Antigen-binding domains and methods of use thereof

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 383,058, filed November 9, 2022, the disclosure of which is incorporated herein 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 hereby incorporated by reference in its entirety. The ASCII copy, created in XX month of 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, which is used to redirect the specificity and function of immunocompetent cells such as T cells, has shown efficacy in patients with malignant tumors (Pule et al., Nat. Med. (14): 1264-1270 (2008) (Non-Patent Document 1); Maude et al., N Engl J Med. (371): 1507-17 (2014) (Non-Patent Document 2); Brentjens et al., Sci Transl Med. (5): 177ra38 (2013) (Non-Patent Document 3)). CAR T cells have been shown to induce complete remissions in patients with CD19-expressing malignant tumors where chemotherapy has led to drug resistance and tumor progression. The success of CD19 CAR therapy provides optimism for treating other malignant tumors, 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 for effectively targeting and treating tumors without damaging normal cells that express the same target antigen. Therefore, there remains a need for CAR-based solid tumor therapies that target tumor cells without targeting normal cells or tissues, such as therapies for the treatment of colorectal cancer (CRC). [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 Provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5; the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8; and the antibody or antigen-binding fragment thereof is humanized.

[0007] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO:2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO:4, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:5; the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:8; and the antibody or antigen-binding fragment thereof is humanized.

[0008] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO:2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO:4, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:5, and the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:8.

[0009] In some embodiments, the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

[0010] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, 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) contained within the VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16, and 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) contained within the VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

[0011] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

[0012] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises an F(ab) fragment, an F(ab') fragment, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding fragment comprises a single-chain variable fragment (scFv). In some embodiments, the VH and VL of the scFv are separated by a peptide linker. In some embodiments, the antigen-binding domain comprises 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: 22-37 and 100. In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99.

[0014] Also provided herein are chimeric proteins comprising an antibody or antigen-binding fragment thereof of any of the isolated antibodies or antigen-binding fragments provided herein and a heterologous molecule or moiety. In some embodiments, the chimeric protein is an antibody-drug conjugate, and the heterologous molecule or moiety comprises a therapeutic agent. In some embodiments, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an activated 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: 41-52.

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

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

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

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

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

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

[0021] Also provided herein are populations of engineered cells that express any one of the engineered nucleic acids or expression vectors provided herein.

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

[0023] Also provided herein are populations of engineered cells that express any one of the antibodies or antigen-binding fragments thereof provided herein, or any one of the chimeric proteins provided herein.

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

[0025] In some 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, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

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

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

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

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

[0030] Also provided herein is a method for 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 any one of the compositions, cells, or pharmaceutical compositions provided herein.

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

[0032] Also provided herein is a method for inhibiting 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 any one of the compositions, cells, or pharmaceutical compositions provided herein.

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

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

[0035] Also provided herein are kits for treating and / or preventing tumors, comprising any one of the chimeric proteins provided herein. In some embodiments, the kits further comprise written instructions for using the chimeric protein to produce one or more antigen-specific cells to treat and / or prevent tumors in a subject.

[0036] Also provided herein are kits for treating and / or preventing tumors, comprising any one of the cells or populations of cells provided herein. In some embodiments, the kits further comprise written instructions for using the cells to treat and / or prevent tumors in a subject.

[0037] Also provided herein are kits for treating and / or preventing tumors, comprising any one of the engineered nucleic acids provided herein. In some embodiments, the kits further comprise written instructions for using the nucleic acid to produce one or more antigen-specific cells to treat and / or prevent tumors in a subject.

[0038] Also provided herein are kits for treating and / or preventing tumors, comprising any one of the vectors provided herein. In some embodiments, the kits further comprise written instructions for using the vector to produce one or more antigen-specific cells to treat and / or prevent tumors in a subject.

[0039] Also provided herein are kits for treating and / or preventing tumors, comprising any one of the compositions provided herein. In some embodiments, the kits further comprise written instructions for using the composition to treat and / or prevent tumors in a subject. [Brief explanation of the drawings]

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0041] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0042] [Figure 1]Shown is CAR expression at day 3 after transduction of a humanized construct based on the parental clone "SB04744" as assessed by flow cytometry. [Figure 2] Shown is CAR expression at day 6 after transduction of a humanized construct based on the parental clone "SB04744" as assessed by flow cytometry. [Figure 3] Shown is CAR expression at day 3 after transduction of a humanized construct based on the parental clone "SB04746" as assessed by flow cytometry. [Figure 4] 1 shows anti-VSIG2 antigen-binding domain-dependent killing of VSIG2-expressing LS174t target cells for various humanized constructs expressed in aCAR format. [Figure 5] 1 shows anti-VSIG2 antigen-binding domain-dependent killing of VSIG2-expressing DLD-1 target cells for various humanized constructs expressed in aCAR format. [Figure 6] Figure 1 shows the time course of anti-VSIG2 antigen-binding domain-dependent killing of VSIG2-expressing LS174t and DLD-1 target cells for various humanized constructs expressed in aCAR format. DETAILED DESCRIPTION OF THE INVENTION

[0043] 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, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Fundamental Virology, 3 rdEdition,vol.I&II(BNFields and DMKnipe,eds.), Handbook of Experimental Immunology,Vols.I-IV(DMWeir and CCBlackwell eds.,Blackwell Scientific Publications), ALLehninger,Biochemistry(Worth Publishers,Inc.,current addition), Sambrook,et al.,Molecular Cloning: A Laboratory Manual(3 rd Edition, 2001), Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0044] definition Unless otherwise defined, all terms, designations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a difference to that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely 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 appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and conditions unless otherwise noted.

[0045] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Terms such as "including," "e.g.," and the like are intended to convey inclusion without limitation unless otherwise indicated.

[0046] As used herein, the term "comprising" also specifically includes embodiments "consisting of" and "consisting essentially of" the listed elements, unless otherwise indicated.

[0047] The term "about" denotes 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.

[0048] As used herein, the terms "stimulating a cell-mediated immune response" or "stimulating an immune response" refer to generating a signal that results in an immune response by one or more cell types or populations of cells. Immunostimulatory activity can include proinflammatory activity. In various embodiments, the immune response occurs following activation of immune cells (e.g., T cells or NK cells) or is co-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 otherwise stimulating proinflammatory polypeptides and / or their ligands can enhance the immune response of immunocompetent cells. Without being bound by theory, receiving multiple stimulatory signals (e.g., costimulation) is important for mounting robust, long-lasting cell-mediated immune responses, such as T cell-mediated immune responses, in which T cells may be inhibited and unresponsive to antigens (also referred to as "T cell anergy") in the absence of costimulatory signals. While the various effects of costimulatory signals, particularly in combination with one another, are diverse and still only partially understood, 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, e.g., in mediating complete and / or sustained elimination of target cells expressing the cognate antigen.

[0049] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain.

[0050] 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.

[0051] As used herein, the term "inhibitory CAR" or "iCAR" refers to a CAR construct / structure that, upon binding to a cognate iCAR ligand, can induce a change in signal transduction or protein expression in an inhibitory CAR-expressing cell that prevents, attenuates, inhibits, reduces, decreases, inhibits, or suppresses an immune response, such as reducing the activation of an immunoresponsive cell that is receiving or has received one or more stimulatory signals, including costimulatory signals.

[0052] 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 through a defined signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0053] As used herein, the term "extracellular antigen-binding domain" or "antigen-binding domain" (ABD) refers to a polypeptide sequence or polypeptide complex that specifically recognizes or binds to a given antigen or epitope, such as the polypeptide sequence or polypeptide complex portion of the chimeric proteins described herein that provide VSIG2-specific binding. The ABD (or antibody, antigen-binding fragment, and / or chimeric protein comprising 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 used herein, "affinity" refers to the strength of interaction of non-covalent intermolecular forces between one molecule and another. Affinity, i.e., the strength of 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 binding molecules that have affinity between the ABD and its cognate antigen or epitope, and KD values ​​are generally in the range of 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, e.g., a biological sample that naturally contains a polypeptide of the present disclosure. In certain embodiments, specifically binding refers to binding of an epitope or antigen or antigenic determinant of an ABD, antibody, or antigen-binding fragment in such a manner that binding can displace or compete with a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0054] The ABD may be an antibody. As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. An antibody may be a tetramer of immunoglobulin molecules.

[0055] 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, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antigen-binding fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., 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 U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0056] 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 may be referred to as "monospecific multivalent" binding molecules. In other multivalent embodiments, at least two of the multiple ABDs have different recognition specificities. Such binding molecules are multivalent and "multispecific." In multivalent embodiments in which the ABDs collectively have two recognition specificities, the binding molecule is "bispecific." In multivalent embodiments in which the ABDs collectively have three recognition specificities, the binding molecule is "trispecific." In multivalent embodiments in which the ABDs collectively have 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 "dualparatopic."

[0057] In various multivalent embodiments, the multivalent binding molecule improves the avidity of the binding molecule for a specific target. As described herein, "avidity" refers to the overall strength of the interaction between two or more molecules, e.g., multivalent binding molecules for 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 method used to determine affinity, as described above. In certain embodiments, the avidity of the binding molecule for a specific target is such that the interaction is a specific binding interaction, and the avidity between the two molecules is 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 particular 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 would be considered to bind each antigen or epitope uniquely and specifically. In certain embodiments, avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs for distinct antigens on a shared specific target or complex, such as distinct antigens found on individual cells. In certain embodiments, avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs for distinct epitopes on a shared individual antigen.

[0058] 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 can be 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- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0059] As used herein, "variable region" refers to the variable region that results 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 associate together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0060] As used herein, the term "complementarity-determining region" or "CDR" refers to sequences within 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 three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of 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) JMB273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids of 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 the 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.

[0061] As used herein, the term "framework region" or "FR" refers to generally conserved sequences within antibody variable regions VH and VL that serve as a scaffold for the interspersed CDRs, typically in the following arrangement (N- 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.

[0062] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations and which usually determine the class to which the antibody belongs.

[0063] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0064] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in 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, which DNA molecule expresses the 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.

[0065] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may 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.

