Antigen-binding domains and methods of use thereof

Abstract

Provided herein is an antibody specific to Endomucin (EMCN) and its antigen-binding fragment. Also provided herein are cells, nucleic acids, vectors, compositions, and methods related to the EMCN-specific antibody or its antigen-binding domain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 333,064, filed April 20, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy was 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, used to redirect the specificity and function of immune-responsive cells such as T cells, has shown efficacy in patients with lymphoid malignancies (Pule et al., Nat. Med. (14):1264-1270 (2008); Maude et al., N Engl J Med. (371):1507-17 (2014); Brentjens et al., Sci Transl Med. (5):177ra38 (2013)). CAR T cells have been shown to induce complete remissions in patients with CD19-expressing malignancies where chemotherapy has led to drug resistance and tumor progression. The success of CD19 CAR therapy provides optimism for treating other hematological malignancies such as acute myeloid leukemia (AML). Acute myeloid leukemia is the most common acute leukemia in adults. AML is a cancer of the blood cells of the myeloid system, characterized by the rapid proliferation of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cells. Sometimes AML can spread to the brain, skin, or gums. Standard chemotherapy treatments for AML have remained virtually unchanged over the past 40 years (Pulte et al., 2008), and overall survival rates remain very poor.

[0004] One challenge in developing CAR therapy for AML is the lack of suitable targets. The ability to identify appropriate CAR targets is important to effectively target and treat tumors without damaging normal cells that express the same target antigen. Thus, there remains a need for CAR-T cell-based AML therapies that target AML cells without targeting normal cells or tissues. Summary of the Invention

[0005] overview Provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), the isolated antibody or antigen-binding fragment thereof comprising: a variable heavy chain (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:6, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:8; a variable light chain (VL) region comprising a VL complementarity region L (CDRL1) having the amino acid sequence of SEQ ID NO:9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:11; wherein the antibody or antigen-binding fragment thereof is humanized.

[0006] The present specification also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human endomucin (EMCN), and is optionally humanized, comprising a variable heavy chain (VH) region comprising a VH complementarity region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 8, and a variable light chain (VL) region comprising a VL complementarity region L (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 11.

[0007] The present specification also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human endomucin (EMCN), comprising a variable heavy chain (VH) region including a VH complementarity region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 8, and a variable light chain (VL) region including a VL complementarity region L (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO: 11.

[0008] In some embodiments, CDRH1 is as set forth in SEQ ID NO:2.

[0009] In some embodiments, the VH region has an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0010] In some embodiments, the VH region has the amino acid sequence set forth in SEQ ID NO:12.

[0011] In some embodiments, the VH region has an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0012] In some embodiments, the VH region has the amino acid sequence set forth in SEQ ID NO:16.

[0013] In some embodiments, the VL has the amino acid sequence set forth in SEQ ID NO:20.

[0014] The present specification also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human endomucin (EMCN), 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 12 to 19 within the VH region, 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 amino acid sequence of SEQ ID NO: 20.

[0015] The present specification also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VL has the amino acid sequence set forth in SEQ ID NO:20.

[0016] The present specification also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0017] In some embodiments, the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the VH region has the amino acid sequence set forth in SEQ ID NO: 12.

[0018] In some embodiments, the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the VH region has the amino acid sequence set forth in SEQ ID NO: 16.

[0019] In some embodiments, the VL has the amino acid sequence set forth in SEQ ID NO:20.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises a F(ab) fragment, a F(ab') fragment, or a single chain variable fragment (scFv). In some embodiments, the antigen-binding fragment comprises a single chain variable fragment (scFv).

[0021] 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: 21-37.

[0022] In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 70, 72, and 74.

[0023] Also provided herein are chimeric proteins comprising an antibody, or antigen-binding fragment thereof, provided herein and a heterologous molecule or moiety.

[0024] In some embodiments, the chimeric protein is an antibody-drug conjugate and the heterologous molecule or moiety comprises a therapeutic agent.

[0025] 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.

