that the VL / VH orientation resulted in a higher mean fluorescence intensity (MFI) than the VH / VL orientation of the scFv. The highest expression was observed for SBSB04750, SB04746, and SB04744.

[0381] VSIG2-CAR NK cells were co-cultured with colorectal cancer (CRC) target cells expressing mKate and VSIG2 (Ls174t, Figure 7A and DLD1, Figure 7B). Target cell proliferation was measured in an Incucyte® via red fluorescence (mKATE). All VSIG2 aCARs tested showed increased killing compared to non-transduced (NV) and control (SB04501, non-targeted aCAR without scFv). The highest killing was observed for SBSB04750, SB04746, and SB04744. Killing by selected scFvs is further shown in Figure 7C (Ls174t cells) and Figure 7D (DLD1 cells).

[0382] VSIG2 as FLT3 NOT gate The use of the VSIG2 iCAR as a FLT3 NOT gate was then tested. NK cells were co-cultured with SEM target cells (generated using the methodology described above) overexpressing FLT3 compared to target cells expressing both FLT3 and VSIG2 at an E:T ratio of 1:4. NK cells were transduced with retroviruses expressing FLT3-aCAR and VSIG2-iCAR with different binders. Figure 8 shows that the VSIG2 CAR shows increased killing in both target cell lines.

[0383] Supernatants from co-culture experiments with NK cells and FLT3 target cells were then analyzed using Luminex to quantify cytokine production. As shown in Figure 9, TNFα production was reduced when NOT-gated CAR-NK cells were co-cultured with VSIG2-positive target cells.

[0384] VSIG2 as CEA NOT gate NK cells were transduced with retroviruses expressing CEA-aCAR and various VSIG2-iCARs constructed using selected VSIG2 binders, and flow cytometry was used to determine the expression of CEA-aCAR and VSIG2-iCAR on NK cells (Figure 10A, control lines shown in Figure 10B).

[0385] The transduced NK cells were then co-cultured with target cells engineered to overexpress human VSIG2 at an E:T ratio of 1:2. The reduction in target cells was compared to target cells that did not express VSIG2 (left column). Importantly, several VSIG2 iCARS significantly reduced NK-mediated killing of target cells in a VSIG2-dependent manner (shown in FIG. 11).

[0386] These data indicate that the tested VSIG2 scFv can be used as a safety antigen, for example for the NOT gate technology in combination with an activating CAR.

[0387] (Incorporated by reference) All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0388] (Equivalent) While various specific embodiments have been shown and described, the above specification is not limiting. It will be understood that various changes can be made without departing from the spirit and scope of the disclosure. Many variations will be apparent to those skilled in the art in light of the scope of this specification.

[0389] (other sequences) Other sequences relevant to this disclosure are set forth below.

[0390] TIFF2024516713000017.tif26158

Claims

1. 1. A chimeric protein comprising an antigen-binding domain specific for a V-set and immunoglobulin domain containing 2 (VSIG2) and a heterologous molecule or moiety, the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) the VH is a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GFTFSNS (SEQ ID NO:2); A heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SDGGLY (SEQ ID NO:3), and Heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence QGVRPFFDY (SEQ ID NO: 4) and The VL is a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence RASENIYSYLA (SEQ ID NO: 11) or RASENLYSYLA (SEQ ID NO: 12); A light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of NAETLPE (SEQ ID NO: 13), and Light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QHHYVIPWT (SEQ ID NO: 14) Contains; or (b) the VH is Heavy chain complementarity determining region 1 (CDR-H1) contained in the VH region amino acid sequence of SEQ ID NO: 1, heavy chain complementarity determining region 2 (CDR-H2) contained in the VH region amino acid sequence of SEQ ID NO: 1, and heavy chain complementarity determining region 3 (CDR-H3) contained in the VH region amino acid sequence of SEQ ID NO: 1 and The VL is Light chain complementarity determining region 1 (CDR-L1) contained in the VL region amino acid sequence of SEQ ID NO: 9, light chain complementarity determining region 2 (CDR-L2) contained in the VL region amino acid sequence of SEQ ID NO: 9, and light chain complementarity determining region 3 (CDR-L3) contained in the VL region amino acid sequence of SEQ ID NO: 9 Contains; or (c) the VH is Heavy chain complementarity determining region 1 (CDR-H1) contained in the VH region amino acid sequence of SEQ ID NO: 1, heavy chain complementarity determining region 2 (CDR-H2) contained in the VH region amino acid sequence of SEQ ID NO: 1, and heavy chain complementarity determining region 3 (CDR-H3) contained in the VH region amino acid sequence of SEQ ID NO: 1 and The VL is Light chain complementarity determining region 1 (CDR-L1) contained in the VL region amino acid sequence of SEQ ID NO: 10, light chain complementarity determining region 2 (CDR-L2) contained in the VL region amino acid sequence of SEQ ID NO: 10, and light chain complementarity determining region 3 (CDR-L3) contained in the VL region amino acid sequence of SEQ ID NO: 10 Including, Optionally, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat or Chothia numbering scheme.

2. The chimeric protein of claim 1 , wherein the VH region comprises the amino acid sequence of SEQ ID NO:

1.

3. The chimeric protein of claim 1, wherein the VL region comprises the amino acid sequence of SEQ ID NO:9, or the VL region comprises the amino acid sequence of SEQ ID NO:

10.

4. the antigen-binding domain comprises a single chain variable fragment (scFv); Optionally, the VH and VL of the scFv are separated by a peptide linker; Optionally, said antigen binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker and VL is a light chain variable domain; and / or Optionally, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-35 and 69-74. The chimeric protein of claim 1.

5. 2. The chimeric protein of claim 1, wherein the chimeric protein is a chimeric antigen receptor (CAR) and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

6. The chimeric protein of claim 5, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response, each of the one or more intracellular inhibitory domains comprising an enzyme inhibitory domain or an intracellular inhibitory co-signaling domain.

7. 2. An engineered polynucleotide encoding the chimeric protein of claim 1.

8. An expression vector comprising the engineered polynucleotide of claim 7.

9. An isolated cell comprising the chimeric protein of claim 1.

10. A population of engineered cells expressing the chimeric protein of claim 1.

11. 10. The cell of claim 9, wherein the cell further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface, and optionally each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

12. 10. The cell of claim 9, selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

13. 10. A pharmaceutical composition comprising an effective amount of the cells of claim 9 and a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, or a combination thereof.

14. A pharmaceutical composition comprising a therapeutically effective dose of the cells of claim 9 for stimulating a cell-mediated immune response against tumor cells in a tumor-bearing subject.

15. 10. A pharmaceutical composition comprising a therapeutically effective dose of the cells of claim 9 for treating a subject having a tumor.

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