[0066] 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 lifespan, 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 in preventing the development of tumors in the first place, such as prophylactic therapy or treatment.

[0067] As used herein, the term "autologous" refers to any material originating from the same subject that is subsequently reintroduced into the subject.

[0068] 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 one another if the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species may be sufficiently genetically different to interact antigenically, e.g., at specific genes, such as MHC alleles. In some embodiments, allogeneic material derived from individuals of the same species may be sufficiently genetically identical to not interact antigenically, e.g., at specific genes, such as MHC alleles.

[0069] Isolated nucleic acid molecules of the present disclosure include any nucleic acid molecule encoding a polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A nucleic acid having "substantial identity" or "substantial homology" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to the pairing to form a double-stranded molecule between a complementary polynucleotide sequence (e.g., a gene described herein) or portion thereof under various stringency conditions. For example, stringent salt concentrations can typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. While low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions typically 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, the concentration of detergents, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed.

[0070] "Substantially identical" or "substantially homologous" means that a polypeptide or nucleic acid molecule exhibits at least about 50% homology or identity to 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 typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705; BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following group: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. An exemplary method for determining the degree of identity may use the BLAST program, where a probability score of e-3 to e-100 indicates closely related sequences.

[0071] As used herein, the term "encoding" 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, and the biological properties 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 shown in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be said to encode a 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 or RNA encoding a protein can also include introns, to the extent that the nucleotide sequence encoding the protein may contain introns in some versions.

[0072] 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 cell-cell recognition and / or interaction.

[0073] 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 bone marrow disorder.

[0074] 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.

[0075] As used herein, the term "immune effector response" or "immune effector function" refers to a function or response, e.g., of an immunocompetent cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response can refer to a property of a T cell or NK cell that promotes the killing or inhibition of growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0076] 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, having the amino acid sequence (Gly-Gly-Gly-Gly-Ser) n or (Gly-Gly-Gly-Ser) n wherein n is a positive integer greater than or equal to 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 includes, but is not limited to, Gly4Ser or (Gly4Ser)3. In other embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). In some embodiments, the flexible polypeptide linker includes a Whitlow linker (e.g., GSTSGSGKPGSGEGSTKG [SEQ ID NO: 36]). For example, linkers described in WO2012 / 138475 are also within the scope of the present disclosure.

[0077] As used herein, the terms "treat," "treatment," and "treating" refer to a 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 the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the present disclosure). In some embodiments, reduction or amelioration refers to an improvement in at least one measurable physical parameter of the proliferative disorder, such as tumor growth, that is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an 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, reduction or amelioration includes a reduction or stabilization of tumor size or cancer cell number.

[0078] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0079] Other aspects of the present disclosure are described in the following sections and are within the scope of the claimed invention.

[0080] Other Rules of Interpretation Ranges recited herein are understood to be shorthand for all values ​​within the range, inclusive of 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.

[0081] Unless otherwise indicated, reference to a compound having one or more stereocenters contemplates each stereoisomer and all stereoisomeric combinations thereof.

[0082] V-set and immunoglobulin domain-containing 2 (VSIG2)-specific 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 proteins bind to human VSIG2 (e.g., Uniprot Q96IQ7, incorporated herein by reference for all purposes) or an epitopic fragment thereof. VSIG2 can be expressed on epithelial cells. VSIG2 can be expressed in cells generally considered healthy, such as healthy epithelial cells. Examples of VSIG2-specific antibodies include OTI2D8 (also known as "2D8" and referred to herein as Ab) and OTI5A10 (also known as "5A10").

[0083] The present disclosure provides a VSIG2-specific antigen-binding domain comprising one or more of the amino acid sequences listed in Table 1.

[0084] Table 1. Humanized VSIG2-specific antigen-binding domains TIFF2025538171000002.tif155161TIFF2025538171000003.tif118161

[0085] 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: 5). 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), 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 within the VH region amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. 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 heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 2, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5. In some embodiments, a VSIG2-specific antigen-binding domain having the above VH sequence can have 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), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of one of SEQ ID NOs: 17 to 21. In some embodiments, a VSIG2-specific antigen-binding domain having the above VH sequence can have a heavy chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO:6, a heavy chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:7, and a heavy chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:8.

[0086] 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), 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 within the VL region amino acid sequence of one of SEQ ID NOs: 17 to 21. 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 SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, a VSIG2-specific antigen-binding domain having the above VL sequence can have 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) amino acid sequences, each having the amino acid sequence of CDR-H1, CDR-H2, and CDR-H3 contained within a VH region amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. In some embodiments, a VSIG2-specific antigen-binding domain having the above VL sequence can have heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 2, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5.

[0087] In some embodiments, a VSIG2-specific antigen-binding domain comprises 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 SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5, and (2) the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8.

[0088] In some embodiments, a VSIG2-specific antigen-binding domain comprises a variable heavy (VH) region comprising a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5, and a variable light (VL) region comprising a VL complementarity region L (CDRL1) having the amino acid sequence of SEQ ID NO: 6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8, wherein the antigen-binding domain is humanized.

[0089] In some embodiments, the VSIG2-specific antigen-binding domain has a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of one of SEQ ID NOs: 9-16.

[0090] 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 one of SEQ ID NOs: 17-21. 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, sdAbs (either VL or VH), single-domain antibodies such as camelid VHH domains, and multispecific formats. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab)2 format. In some embodiments, the VSIG2-specific antigen-binding domain is in F(ab')2 format.

[0091] 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 2). 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.

[0092] In some embodiments, the scFV has an amino acid sequence selected from SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99.

[0093] In some embodiments, the VSIG2-specific antigen-binding domain is humanized.

[0094] The present disclosure also provides a VSIG2-specific antigen-binding domain that competes with 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 SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5, and (2) the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8.

[0095] The present disclosure also provides a VSIG2-specific antigen-binding domain that competes with 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 SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5, and (2) the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8.

[0096] In some embodiments, a VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a different 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 SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5, and (2) the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, a VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a different 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 SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 5, and (2) the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, a VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a different human VSIG2 epitope) as a reference antibody or antigen-binding fragment thereof having a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. In some embodiments, a VSIG2-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a different human VSIG2 epitope) as a reference antibody or antigen-binding fragment thereof having a VL comprising the amino acid sequence of one of SEQ ID NOs: 17 to 21.

[0097] The present disclosure also provides chimeric proteins, and nucleic acids encoding such chimeric proteins, that include a VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 1. The chimeric proteins can include any of the foregoing VSIG2-specific antigen-binding domains.

[0098] Chimeric antigen receptor (CAR) Certain aspects of the present disclosure relate to chimeric receptors 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). Generally, a CAR is a chimeric protein comprising an antigen-binding domain and a polypeptide molecule heterologous to the antigen-binding domain, such as a peptide heterologous to the antibody from which the antigen-binding domain can be derived. The polypeptide molecule heterologous to the antigen-binding domain can include, 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.

[0099] In some embodiments, a CAR is an engineered receptor (e.g., VSIG2) that grafts or confers a desired specificity onto an immune effector cell. In certain embodiments, a CAR can be used to graft the specificity of an antibody onto an immunoresponsive cell, such as a T cell. In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0100] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, 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.

[0101] The CARs of the present disclosure can be first-, second-, or third-generation CARs. 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 primary transmitter of signals from endogenous TCRs. A "first-generation" CAR provides de novo antigen recognition and transduces CD4 T cells via the CD3ζ chain signaling domain within a single fusion molecule, independent of HLA-mediated antigen presentation. + 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 anti-tumor activity of immunoresponsive 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).

[0102] 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 the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0103] In some embodiments, a chimeric inhibitory receptor (a VSIG2-specific chimeric inhibitory receptor) can be used, for example, as a NOT logic gate to control, regulate, or otherwise inhibit one or more activities of 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, 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 can 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 chimeric inhibitory receptor decreases, reduces, prevents, or inhibits activation of the immunoresponsive cell via the tumor-targeting chimeric receptor ("NOT logic gating"). In some embodiments, a chimeric inhibitory receptor of the present disclosure can inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, immunoresponsive cells may comprise one or more tumor-targeting chimeric receptors and one or more chimeric inhibitory receptors that target antigens that are not expressed or thought to be generally not expressed on tumors (e.g., VSIG2). A combination of tumor-targeting chimeric receptors and chimeric inhibitory receptors on the same immunoresponsive cell may be used to reduce on-target off-tumor toxicity.

[0104] In some embodiments, the extracellular antigen binding domain of a CAR of the disclosure is in a concentration of 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 dIn some embodiments, K d The range is approximately 2 × 10 -7 M ~ approx. 1×10 -9 In some embodiments, the VSIG2-specific antigen-binding domain of the aCAR format can be selected based on affinity, including selecting based on having a higher or lower affinity compared to other VSIG2-specific antigen-binding domains. In some embodiments, the VSIG2-specific antigen-binding domain of the iCAR format can be selected based on affinity, including selecting based on having a higher or lower affinity compared to other VSIG2-specific antigen-binding domains.

[0105] Binding of the extracellular antigen-binding domain of a 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 labeled reagent (e.g., antibody or scFv) specific for the complex of interest. For example, an scFv can be radiolabeled and used in an RIA assay. The radioisotope 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).

[0106] 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 cross-linked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment. 。

[0107] Extracellular antigen-binding domain The extracellular antigen-binding domain of a 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 hematopoietic stem cells. In certain embodiments, the extracellular antigen-binding domain binds to VSIG2 expressed on cells generally considered to be healthy, such as healthy HSCP. In some embodiments, the VSIG2 is human VSIG2.

[0108] The antigen-binding domain of the present disclosure can comprise any domain that binds to an antigen, including, but not limited to, 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 alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCR), affinity-enhanced recombinant 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.

[0109] 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.

[0110] 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.

[0111] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to a distinct epitope 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).

[0112] 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 2. 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 aspects, 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 together, each scFv can be linked to the next scFv linked by a peptide. In some embodiments, each of the one or more scFvs is separated by a peptide linker.