[0026] In some embodiments, the CAR is an activated CAR that contains one or more intracellular signaling domains that stimulate an immune response.

[0027] In some embodiments, the CAR is an inhibitory CAR that comprises one or more intracellular inhibitory domains that inhibit immune responses. 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.

[0028] 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.

[0029] Also provided herein are compositions comprising an antibody or antigen-binding fragment thereof provided herein, or a chimeric protein provided herein, and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0030] Also provided herein are engineered nucleic acids encoding the antibodies or antigen-binding fragments provided herein, or the chimeric proteins provided herein.

[0031] Also provided herein is an expression vector comprising an engineered nucleic acid encoding any one of the antibodies or antigen-binding fragments thereof provided herein.

[0032] Also provided herein are compositions comprising an engineered nucleic acid provided herein, or an expression vector provided herein, and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0033] Also provided herein is a method of making an engineered cell, the method comprising transducing an isolated cell with an engineered nucleic acid provided herein or an expression vector provided herein.

[0034] Also provided herein is an isolated cell comprising an engineered nucleic acid provided herein, an expression vector provided herein, or a composition provided herein.

[0035] Also provided herein are populations of engineered cells that express the engineered nucleic acids provided herein, the expression vectors provided herein.

[0036] Also provided herein is an isolated cell comprising an antigen-binding fragment provided herein, or a chimeric protein provided herein.

[0037] Also provided herein are populations of engineered cells that express an antigen-binding fragment provided herein, or a chimeric protein provided herein.

[0038] In some aspects, the chimeric protein is recombinantly expressed by a cell or population of cells, hi some aspects, the chimeric protein is expressed from a vector or from a locus selected from the genome of the cell.

[0039] 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.

[0040] In some embodiments, the cell or population of cells is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0041] In some aspects, the cells are autologous. In some aspects, the cells are allogeneic.

[0042] Also provided herein are pharmaceutical compositions comprising an effective amount of the cells or engineered cell populations provided herein and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0043] Also provided herein is a pharmaceutical composition comprising an effective amount of a genetically modified cell expressing an antigen-binding fragment provided herein or a chimeric protein provided herein, and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0044] In some embodiments, the pharmaceutical composition is for treating and / or preventing a tumor.

[0045] Also provided herein are methods of treating a subject in need thereof, comprising administering a therapeutically effective dose of any of the compositions provided herein, or cells provided herein.

[0046] Also provided herein is a method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective dose of any of the compositions provided herein, or cells provided herein.

[0047] In some embodiments, the methods include administering to a subject a cell or population of cells provided herein, wherein the cell or population of cells expresses a chimeric protein comprising an activated CAR provided herein.

[0048] Also provided herein is a method of inhibiting a cell-mediated immune response to tumor cells in a subject, comprising administering to a tumor-bearing subject a therapeutically effective dose of any of the compositions provided herein, or cells provided herein. In some aspects, the method comprises administering to the subject a cell or population of cells provided herein, wherein the cell or population of cells expresses an inhibitory chimeric protein provided herein.

[0049] Also provided herein are methods of treating a subject having a tumor, comprising administering a therapeutically effective dose of any of the compositions provided herein, or cells provided herein.

[0050] Also provided herein is a kit for treating and / or preventing tumors, comprising the chimeric protein provided herein.

[0051] In some embodiments, the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0052] Also provided herein is a kit for treating and / or preventing tumors, comprising the cells or populations of cells provided herein. In some embodiments, the kit further comprises written instructions for using the cells to treat and / or prevent tumors in a subject.