[0113] Table 2: Peptide linkers TIFF2025538171000004.tif120128

[0114] In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domain of the first chimeric receptor and the antigen-binding domain of the second chimeric receptor can be any suitable antigen-binding domain described herein or known in the art. For example, the first or second antigen-binding domain can be one or more of an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor 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 chimeric receptor can be specific for VSIG2, and the chimeric receptor can be specific for a second, different antigen, such as a cancer antigen (e.g., an antigen expressed on CRC tumor cells).

[0115] 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.

[0116] 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.

[0117] 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-enhanced 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).

[0118] In certain embodiments, a bispecific CAR or tanCAR comprises an antigen-binding domain comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with the VH (VH1) upstream of the VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with the VL (VL2) upstream of the VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged in a VL (VL1) configuration upstream of the VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged in a VH (VH2) configuration upstream of the VL (VL2), with the overall bispecific antibody molecule having the configuration VL1-VH1-VH2-VL2. In some embodiments, a linker is placed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker described herein, e.g., a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. Generally, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is disposed between the VL and VH of a first scFv. In some embodiments, the linker is disposed between the VL and VH of a 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 the arrangements described herein.

[0119] In some embodiments, the chimeric receptor 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 1. In some embodiments, the ABDs of the bivalent VSIG2 CAR each comprise the same ABD.

[0120] 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 1.

[0121] Transmembrane domain In some embodiments, the transmembrane domain of a CAR of the present disclosure (e.g., a 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, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of a CAR of the present disclosure can include the 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, an LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0122] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide 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 can 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.

[0123] 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_0 NP_05009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions.In some embodiments, the polypeptide 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 Nos. NP_001139345, AAA92533.1, NP_006130 ... NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0124] Further examples of suitable polypeptides from which transmembrane domains 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 regions of the alpha, beta, or zeta chains of NG2C.

[0125] In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 38). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 39). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 40).

[0126] Spacer region In some embodiments, a CAR of the present disclosure (e.g., a VSIG2-specific CAR described herein) can also include a spacer region linking 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, a spacer region may comprise any of the amino acid sequences listed in Table 3 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 3. In some embodiments, a nucleic acid encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table 4 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 4.

[0127] (Table 3) Spacer amino acid sequences TIFF2025538171000005.tif119164

[0128] Table 4: Spacer nucleic acid sequences TIFF2025538171000006.tif160166

[0129] 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. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO:49. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO:50. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO:51. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO:52.

[0130] In some embodiments, a CAR of the present disclosure may further comprise a short oligopeptide or polypeptide linker between 2 and 10 amino acid residues in length that 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 GGCKJSGGCKJS (SEQ ID NO: 62).

[0131] In some embodiments, the transmembrane domain further comprises at least a portion of the extracellular domain of the same protein.

[0132] Intracellular signaling domains In some embodiments, a CAR of the disclosure (e.g., a VSIG2-specific CAR described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of a CAR can comprise an intracellular signaling domain.

[0133] Examples of suitable intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to regulate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capabilities.

[0134] 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 therefore secondary and / or costimulatory signals are also typically 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 regulated by inhibitory receptors present in T cells through intracellular inhibitory co-signaling domains.

[0135] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise an inhibitory intracellular signaling domain. Examples of inhibitory intracellular domains that can be used include PD-1, CTLA4, TIGIT, BTLA, and LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10. In some embodiments, the inhibitory intracellular signaling domain comprises one or more intracellular inhibitory co-signaling domains. In some embodiments, one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., transmembrane domains) through peptide linkers (see, e.g., Table 2) or spacer or hinge sequences (see, e.g., Table 3). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains can be linked through peptide linkers (see, e.g., Table 2) or spacer or hinge sequences (see, e.g., Table 3). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains of the chimeric protein comprise one or more ITIM-containing proteins, or fragments thereof. ITIMs are conserved amino acid sequences found in the cytoplasmic tails of many inhibitory immunoreceptors. Examples of ITIM-containing proteins include PD-1, TIGIT, BTLA, and LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffolding proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffolding 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 selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP. Examples of enzymatic regulation of signal transduction are described in detail by Pavel Otahal et al. (Biochim Biophys Acta. 2011 Feb;1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 Jun;14(6):669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19), each of which is incorporated herein by reference for all purposes.

[0136] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a primary signaling domain that regulates primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in a CAR of the present disclosure include, but are not limited to, those of CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0137] In some embodiments, a CAR of the present disclosure (a VSIG2-specific CAR described herein) comprises an intracellular signaling domain, e.g., the primary signaling domain of a CD3-zeta polypeptide. The CD3-zeta polypeptide of the present disclosure can 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 numbers NP_932170 or NP_001106864.2, or a fragment thereof. In some embodiments, the CD3-zeta polypeptide can include one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a 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.

[0138] 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.

[0139] In some embodiments, the intracellular signaling domain of a 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 a CAR of the present disclosure. For example, the intracellular signaling domain of a CAR can comprise a portion of a CD3-zeta chain and a costimulatory signaling domain. A costimulatory signaling domain can refer to a portion of a CAR that includes 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 may be required for an 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 to CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin G receptor protein, and the like. Cholesterol-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, 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, etc.

[0140] In some embodiments, intracellular signaling sequences within the cytoplasmic portion of a CAR of the present disclosure can be linked to each other in random or specified order. In some embodiments, a short oligopeptide or polypeptide linker, e.g., 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form the linkage between intracellular signaling sequences. In one embodiment, a glycine-serine duplex can be used as a suitable linker. In one embodiment, a single amino acid, e.g., alanine or glycine, can be used as a suitable linker.

[0141] 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 2). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0142] In some embodiments, the cells of the present disclosure express a CAR comprising an antigen binding domain that binds VSIG2, a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0143] In some embodiments, cells of the present disclosure express an iCAR that includes 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 1), a transmembrane domain of the present disclosure, and one or more intracellular inhibitory co-signaling domains. In some embodiments, the VSIG2-specific antigen binding domain of the iCAR format can be selected based on a higher affinity for VSIG2 compared to other VSIG2-specific antigen binding domains. In some embodiments, the VSIG2-specific antigen binding domain of the iCAR format can be selected based on a lower affinity for VSIG2 compared to other VSIG2-specific antigen binding domains. In some embodiments, cells of the present disclosure express a CAR that includes 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 1), a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains. In some embodiments, a cell of the present disclosure expresses a CAR that includes an antigen binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen binding domain having one or more of the amino acid sequences listed in Table 1), a transmembrane domain of the present disclosure, a hinge positioned between the antigen binding domain and the transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0144] In some embodiments, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR, each comprising a transmembrane domain and at least one intracellular inhibitory co-signaling domain derived from a protein selected from PD-1, CTLA4, TIGIT, BTLA, and LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

[0145] In some embodiments, the transmembrane domain is derived from a first protein and the one or more intracellular signaling domains are derived from a second protein that is different from the first protein.

[0146] Natural killer cell receptor (NKR) CAR In some embodiments, a CAR of the present disclosure (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 KIR3S1. The transmembrane domain, hinge domain, or cytoplasmic domain may be 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, and NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing an NKR receptor component are described in International Patent Publication No. 2014 / 145252, published September 18, 2014.

[0147] Further chimeric receptor targets Certain aspects of the present disclosure relate to chimeric receptors that bind to an antigen of interest in addition to VSIG2 and nucleic acids encoding such chimeric receptors. Certain aspects of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, that have been genetically modified to express one or more such chimeric receptors that bind to an antigen of interest in addition to VSIG2, as well as methods of using such receptors and cells to treat and / or prevent myeloid malignancies, such as CRC, and other conditions in which an antigen-specific immune response is desired. Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination. The present disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0148] In some embodiments, a 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 1), and a second chimeric receptor comprises an additional antigen-binding domain that binds a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, a cell can express a first chimeric receptor specific for VSIG2 (e.g., a CAR comprising a VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 1) and a second chimeric receptor specific for a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, a cell can express a first chimeric inhibitory receptor specific for VSIG2 (e.g., an inhibitory CAR 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 CRC-associated antigen). For example, cells (e.g., immunoresponsive cells) can be engineered to co-express or co-express an iCAR comprising an antigen-binding domain that binds to VSIG2 (e.g., a VSIG2-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 1) and an aCAR that targets a tumor-associated antigen (e.g., a CRC-associated antigen). Suitable antibodies that bind to antigens in addition to VSIG2 include any antibody, whether natural or synthetic, full-length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). In some embodiments, commercially available antibodies can be used to bind to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). The CDRs of commercially available antibodies are readily accessible by those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art can construct nucleic acids encoding scFvs and chimeric receptors (e.g., CARs and TCRs) based on the CDRs of such commercially available antibodies.

[0149] 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 CRC-associated antigen), where the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). The TCRs of the present disclosure are disulfide-linked heterodimeric proteins comprising two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCRs of the present disclosure comprise an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCRs comprise a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0150] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each variable region has three complementarity-determining regions (CDRs).

[0151] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. Upon complexation of the TCR complex with its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0152] 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 a naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from a naturally occurring TCR by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least 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 two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least eleven amino acid residues, at least 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.

[0153] Chimeric TCR In some embodiments, the TCRs of the present disclosure comprise 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 CRC-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., an scFv) can be grafted onto the constant domains of a TCR chain, such as the TCR alpha chain and / or the TCR beta chain, e.g., the extracellular constant domain, the transmembrane domain, and at least a portion of the cytoplasmic domain. As another example, the CDRs of an antibody or antibody fragment can be grafted onto the TCR alpha chain and / or the beta chain to create a chimeric TCR that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., a CRC-associated antigen). Such chimeric TCRs can be produced by methods known in the art (e.g., Willemsen RA et al., Gene Therapy 2000;7:1369-1377, Zhang T et al., Cancer Gene Ther 2004 11:487-496, and Aggen et al., Gene Ther. 2012 Apr;19(4):365-74).

[0154] immunoresponsive cells Certain aspects of the present disclosure relate to cells, such as immunoresponsive cells, that have been genetically engineered to contain one or more chimeric receptors of the present disclosure or one or more nucleic acids encoding such chimeric receptors, and methods of using such cells, for example, to treat malignancies (e.g., CRC).

[0155] 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 stromal cells (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is derived from or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. The immune 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 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 neural cell. The neural cells of the present disclosure can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary neural 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.

[0156] In some embodiments, the cell is an immunoresponsive cell. The immunoresponsive cells of the present disclosure can be isolated or differentiated from 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 the lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, provides antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, etc. Examples of immunoresponsive cells of the 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., from which lymphoid cells can be derived or differentiated). 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 γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells 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.

[0157] Natural killer (NK) cells are 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.

[0158] In some embodiments, the immunoresponsive cells of the present disclosure are T cells. The T cells of the present disclosure can be derived in vitro from autologous, allogeneic, or engineered progenitor or stem cells.

[0159] In some embodiments, the immunoresponsive cells of the present disclosure are universal T cells 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.

[0160] 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.

[0161] In some embodiments, the immunoresponsive cells are T cells. In some embodiments, the immunoresponsive cells are natural killer (NK) cells.

[0162] In some embodiments, immunoresponsive cells express or are capable of expressing an immunoreceptor. Immunoreceptors generally can induce signal transduction or protein expression changes in immunoreceptor-expressing cells that result in modulation of an immune response (e.g., modulation, activation, initiation, stimulation, augmentation, prevention, attenuation, inhibition, reduction, decrease, inhibition, or suppression of an immune response) upon binding to a cognate ligand. For example, clustering of CD3 chains present in a TCR / CAR cluster in response to ligand binding produces an immunoreceptor tyrosine-based activation motif (ITAM) meditative signaling cascade. In particular, in certain embodiments, binding of an endogenous TCR, an exogenous TCR, a chimeric TCR, or a CAR (particularly an activating CAR) to its respective antigen results in the formation of an immunological synapse, which involves the clustering of many molecules in the vicinity of the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the phosphorylation of ITAM motifs contained within the CD3 chains, which then initiate the T cell activation pathway and ultimately activate transcription factors such as NF-κβ and AP-1. These transcription factors can increase IL-2 production for proliferation, induce expression of master regulator T cell proteins, and induce general gene expression in T cells to initiate T cell-mediated immune responses such as cytokine production and / or T cell-mediated killing.

[0163] Cells expressing multiple chimeric receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors of the present disclosure. In some embodiments, the cell comprises two or more chimeric receptors, and one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, and 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, and 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, and at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0164] In some embodiments, each of the two or more chimeric receptors comprises a different antigen-binding domain, e.g., antigen-binding domains that bind to the same antigen or different antigens. In some embodiments, each of the antigens bound by the two or more chimeric receptors is expressed on the same cell, such as a tumor cell (e.g., the same CRC tumor cell).

[0165] 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 the first chimeric receptor that comprises an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor can comprise an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can comprise a VHH.

[0166] Without wishing to be bound by theory, it is believed that in cells bearing multiple chimeric membrane-embedded receptors, each comprising an antigen-binding domain, interactions between the antigen-binding domains of each of the receptors may be undesirable because such interactions may inhibit the ability of one or more of the antigen-binding domains to bind to 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 comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv, and the antigen-binding domain of 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.

[0167] In some embodiments, when present on the surface of a cell, binding of the antigen-binding domain of a first chimeric receptor to its cognate antigen (e.g., binding of a VSIG2-specific chimeric receptor to VSIG2) is not substantially reduced by the presence of a second chimeric receptor. In some embodiments, binding of the antigen-binding domain of a 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 if both were scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen-binding domains.

[0168] Chimeric inhibitory receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises one or more chimeric inhibitory receptors of the present disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen-binding domain that binds to an antigen that is generally expressed on normal cells (e.g., cells generally considered to be healthy) but not on tumor cells, such as CRC cells. In some embodiments, the chimeric inhibitory 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 1).

[0169] 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 the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0170] In some embodiments, a chimeric inhibitory receptor (a VSIG2-specific chimeric inhibitory receptor) can be used, for example, as a NOT logic gate to control, modulate, or otherwise inhibit one or more activities of 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. In some embodiments, a chimeric inhibitory receptor of the present disclosure can inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure.

[0171] In some embodiments, the cells of the present disclosure comprise one or more chimeric inhibitory receptors of the present disclosure and further comprise a tumor-targeting chimeric receptor that binds to one or more tumor-associated antigens, in some embodiments, the one or more tumor-associated antigens comprise CRC-associated antigens.

[0172] Costimulatory Ligands In some embodiments, cells (e.g., immunoresponsive cells) of the present disclosure can further comprise one or more recombinant or exogenous costimulatory ligands. For example, 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., a VSIG2-specific CAR 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 important for the full activation of 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 primary role is to regulate immune cells. Members of the TNF superfamily share several common characteristics. Most TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, 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 cell recognition, binding, or adhesion processes. These proteins share structural characteristics with immunoglobulins and possess 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.

[0173] chemokine receptors In some embodiments, 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 the 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 chemokine receptors expressed on chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by tumors and improve tumor targeting of the cells, which may promote tumor infiltration and enhance the anti-tumor effects of the cells. The chemokine receptor of the present disclosure may include a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment 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 chemokines secreted by tumors.

[0174] Chimeric receptor regulation Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities (e.g., VSIG2-specific CARs described herein) of a chimeric receptor-expressing cell of the present disclosure. There are several ways to regulate chimeric receptor activity. In some embodiments, a regulatable chimeric receptor that can regulate 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 containing a chemical inducer (CID) that mediates dimerization in the presence of a CID, resulting in the inducible and selective depletion of chimeric receptor-expressing cells.

[0175] Alternatively, in some embodiments, the chimeric receptors of the present disclosure can be controlled by utilizing small molecules or antibodies that inactivate or otherwise inhibit chimeric receptor activity. For example, antibodies can delete chimeric receptor-expressing cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the chimeric receptor-expressing cells of the present disclosure can further express an antigen recognized by a molecule capable of inducing ADCC-mediated cell death or complement-induced cell death. For example, the chimeric receptor-expressing cells of the present disclosure can 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, These include, but are not limited to, CD5, 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.

[0176] 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).

[0177] 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 the CD32 and CD20 antigens in the chimeric receptor-expressing cells, which binds to 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 conjugated 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 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.

[0178] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides in which the components of a chimeric receptor of the present disclosure are 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, allows the polypeptides to bind to each other to form a functional chimeric receptor.

[0179] Nucleic acid constructs encoding VSIG2-specific proteins Certain aspects of the present disclosure pertain to nucleic acids (e.g., isolated nucleic acids) encoding one or more VSIG2-specific proteins of the present disclosure (e.g., VSIG2-specific CARs described herein). In some embodiments, the nucleic acid is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the nucleic acid is a DNA construct.

[0180] In some embodiments, a nucleic acid of the present disclosure encodes a chimeric receptor comprising one or more antigen binding domains, each domain binding to a target antigen (e.g., VSIG2), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the nucleic acid 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 nucleic acid further comprises a nucleotide sequence encoding a spacer region. In some embodiments, the antigen binding domain is connected to the transmembrane domain by 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.

[0181] The nucleic acids 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, deriving 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.

[0182] In some embodiments, the nucleic acids of the disclosure are contained within a vector, hi some embodiments, the nucleic acids of the disclosure are expressed in cells via transposons, CRISPR / Cas9 systems, TALENs, or zinc finger nucleases.

[0183] In some embodiments, expression of a nucleic acid 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.

[0184] 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.

[0185] The nucleic acids of the present disclosure can be cloned into several types of vectors. For example, the nucleic acids 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.

[0186] In some embodiments, the plasmid vector comprises a transposon / transposase system for integrating a nucleic acid 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 by reference in its entirety. In some embodiments, the transposon system is a Sleeping Beauty transposon / transposase or a piggyBac transposon / transposase.

[0187] 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 they 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). A number of viral-based systems have been developed for gene transfer into mammalian cells. A selected gene can be inserted into a vector and packaged 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.

[0188] In some embodiments, vectors of the present disclosure contain additional promoter elements, such as enhancers, to control the frequency of transcription initiation. Enhancers are typically located 30 to 110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site. Spacing between promoter elements can be flexible so that promoter function is maintained when elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter, spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Exemplary promoters 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.

[0189] In some embodiments, the promoter capable of expressing the nucleic acid of the present disclosure in mammalian cells, such as the immunoresponsive cells of the present disclosure, is the EF1a promoter.The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes.The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in promoting the expression of chimeric receptors from nucleic acids cloned into lentiviral vectors.

[0190] In some embodiments, a promoter capable of expressing a nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is a constitutive promoter. For example, a suitable constitutive promoter is the immediate-early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter capable of driving 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.

[0191] In some embodiments, a promoter capable of expressing a nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is an inducible promoter. The use of an inducible promoter can provide a molecular switch that can induce or repress expression of a nucleic acid of the present disclosure when the promoter is operably linked to the nucleic acid. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0192] 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 episomal replication, and / or elements that allow selection.

[0193] In some embodiments, vectors of the present disclosure may further comprise a selectable marker gene and / or reporter gene to facilitate identification and selection of chimeric receptor-expressing cells from a population of cells transduced with the vector. In some embodiments, the selectable marker may be separate from the vector and encoded by a nucleic acid used in the co-transfection procedure. Either the selectable marker or reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Examples of selectable markers include, but are not limited to, antibiotic resistance genes, such as neo.

[0194] In some embodiments, reporter genes can be used to identify transduced cells and evaluate the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene encoding 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 enzymatic activity. Expression of the reporter gene can be assayed at an appropriate time after the nucleic acid is introduced into the recipient cells. Examples of reporter genes include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein. Suitable expression systems are well known in the art and can be prepared using known techniques or commercially obtained. In some embodiments, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0195] In some embodiments, a vector comprising a nucleic acid sequence encoding a VSIG2-specific protein (e.g., a chimeric receptor) of the present disclosure further comprises a second nucleic acid encoding a polypeptide that increases the activity of the chimeric receptor.