[0053] Also provided herein is a kit for treating and / or preventing a tumor, comprising the engineered nucleic acid provided herein. In some embodiments, the kit further comprises written instructions for using the nucleic acid to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0054] Also provided herein is a kit for treating and / or preventing tumors, comprising the vector provided herein.In some embodiments, the kit further comprises a written instruction manual for using the vector to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0055] Also provided herein is a kit for treating and / or preventing tumors, comprising the composition provided herein. In some embodiments, the kit further comprises a written instruction manual for using the composition to treat and / or prevent tumors in a subject. [Brief description of the drawings]

[0056] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0058] [Figure 1] FIG. 1 shows the killing activity of T cells expressing a humanized anti-EMCN CAR of the present disclosure against EMCN-expressing target cells. [Diagram 2] FIG. 2 shows CAR expression of NK cells transduced with various anti-EMCN CARs. [Diagram 3] FIG. 3 shows the killing activity of NK cells transduced with various anti-EMCN CARs against EMCN-expressing target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Detailed Description The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SL Tan ed., Taylor & Francis, 2006); Fundamental Virology, 3 rd Edition, vol. I & II (BN Fields and DM Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (3 rdEdition, 2001); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0060] definition Unless otherwise defined, all terms, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference to what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well known and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and conditions defined by the manufacturer, unless otherwise stated.

[0061] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Terms such as "including," "etc," and the like are intended to convey inclusion without limitation unless otherwise indicated.

[0062] As used herein, the term "comprising" also specifically includes embodiments "consisting of" and "consisting essentially of" the listed elements, unless otherwise indicated.

[0063] The term "about" refers to and includes the indicated value and the 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.

[0064] As used herein, the term "stimulating a cell-mediated immune response" or "stimulating an immune response" refers to the generation of a signal by one or more cell types or cell populations that results in an immune response. Immunostimulatory activity may include proinflammatory activity. In various embodiments, the immune response occurs after activation of immune cells (e.g., T cells or NK cells) or is simultaneously mediated through receptors, including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides may be present in the tumor microenvironment and may activate an immune response against tumor cells. In various embodiments, promoting, stimulating, or ligand-stimulating receptors of proinflammatory polypeptides and / or their ligands may enhance the immune response of immunoresponsive cells. Without being bound by any particular theory, receiving multiple stimulatory signals (e.g., costimulation) is important to support robust, long-lasting cell-mediated immune responses, such as T cell-mediated immune responses in which T cells are inhibited and may not respond to antigens in the absence of costimulatory signals (also referred to as "T cell anergy"). Although the various effects of costimulatory signals vary and are only partially understood, especially in combination with one another, costimulation generally results in increased gene expression to generate long-lived, proliferative, and apoptosis-resistant cells, such as T cells or NK cells, that respond potently to antigens, for example, in mediating complete and / or sustained elimination of target cells expressing the cognate antigen.

[0065] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain that includes a functional signaling domain (also referred to herein as an "intracellular signaling domain").

[0066] 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.

[0067] As used herein, the term "inhibitory CAR" or "iCAR" refers to a CAR construct / structure capable of inducing a change in signal transduction or protein expression in an inhibitory CAR-expressing cell that inhibits, attenuates, reduces, diminishes, suppresses, or inhibits an immune response upon a cognate iCAR ligand, such as reducing activation of an immunoresponsive cell that is receiving or has received one or more stimulatory signals, including costimulatory signals.