[0196] In embodiments in which a VSIG2-specific protein-expressing cell comprises two or more heterologous proteins (e.g., two or more chimeric receptors), a single nucleic acid may encode the two or more proteins under a single regulatory control element (e.g., a promoter) or under separate regulatory control elements for each protein-encoding nucleotide sequence contained in the nucleic acid. In some embodiments in which a VSIG2-specific protein-expressing cell comprises two or more heterologous proteins, each heterologous protein may be encoded by a separate nucleic acid. In some embodiments, each separate nucleic acid comprises its own regulatory element (e.g., a promoter). In some embodiments, a single nucleic acid encodes two or more chimeric receptors, and the nucleotide sequences encoding the chimeric receptors are in the same reading frame and 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, e.g., autocleavage sites or substrates for intracellular proteases. Suitable peptide cleavage sites may include, but are not limited to, a T2A peptide cleavage site, a P2A peptide cleavage site, an E2A peptide cleavage site, and an F2A peptide cleavage site. In some embodiments, 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.

[0197] 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 nucleic acids into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Examples of chemical means for introducing nucleic acids 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 nucleic acids into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0198] In some embodiments, liposomes can be used as a non-viral delivery system for introducing the disclosed nucleic acids or vectors into host cells in vitro, ex vivo, or in vivo. In some embodiments, nucleic acids can be associated with lipids, for example, by being encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the nucleic acid, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contained or complexed in a micelle, or otherwise associated with the lipid. As disclosed herein, lipid-associated nucleic acid or vector compositions are not limited to any particular structure in solution. In some embodiments, such compositions can exist as micelles or in bilayer structures with a "collapsed" structure. Such compositions can also be interspersed in 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 naturally occurring in the cytoplasm, or classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Suitable lipids can 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, "liposome" can encompass a variety of mono- and multi-layered lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates.In some embodiments, liposomes can be characterized as having vesicular structures 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 forming 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.

[0199] In some embodiments, a nucleic acid or vector of the present disclosure is introduced into a mammalian host cell, such as an immunoresponsive cell of the present disclosure. In some embodiments, the presence of a nucleic acid or vector of the present disclosure in 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.

[0200] In some embodiments, a nucleic acid or vector of the disclosure is stably transduced into an immunoresponsive cell of the disclosure. In some embodiments, cells exhibiting stable expression of the nucleic acid or vector express the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months after transduction.

[0201] In embodiments in which a VSIG2-specific protein (e.g., a chimeric receptor) of the present disclosure is transiently expressed in a cell, a nucleic acid or vector encoding a VSIG2-specific protein of the present disclosure is transfected into an immunoresponsive cell of the present disclosure. In some embodiments, the immunoresponsive cell expresses the VSIG2-specific protein for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after transfection.

[0202] In some embodiments, the nucleic acid construct encodes a bicistronic-encoded chimeric antigen receptor. In some embodiments, the encoded bicistronic chimeric antigen receptor comprises a VSIG2 CAR (such as a VSIG2-inhibitory CAR) and a CAR specific for a second antigen (such as a tumor-targeting chimeric receptor).

[0203] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a VSIG2 antigen binding domain and a second antigen binding domain.

[0204] Pharmaceutical Compositions and Administration Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising one or more VSIG2-specific proteins (e.g., chimeric receptors) of the present disclosure or immunoresponsive cells of the present disclosure expressing such one or more VSIG2-specific proteins. In some embodiments, compositions comprising such VSIG2-specific proteins (e.g., chimeric receptors) or genetically modified immunoresponsive cells expressing such VSIG2-specific proteins 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 the organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Expansion 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.

[0205] Compositions comprising 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 x 10 5 cells can be administered, ultimately resulting in 1 x 10 10The 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. Dosage can be easily adjusted by one of skill in the art (e.g., a decrease in purity may require an increased dosage). Cells can be introduced by injection, catheter, etc. In some embodiments, factors such as 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 may also be included.

[0206] In certain embodiments, the composition is a pharmaceutical composition comprising genetically modified cells, such as immunoresponsive cells or their progenitor cells, and a pharmaceutically 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 a different compatible subject. In some embodiments, the immunoresponsive cells of the present disclosure or their progeny can be derived from peripheral blood cells (e.g., from in vivo, ex vivo, or in vitro sources) and can be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition comprising genetically modified cells of the present disclosure), it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0207] formulation Certain aspects of the present disclosure relate to formulations of compositions comprising a VSIG2-specific protein (e.g., a chimeric receptor) of the present disclosure or genetically modified cells expressing such a protein (e.g., an immunoresponsive cell of the present disclosure). In some embodiments, 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 can be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions may be more convenient to administer, particularly by injection. In some embodiments, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with specific tissues. 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.

[0208] In some embodiments, sterile injectable solutions can be prepared by incorporating the genetically modified cells of the present disclosure in a sufficient amount of an appropriate solvent with various amounts of any other ingredients, as needed. Such compositions can be in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, or the like. In some embodiments, the compositions can also be lyophilized. The compositions can contain auxiliary substances, such as wetting agents, dispersing agents, pH buffering agents, and antibacterial agents, depending on the route of administration and the desired preparation.

[0209] In some embodiments, the compositions of the present disclosure may further contain various additives that can improve the stability and sterility of the composition. Examples of such additives include, but are not limited to, antimicrobial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination can be prevented by the inclusion of any of a variety of antibacterial and antifungal agents, including, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical formulations of the present disclosure can be achieved by the use of suitable agents that delay absorption, such as aluminum monostearate and gelatin.

[0210] In some embodiments, the compositions of the present disclosure can be isotonic, i.e., have the same osmotic pressure as blood and tear fluid. 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.

[0211] 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.

[0212] One consideration regarding the therapeutic use of the genetically modified cells of the present disclosure is the amount of cells required to achieve optimal efficacy. In some embodiments, the amount of cells administered will vary depending on the subject being treated. In certain embodiments, the amount of genetically modified cells administered to a subject in need of treatment is 1 x 10 4 cells ~1×10 10 In some embodiments, the exact amount of cells that constitutes an effective dose may be based on factors individual to each subject, including the size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by one of ordinary skill in the art based on this disclosure and the knowledge of those skilled in the art.

[0213] Heterologous moieties and modifications In a further set of embodiments, the VSIG2-specific chimeric 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 1) comprise additional moieties and / or modifications.

[0214] Drug conjugates In various embodiments, proteins comprising a VSIG2-specific antigen-binding domain described herein are 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), immunomodulatory agents (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 through a linker peptide, as discussed in more detail herein.

[0215] Methods for preparing antibody-drug conjugates (ADCs) (e.g., having one or more of the amino acid sequences listed in Table 1) that can be adapted to conjugate drugs to the antigen-binding domains disclosed herein are described, for example, in U.S. Pat. No. 8,624,003 (pot 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. 8,337,856, U.S. Pat. No. 5,773,001, U.S. Pat. No. 7,829,531, U.S. Pat. No. 5,208,020, U.S. Pat. No. 7,745,394, WO2017 / 136623, WO2017 / 015502, WO2017 / 015496, WO2017 / 015495, WO2004 / 01095 7, WO2005 / 077090, WO2005 / 082023, WO2006 / 065533, WO2007 / 030642, WO2007 / 103288, WO2013 / 17 3337, WO2015 / 057699, WO2015 / 095755, WO2015 / 123679, WO2015 / 157286, WO2017 / 165851, WO2009 / 073445, WO2010 / 068759, WO2010 / 138719, WO2012 / 171020, WO2014 / 008375, WO2014 / 093394, WO2014 / 093640, WO2014 / 160360, WO2015 / 054659, WO2015 / 195925, WO2017 / 160754, Storz (MAbs.2015 November-December;7(6):989-1009), Lambert et al. (Adv Ther2017 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., 201324:846-851), 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 by reference in its entirety for all it teaches.

[0216] Additional binding moieties In various embodiments, the VSIG2-specific protein comprises an antigen-binding domain having one or more of the amino acid sequences listed in Table 1 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, an scDiabody, 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 Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), which is incorporated herein by reference for all that it teaches.

[0217] In certain embodiments, 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, 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.

[0218] 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.

[0219] 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 1) 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 1) through 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 1) 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 on the same expression vector (e.g., plasmid).

[0220] 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 1) have modifications that include functional or chemically reactive groups that can be used in downstream processes, such as linking to additional moieties (e.g., drug conjugates and additional binding moieties) and downstream purification processes.

[0221] In certain embodiments, the modification is a chemically reactive group, including, but not limited to, a reactive thiol (e.g., maleimide-based reactive group), a reactive amine (e.g., N-hydroxysuccinimide-based reactive group), a "click chemistry" group (e.g., a reactive alkyne group), and an aldehyde with formylglycine (FGly). In certain embodiments, the modification is a functional group, including, but not limited to, an affinity peptide sequence (e.g., HA, HIS, FLAG, GST, MBP, Strep, etc.). In certain embodiments, the functional group or chemically reactive group comprises 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-associated protease. ADC therapy employing protease cleavage is described in detail by Choi et al. (Theranostics, 2012;2(2):156-178.), the entire contents of which are incorporated herein by reference for all that it teaches.

[0222] Treatment method Certain aspects of the present disclosure relate to methods of using VSIG2-specific proteins (e.g., chimeric receptors) and genetically modified cells (e.g., immunoresponsive cells) of the present disclosure expressing such proteins to treat a subject in need of treatment. In some embodiments, the methods of the present disclosure are useful for treating cancer in a subject, such as colorectal cancer (CRC). Another aspect of the present disclosure relates to the use of VSIG2-specific chimeric receptors and genetically modified cells (e.g., immunoresponsive cells) of the present disclosure expressing such chimeric receptors in methods of 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 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, an effective amount may be provided in a single or series of administrations of genetically modified cells (e.g., immunoresponsive cells) of the present disclosure. In some embodiments, an effective amount may be provided by bolus or continuous perfusion.

[0223] As disclosed herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to affect a beneficial or desired clinical outcome upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to palliate, ameliorate, 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 one of ordinary skill in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective amount. 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 administered.

[0224] For adoptive immunotherapy using antigen-specific cells (e.g., immunoresponsive cells such as T cells), approximately 1 × 10 6 ~1×10 10 cells (e.g., approximately 1 x 10 9Cell doses ranging from 0.1 to 1.5 cells are typically injected. Upon administration of the cells to a subject and subsequent differentiation, immunoresponsive cells specifically directed against a specific antigen are induced. 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 in autoimmune diseases and the like. Genetically modified cells can be administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

[0225] In some embodiments, the methods of use include methods of inhibiting an immune response. Inhibiting an immune response can refer to preventing, attenuating, or inhibiting a cell-mediated immune response, such as that induced by a chimeric receptor expressed on the surface of an immunomodulatory cell. In embodiments, the methods include preventing, attenuating, or inhibiting activation of an activating chimeric receptor expressed on the surface of an immunomodulatory cell.

[0226] In some embodiments, the chimeric inhibitory receptors of the present disclosure are used to prevent, attenuate, inhibit, or suppress an immune response initiated by a tumor-targeting chimeric receptor (e.g., an activating CAR). For example, an immunomodulatory cell expresses an inhibitory chimeric antigen that recognizes antigen target 1 (e.g., a non-tumor antigen) and a tumor-targeting chimeric receptor that recognizes a different antigen target 2 (e.g., a tumor target). In this example, when the immunomodulatory cell contacts the target cell, the inhibitory chimeric receptor and the tumor-targeting chimeric receptor may or may not bind to their cognate antigens. In this example scenario, if the target cell is a non-tumor cell that expresses both antigen target 1 and antigen target 2, both the inhibitory chimeric receptor and the tumor-targeting receptor can be activated. In such a case, activation of the inhibitory chimeric receptor results in the prevention, attenuation, or inhibition of tumor-targeting chimeric receptor signaling, and the immunomodulatory cell is not activated. Similarly, in the exemplary case where the target cell is a non-tumor cell that expresses only antigen target 1, only the inhibitory chimeric receptor can be activated. In contrast, in the exemplary case where the target cell is a tumor cell that expresses only antigen target 2, the inhibitory chimeric receptor cannot be activated, whereas the tumor-targeting chimeric receptor can be activated, resulting in signaling that leads to the activation of immune regulatory cells.

[0227] Inhibition of an immune response initiated by a tumor-targeting chimeric receptor can be inhibition or reduction of activation of the tumor-targeting chimeric receptor, inhibition or reduction of signal transduction by the tumor-targeting chimeric receptor, or inhibition or reduction of activation of immunoregulatory cells. An inhibitory chimeric receptor can inhibit activation of the tumor-targeting chimeric receptor, signal transduction by the tumor-targeting chimeric receptor, or activation of immunoregulatory cells by the tumor-targeting chimeric receptor by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, compared to immunoregulatory cells lacking the inhibitory chimeric receptor. In some embodiments, inhibition refers to a decrease or reduction in the activity of the tumor-targeting chimeric receptor before or after the chimeric receptor is activated.

[0228] The immune response may be the production and secretion of cytokines or chemokines from activated immunoregulatory cells. The immune response may be a cell-mediated immune response against target cells.

[0229] In some embodiments, the chimeric inhibitory receptor can suppress cytokine production from activated immunomodulatory cells, hi some embodiments, the chimeric inhibitory receptor can suppress a cell-mediated immune response against a target cell, the immune response being induced by activation of the immunomodulatory cells.

[0230] Treatment In some embodiments, the disclosed methods increase an immune response in a subject in need thereof. In some embodiments, the disclosed methods include methods for treating and / or preventing bone marrow disorders in a subject. In some embodiments, the subject is human. In some embodiments, human subjects suitable for therapy may include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced 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 the percentage of tumor 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, pharmaceutical compositions of the present disclosure are administered to these subjects to elicit 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, although any clinical improvement would constitute a benefit. In some embodiments, clinical improvement includes a reduced risk or reduced rate of progression of the pathological consequences of the tumor. In some embodiments, a second group of suitable human subjects is the "adjuvant group" subjects. These subjects are individuals with a history of bone marrow disorder but who have responded to other treatments. 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 but are suspected of being at risk for disease progression near the original tumor site or through 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 disorders. Typical characteristics of the high-risk subgroup are tumor invasion into adjacent tissues or lymph node involvement.

[0231] In any and all aspects of increasing an 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 described herein.

[0232] Increasing an immune response can be both enhancing an immune response or inducing 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 increased immune response is an adaptive immune response. In some embodiments, the increased immune response is an innate immune response.

[0233] In some embodiments, a further group of subjects are those who have a genetic predisposition to bone marrow disorders but have not yet demonstrated clinical signs of bone marrow disorders. For example, women who have tested positive for a genetic mutation associated with CRC but are also 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 development of CRC until it is appropriate to perform prophylactic surgery. In some embodiments, subjects may have an advanced form of the disease, in which case the treatment goal may include alleviating or reversing disease progression and / or ameliorating side effects. In some embodiments, subjects may have a history of a condition for which they have already been treated, in which case the treatment goal may typically include reducing or delaying the risk of recurrence.

[0234] Combination therapy In some embodiments, genetically modified cells (e.g., immunoresponsive cells) of the present disclosure that express one or more proteins comprising an antigen binding domain (e.g., scFv) of the present disclosure, such as a chimeric receptor of the present disclosure, can be used in combination with other known drugs and therapies. In some embodiments, combination therapies of the present disclosure include genetically modified cells of the present disclosure that can be administered in combination with one or more additional therapeutic agents. In some embodiments, the genetically modified cells and one or more additional therapeutic agents can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the genetically modified cells can be administered first and the one or more additional agents can be administered second, or the order of administration can be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[0235] 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, mycophenolic acid, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH or anti-CD3 antibodies), cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, and peptide vaccines.

[0236] In some embodiments, the genetically modified cells of the present disclosure may be used in combination with a lymphodepleting agent. Suitable lymphodepleting agents reduce or deplete lymphocytes, e.g., B-cell lymphocytes and / or T-cell lymphocytes, prior to immunotherapy. Examples of suitable lymphodepleting agents include, but are not limited to, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[0237] 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, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors such as fludarabine), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulatory agents such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0238] Examples of common chemotherapeutic agents suitable for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), and ribosomal steroids (RIs). trademark), chlorambucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegen), daunorubicin hydrochloride (Cerubicin ine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluoxetine), rhodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idaniycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg,These include, but are not limited to, paclitaxel (Taxol®), phoenix (Yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 implant with carmustine (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0239] Examples of suitable alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®), Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Rev), immune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional exemplary alkylating agents include oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin-D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®), altretame (also known as hexamethylmelamine (HMMM), Hexalen®), carmustine (Bi CNU®), bendamustine (Treanda®), busulfan (Busulfex® and Myleran®), carboplatin (Paraplatin®), lomustine (also known as CCNU, CeeNU®), cisplatinomastrin (also known as CDDP, Platinol® and Platino®-AQ), chlorambucil (Leukeran®), cyclophosphamide (Cytoxan® and and Neosar®), Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®), Itretamine (also known as hexamethylmelamine (HMM), Hexalen®), Ifosfamide (Ifex®), Prednimustine, Procarbazine (Matulane®), Mechlorethamine (also known as nitrogen mustard, mustine, and mechloroethamine hydrochloride) Examples of antihistamines that may be used include, but are not limited to, Mustargen®, streptozocin (Zanosar®), thiotepa (also known as thiophosphamide, TESPA, and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine HCl (Treanda®).

[0240] Examples of suitable mTOR inhibitors include, but are not limited to, temsirolimus, ridaforolimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RADOOl), rapamycin (AY22989, Sirolmius®), and XL765.

[0241] 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.

[0242] Examples of suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin® and Rubex®), bleomycin (lenoxane®), daunorubicin (daunorubicin hydrochloride, daunomycem, 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 deacetirabidomycin.

[0243] Examples of suitable vinca alkaloids include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®), vinblastine (also known as vinblastine sulfate, vincaleucoblastine, and VLB, Alkaban-AQ® and Velban®), and vinorelbine (Navelbine®).

[0244] 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.

[0245] 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 fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Prol31921.

[0246] 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 cancer cell death or slow cancer cell growth. In some cases, the oncolytic virus has no effect or only minimal effect on non-cancerous 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.

[0247] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, such as an SHP-I 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.

[0248] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and at the tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the efficacy of chimeric receptor-expressing cell therapy. Without being bound by theory, it is believed that administration of an MDSC modulator enhances the efficacy of the genetically modified cells of the present disclosure. Examples of suitable modulators of MDSCs include, but are not limited to, MCS110 and BLZ945.

[0249] 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 interfere with 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.

[0250] In some embodiments, the genetically modified cells of this disclosure are administered to a subject in combination with an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide. In some embodiments, the genetically modified cells of this disclosure are further modified to express an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide.

[0251] In some embodiments, a subject with cancer (e.g., CRC) 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, a 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 comprising cytarabine and daunorubicin at a 5:1 molar ratio. In some embodiments, a 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, a subject is administered the genetically modified cells of the present disclosure in combination with a biologic 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, a subject is administered the genetically modified cells of the present 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), or ASP2215 (Astelias). In some embodiments, subjects are 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 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 present 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 Button tyrosine kinase (Btk), e.g., ibrutinib (Pharmacyclics / Johnson & Johnson Janssen Pharmaceutical). In some embodiments, subjects are administered genetically modified cells of the present 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 peptide-based CXCR4 inverse agonist, e.g., BL-8040 (BioLineRx).

[0252] In some embodiments, a subject can be administered an agent that enhances the activity or compatibility of the genetically modified cells of the present disclosure. For example, the agent can inhibit a molecule that regulates or controls, e.g., inhibits, T cell function. In some embodiments, the molecule that regulates or controls T 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 mount 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, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LIR-1 (LILRB1), 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 regulate or control, e.g., inhibit, T 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, an agent, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the genetically modified cell. In one embodiment, the inhibitor is an shRNA. In some embodiments, the genetically modified cells of the present disclosure can be further modified to express an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, 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.

[0253] In one embodiment, an agent that modulates or regulates (e.g., inhibits) T cell function is inhibited in the genetically modified cell of the present disclosure. In such an embodiment, a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is linked to a nucleic acid encoding a component of a chimeric receptor of the present disclosure, e.g., all of the components. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is operably linked to a promoter, e.g., an HI- or U6-derived promoter, such that the dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is expressed, e.g., expressed in the genetically modified cell. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is present on the same vector, e.g., a lentiviral vector, that includes nucleic acid molecules encoding a component of a chimeric receptor, e.g., all of the components. In such embodiments, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that regulates or controls (e.g., inhibits) T cell function is located on a vector, e.g., a lentiviral vector, 5' or 3' to the nucleic acid encoding a component of the chimeric receptor, e.g., all of the components. The nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that regulates or controls (e.g., inhibits) T cell function can be transcribed in the same or a different orientation as the nucleic acid encoding the component of the chimeric receptor, e.g., all of the components. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that regulates or controls (e.g., inhibits) T cell function is present on a vector other than the vector containing the nucleic acid molecules encoding the component of the chimeric receptor, e.g., all of the components. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that regulates or controls (e.g., inhibits) T cell function is transiently expressed in the genetically modified cell. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that regulates or controls (e.g., inhibits) T cell function is stably integrated into the genome of the genetically modified cell of the present disclosure.

[0254] In one embodiment, the agent that modulates or regulates, e.g., inhibits, T cell function can be an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-1, PD-L1, PD-L2, or CTLA4. In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

[0255] 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.

[0256] 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 immunoresponsive cell proliferation, differentiation, survival, and the homeostat. 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.

[0257] In some embodiments, the cytokine may be administered simultaneously or concurrently with the genetically modified cells, e.g., on the same day. The cytokine may be prepared in the same pharmaceutical composition as the genetically modified cells, or in a separate pharmaceutical composition. Alternatively, the cytokine may be administered shortly after administration of the genetically modified cells, e.g., 1, 2, 3, 4, 5, 6, or 7 days after administration of the genetically modified cells. In some embodiments in which the cytokine is administered in a dosing regimen that occurs over more than one day, day 1 of the cytokine dosing regimen can be the same day as administration with the genetically modified cells, or day 1 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 day 1, the genetically modified cells are administered to a subject, and on day 2, the cytokine is administered once daily for the next 7 days. In some embodiments, the cytokine is administered for a period of time after 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 1 year or more after administration of the genetically modified cells. In one embodiment, the cytokine is administered after assessment of the subject's response to the genetically modified cells.

[0258] kit Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of cancer (e.g., CRC) or other diseases (e.g., immune-related or autoimmune diseases). In certain embodiments, the kits comprise a therapeutic or prophylactic composition comprising an effective amount of one or more proteins, including an antigen-binding domain (e.g., scFv) of the present disclosure, such as a chimeric receptor of the present disclosure, an isolated nucleic acid of the present disclosure, a vector of the present disclosure, and / or a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, the kits comprise 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 can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding pharmaceutical products.

[0259] In some embodiments, the therapeutic or prophylactic composition is provided along with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., CRC). In some embodiments, the instructions may include information 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, administration schedules for the treatment or prevention of a disease or its symptoms, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical trials, and / or bibliographic references. In some embodiments, the instructions may be printed directly on the container (if present), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0260] Enumerated embodiments: Embodiment 1: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a. the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5; b. the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:8; c. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is humanized. Embodiment 2: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a. the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5; b. the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:8; c. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is humanized. Embodiment 3: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a. the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 4, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5; b. An antibody or antigen-binding fragment thereof, wherein the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 8. Embodiment 4: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. Embodiment 5: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21. Embodiment 6: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, 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) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16; An antibody or antigen-binding fragment thereof, comprising 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 VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21. Embodiment 7: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21. Embodiment 8: An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16. Embodiment 9: The antibody or antigen-binding fragment thereof of embodiment 6 or 8, wherein VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21. Embodiment 10: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment. Embodiment 11: The antibody or antigen-binding fragment thereof of embodiment 10, wherein the antigen-binding fragment comprises a F(ab) fragment, a F(ab') fragment, or a single-chain variable fragment (scFV). Embodiment 12: The antibody or antigen-binding fragment thereof of embodiment 11, wherein the antigen-binding fragment comprises a single-chain variable fragment (scFv). Embodiment 13: The antibody or antigen-binding fragment thereof of embodiment 12, wherein the VH and VL of the scFv are separated by a peptide linker. Embodiment 14: The antibody or antigen-binding fragment thereof of embodiment 13, 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. Embodiment 15: The antibody or antigen-binding fragment thereof of embodiment 13 or 14, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 37 and 100. Embodiment 16: The antibody or antigen-binding fragment thereof described in any one of embodiments 12 to 15, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99. Embodiment 17: A chimeric protein comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16 and a heterologous molecule or moiety. Embodiment 18: The chimeric protein of embodiment 17, wherein the chimeric protein is an antibody-drug conjugate and the heterologous molecule or moiety comprises a therapeutic agent. Embodiment 19: The chimeric protein of embodiment 17, 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. Embodiment 20: The chimeric protein of embodiment 19, wherein the CAR comprises a transmembrane domain. Embodiment 21: The chimeric protein of embodiment 19 or 20, wherein the CAR comprises one or more intracellular signaling domains. Embodiment 22: The chimeric protein of any one of embodiments 19 to 21, wherein the CAR is an activated CAR comprising one or more intracellular signaling domains that stimulate an immune response. Embodiment 23: A chimeric protein described in any one of embodiments 19 to 21, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. Embodiment 24: The chimeric protein of embodiment 23, wherein the intracellular inhibitory domain comprises an enzyme inhibitory domain. Embodiment 25: The chimeric protein of embodiment 23 or 24, wherein the intracellular inhibitory domain comprises an intracellular inhibitory co-signalling domain. Embodiment 26: The chimeric protein of any one of embodiments 19 to 25, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain. Embodiment 27: The chimeric protein of embodiment 26, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52. Embodiment 28: A composition comprising the antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 16, or the chimeric protein described in any one of embodiments 17 to 27, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. Embodiment 29: An engineered nucleic acid encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 16, or the chimeric protein of any one of embodiments 17 to 27. Embodiment 30: An expression vector comprising the engineered nucleic acid of embodiment 29. Embodiment 31: A composition comprising the engineered nucleic acid of embodiment 29 or the expression vector of embodiment 30, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. Embodiment 32: A method of producing an engineered cell, comprising transducing an isolated cell with the engineered nucleic acid of embodiment 29 or the expression vector of embodiment 30. Embodiment 33: An isolated cell comprising the engineered nucleic acid of embodiment 29, the expression vector of embodiment 30, or the composition of embodiment 31. Embodiment 34: A population of engineered cells expressing the engineered nucleic acid of embodiment 29 or the expression vector of embodiment 30. Embodiment 35: An isolated cell comprising the antigen-binding fragment of any one of embodiments 1 to 16 or the chimeric protein of any one of embodiments 17 to 27. Embodiment 36: A population of engineered cells expressing the antigen-binding fragment of any one of embodiments 1 to 16 or the chimeric protein of any one of embodiments 17 to 27. Embodiment 37: The cell or population of cells according to any one of embodiments 33 to 36, wherein the chimeric protein is recombinantly expressed. Embodiment 38: The cell or population of cells according to any one of embodiments 33 to 37, wherein the chimeric protein is expressed from a vector or a selected locus from the genome of the cell. Embodiment 39: The cell or population of cells according to any one of embodiments 33 to 38, wherein the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface. Embodiment 40: The cell or population of cells of embodiment 39, wherein each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor. Embodiment 41: The cell or population of cells according to any one of embodiments 33 to 40, 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, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. Embodiment 42: The cell or population of cells according to any one of embodiments 33 to 41, wherein the cell is autologous. Embodiment 43: The cell or population of cells according to any one of embodiments 33 to 41, wherein the cells are allogeneic. Embodiment 44: A pharmaceutical composition comprising an effective amount of the cells or population of engineered cells of any one of embodiments 33 to 43, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. Embodiment 45: A pharmaceutical composition comprising an effective amount of a genetically modified cell expressing the antigen-binding fragment of any one of embodiments 1 to 16 or the chimeric protein of any one of embodiments 17 to 27, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. Embodiment 46: A pharmaceutical composition according to embodiment 44 or 45 for treating and / or preventing tumors. Embodiment 47: A method of treating a subject in need thereof, comprising administering a therapeutically effective dose of a composition according to embodiment 28 or 31, or a cell according to any one of embodiments 33 to 43, or a pharmaceutical composition according to embodiment 44 or 45. Embodiment 48: A method for stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering a therapeutically effective dose of the composition of embodiment 28 or 31, or any of the cells of any one of embodiments 33 to 43, or the composition of embodiment 44 or 45 to a subject having a tumor. Embodiment 49: The method of embodiment 48, comprising administering to a subject any of the cells of any one of embodiments 33 to 43, wherein the isolated cell or population of cells expresses a chimeric protein comprising an activating CAR of embodiment 23. Embodiment 50: A method for inhibiting a cell-mediated immune response against tumor cells in a subject, the method comprising administering a therapeutically effective dose of the composition of embodiment 28 or 31, or any of the cells of any one of embodiments 33 to 43, or the composition of embodiment 44 or 45 to a subject having a tumor. Embodiment 51: The method of embodiment 50, comprising administering to a subject any of the cells described in any one of embodiments 33 to 43, wherein the isolated cell or population of cells expresses a chimeric protein comprising the inhibitory CAR described in embodiment 23. Embodiment 52: A method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of the composition of embodiment 28 or 31, or any of the cells of any one of embodiments 33 to 43, or the composition of embodiment 44 or 45. Embodiment 53: A kit for treating and / or preventing tumors, comprising a chimeric protein according to any one of embodiments 17 to 27. Embodiment 54: The kit of embodiment 53, wherein the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells to treat and / or prevent a tumor in a subject. Embodiment 55: A kit for treating and / or preventing tumors, comprising a cell or population of cells according to any one of embodiments 33 to 43. Embodiment 56: The kit of embodiment 55, wherein the kit further comprises written instructions for using the cells to treat and / or prevent a tumor in a subject. Embodiment 57: A kit for treating and / or preventing tumors, comprising an engineered nucleic acid according to embodiment 41. Embodiment 58: The kit of embodiment 57, wherein the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells to treat and / or prevent a tumor in a subject. Embodiment 59: A kit for treating and / or preventing tumors, comprising the vector of embodiment 30. Embodiment 60: The kit of embodiment 59, wherein the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells to treat and / or prevent a tumor in a subject. Embodiment 61: A kit for treating and / or preventing tumors, comprising a composition according to embodiment 28, embodiment 31, embodiment 44, or embodiment 45. Embodiment 62: The kit of embodiment 61, wherein the kit further comprises written instructions for using the composition to treat and / or prevent a tumor in a subject. [Example]

[0261] 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.

[0262] 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 have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0263] Example 1: Construction of a humanized anti-VSIG2 antigen-binding domain To generate humanized anti-VSIG2 antigen-binding domains (e.g., scFvs), the VH and VL sequences from human germline antibodies were first aligned to the murine anti-VSIG2 VH and VL sequences of SEQ ID NO: 63 and SEQ ID NO: 64, respectively. The murine anti-VSIG2 VH and VL sequences are shown in Table 5.

[0264] Table 5. Anti-VSIG2 VH and VL sequences from mouse TIFF2025538171000007.tif36163

[0265] Next, the framework regions surrounding the CDRs (as specified by the Kabat annotation and numbering scheme) were replaced with human germline antibody sequences. For the heavy chain human sequences, the variable heavy chains IGHV3-21 and IGHV3-11 were selected as framework regions with greater than 70% sequence identity to the mouse heavy chain sequence. For the light chain, the variable kappa chain IGKV1-5 was selected as framework regions with 65% sequence identity to the mouse light chain sequence. The germline sequences of IGHV3-21 and IGHV3-11, and IGKV1-5, from framework region 1 (FR1) to framework region 3 (FR3), are shown in Table 6. The heavy chain J segment IGHJ4*02 (WGQGTLVTVSS, SEQ ID NO: 101) and the light chain J segment IGKJ1*01 (FGQGTKVEIK, SEQ ID NO: 102) were also used as framework region 4 (FR4) following the respective CDR3 sequences.

[0266] Table 6: Germline sequences of IGHV3-21 and IGHV3-11, and IGKV1-5 TIFF2025538171000008.tif49164

[0267] Using Kabat annotations, the murine CDRs were grafted onto the human frameworks by replacing the human CDRs with the murine CDR sequences of both the heavy and light chains. By grafting the complete murine CDRs onto these human frameworks, this resulted in two different scFv humanized sequences: SB05441, which contains a Whitlow linker, and SB05443, which contains a GGGGS linker.

[0268] To further optimize the humanized scFvs and ensure proper scFv expression and binding, individual residues in the human framework regions were then mutated to their murine counterparts. Two Vernier residues were identified in the heavy chain framework region (H49 and H76), and three were similarly identified in light chain framework region 2 (L43, L48, L49). All combinations of fully human frameworks and grafted murine CDRs with these two or three mutations in the heavy and light frameworks were then combinatorially spliced ​​together (SB05442, SB05444, and SB05445-5448). Two different linkers were also used to connect the heavy and light chains of these humanized scFv designs (SB05441-SB05448, and SB05449-SB05456). The sequences are presented in Table 7.

[0269] Table 7. Humanized anti-VSIG2 antibody scFv sequences TIFF2025538171000009.tif234165TIFF2025538171000010.tif255165TIFF2025538171000011.tif255165TIFF2025538171000012.tif25516 5TIFF2025538171000013.tif253165TIFF2025538171000014.tif255164TIFF2025538171000015.tif255165TIFF2025538171000016.tif49165

[0270] Activating chimeric antigen receptors (aCARs) were engineered to contain a humanized anti-VSIG2 antigen-binding domain. The architecture of the produced humanized anti-VSIG2 aCARs is shown in Table 8. Each aCAR contained a CD8 signal sequence and a FLAG tag N-terminal to the scFV, a CD28 transmembrane (TM) domain, a CD28 intracellular domain (ICD), and a CD3zeta activation domain.

[0271] Table 8. CARs containing humanized anti-VSIG2 scFv TIFF2025538171000017.tif116142

[0272] Example 2: Evaluation of humanized anti-VSIG2 CAR expression The CAR construct shown in Example 1 was cloned into a retroviral vector. Additionally, as a control, an equivalent aCAR containing the antigen-binding domain of the parental murine anti-VSIG2 clone ("SB04744" or "SB04746") was also cloned into a retroviral vector. Primary NK cells from donors were expanded for 10 days, grown with K562 feeder cells in IL21 and IL15, and then transduced.

[0273] The aCAR constructs were evaluated for expression after transduction. As shown in Figure 1 (Day 3 "SB04744" parental), Figure 2 (Day 6 "SB04744" parental), and Figure 3 (Day 3 "SB04746" parental), cells expressed the humanized CAR with an MFI similar to the mouse parental sequence.

[0274] Example 3: Evaluation of humanized anti-VSIG2 function in NK cell killing assays A CAR cell killing assay was performed to evaluate whether the humanized anti-VSIG2 antigen-binding domain effectively targets the CAR to VSIG2-expressing cells. The function of the humanized anti-VSIG2 scFv was compared with that of an activated CAR with the parent mouse anti-VSIG2 antigen-binding domain. The CAR was cloned and transduced as described in Example 2. A co-culture killing assay was then performed. Target cells (LS174t mKate cell line or DLD-1 mKate cell line, each engineered to express VSIG2) were seeded into 96-well plates. Effector cells (NK cells expressing each construct) were added to the plate at a defined effector-to-target (E-to-T) cell ratio of 1:1, and the cells were co-cultured. Following co-culture, a real-time fluorescence-based assay to measure mKate levels was performed to evaluate target cell killing.

[0275] As shown in Figure 4 (LS174t target cells) and Figure 5 (DLD-1 target cells), aCARs containing each of the humanized anti-VSIG2 antigen-binding domains induced killing of VSIG2-expressing cell lines comparable to CARs containing the parental murine anti-VSIG2 antigen-binding domain. Figure 6 shows the time course for killing DLD-1 VISG+ target cells (right panel) and LS174t VISG+ target cells (left panel).

[0276] Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are incorporated herein 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.

[0277] equivalent While various specific embodiments have been illustrated 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 present disclosure. Many variations will be apparent to those skilled in the art upon consideration of this specification.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human V-set and immunoglobulin domain containing 2 (VSIG2), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the VH comprises a VH complementarity region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1 or 2, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 3 or 4, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 5; the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:6, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:7, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:8; Optionally, the antibody or antigen-binding fragment thereof is humanized. An antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 16.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

4. An isolated antibody or antigen-binding fragment thereof that specifically binds to human V set and immunoglobulin domain containing 2 (VSIG2), comprising a variable heavy (VH) region and a variable light (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

6. 6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment, optionally comprising an F(ab) fragment, an F(ab') fragment, or a single-chain variable fragment (scFv), optionally wherein the VH and VL of the scFv are separated by a peptide linker, and optionally wherein the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is the peptide linker, and VL is a light chain variable domain, and optionally wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-37 and 100.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99.

8. A chimeric protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 and a heterologous molecule or moiety, optionally wherein the chimeric protein is an antibody-drug conjugate, and the heterologous molecule or moiety comprises a therapeutic agent, and optionally 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, and optionally wherein the CAR comprises a transmembrane domain, and optionally wherein the CAR comprises one or more intracellular signaling domains. a chimeric protein comprising an antigen-binding domain and a transmembrane domain; optionally, the CAR is an activating CAR comprising one or more intracellular signaling domains that stimulate an immune response; optionally, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response; optionally, the intracellular inhibitory domain comprises an enzyme inhibitory domain; optionally, the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain; and optionally, the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52.

9. A composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, or the chimeric protein described in claim 8, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

10. An engineered nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 7, or the chimeric protein of claim 8.

11. 11. An expression vector comprising the engineered nucleic acid of claim 10.

12. 12. A composition comprising the engineered nucleic acid of claim 10 or the expression vector of claim 11, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

13. 12. A method of producing an engineered cell, comprising transducing an isolated cell with the engineered nucleic acid of claim 10 or the expression vector of claim 11.

14. 13. An isolated cell comprising the engineered nucleic acid of claim 10, the expression vector of claim 11, the composition of claim 12, the antigen-binding fragment of any one of claims 1 to 7, or the chimeric protein of claim 8.

15. A population of engineered cells that express the engineered nucleic acid of claim 10, the expression vector of claim 11, the antigen-binding fragment of any one of claims 1 to 7, or the chimeric protein of claim 8.

16. The chimeric protein is recombinantly expressed, optionally the chimeric protein is expressed from a vector or a selected locus from the genome of the cell, and optionally 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, and optionally 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), and the like. ), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TIL), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells, optionally wherein the cells are autologous or allogeneic.

17. 13. A pharmaceutical composition comprising an effective amount of the cell or population of engineered cells of claim 14, the genetically modified cell expressing the antigen-binding fragment of any one of claims 1 to 7, or the chimeric protein of claim 8, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, optionally for treating and / or preventing tumors.

18. A method for stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering a therapeutically effective dose of a composition described in claim 9 or 12, or any of the cells described in any one of claims 14, or the pharmaceutical composition described in claim 17 to a subject having a tumor.

19. 19. The method of claim 18, wherein the method comprises administering to the subject any of the cells of claim 14, wherein the isolated cell or population of cells expresses a chimeric protein comprising the activated CAR of claim 8.

20. 10. A kit for treating and / or preventing tumors comprising the chimeric protein, cell or population of cells, engineered nucleic acid, vector, or composition of claim 8, optionally further comprising written instructions for using the chimeric protein to produce one or more antigen-specific cells to treat and / or prevent tumors in a subject.

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