[0068] As used herein, the term "intracellular signaling domain" refers to a functional portion of a protein that acts by transmitting information intracellularly to regulate cellular activity via a defined signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0069] As used herein, the term "extracellular antigen-binding domain" or "antigen-binding domain" (ABD) refers to a polypeptide sequence or polypeptide complex that specifically recognizes or binds a given antigen or epitope, such as the polypeptide sequence or polypeptide complex portion of the chimeric proteins described herein that result in EMCN-specific binding. The ABD (or antibody, antigen-binding fragment, and / or chimeric protein containing it) is said to "recognize" the epitope (or more generally, the antigen) to which the ABD specifically binds, and the epitope is said to be the "recognition specificity" or "binding specificity" of the ABD. The ABD is said to bind its specific antigen or epitope with a particular affinity. As described herein, "affinity" refers to the strength of interaction of non-covalent intermolecular forces between one molecule and another molecule. Affinity, i.e., the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), with a lower KD value indicating a stronger interaction between the molecules. The KD values ​​of antibody constructs are measured by methods well known in the art, including, but not limited to, biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays (e.g., flow cytometry). Specific binding, as assessed by affinity, may refer to a binding molecule that has affinity between the ABD and its cognate antigen or epitope, and the KD value is greater than or equal to 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. Specific binding can also include recognition and binding of a biological molecule (e.g., a polypeptide) of interest, while not specifically recognizing and binding to other molecules in a sample, such as a biological sample that naturally contains the polypeptide of the present disclosure. In certain embodiments, specifically binding refers to the binding of an epitope or antigen or antigenic determinant of an ABD, antibody, or antigen-binding fragment in such a way that the binding can be displaced or competed with a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0070] The ABD may be an antibody. The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0071] ABD can 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 (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, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, 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. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0072] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the "valency" of the binding molecule. A binding molecule with a single ABD is "monovalent". A binding molecule with multiple ABDs is said to be "multivalent". A multivalent binding molecule with two ABDs is "bivalent". A multivalent binding molecule with three ABDs is "trivalent". A multivalent binding molecule with four ABDs is "tetravalent". In various multivalent embodiments, all of the multiple ABDs have the same recognition specificity and can be referred to as a "monospecific multivalent" binding molecule. In other multivalent embodiments, at least two of the multiple ABDs have different recognition specificities. Such a binding molecule is multivalent and "multispecific". In multivalent embodiments where the ABDs collectively have two recognition specificities, the binding molecule is "bispecific". In multivalent embodiments where the ABDs collectively have three recognition specificities, the binding molecule is "trispecific". In multivalent embodiments where the ABDs collectively have multiple recognition specificities for different epitopes present on the same antigen, the binding molecule is "multiparatopic." Multivalent embodiments where the ABDs collectively recognize two epitopes on the same antigen are "dual paratopic."

[0073] In various multivalent embodiments, the multivalent binding molecule improves the avidity of the binding molecule to a specific target. As described herein, "avidity" refers to the overall strength of the interaction between two or more molecules, e.g., a multivalent binding molecule to a specific target, and avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs. Avidity can be measured by the same methods used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule to a specific target is measured by measuring the strength of the interaction between two molecules, where 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 specific target has a KD value such that the interaction is a specific binding interaction, and the affinity of one or more of the individual ABDs does not have a KD value that recognizes the specific binding to their own respective antigen or epitope.In certain embodiments, the avidity is the cumulative strength of the interaction caused by the affinity of multiple ABDs for separate antigens on a shared specific target or complex, such as separate antigens found on individual cells.In certain embodiments, the avidity is the cumulative strength of the interaction caused by the affinity of multiple ABDs for separate epitopes on a shared individual antigen.

[0074] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising at least one antigen-binding fragment comprising a variable region of a light chain and at least one antigen-binding fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are linked via a short flexible polypeptide linker and capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless specified, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and the scFv can comprise a VL-linker-VH or a VH-linker-VL.

[0075] As used herein, "variable region" refers to the variable region resulting from a recombination event, for example, after V, J, and / or D segment recombination in immunoglobulin genes in B cells or T cell receptor (TCR) genes in T cells. In immunoglobulin genes, variable regions are typically defined from the antibody chain from which they are derived, e.g., VH refers to the variable region of an antibody heavy chain and VL refers to the variable region of an antibody light chain. A selected VH and a selected VL can combine together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0076] The term "complementarity determining region" or "CDR" as used herein 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 there are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al, (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acid residues of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of a VH, e.g., a mammalian VH, e.g., a human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of a VL, e.g., a mammalian VL, e.g., a human VL. In various embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences.

[0077] As used herein, the term "framework region" or "FR" refers to the generally conserved sequences in antibody variable regions VH and VL that typically serve as scaffolds for interspersed CDRs, in the following configuration (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In various embodiments, the FRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In certain embodiments, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthetic sequences, including but not limited to rationally designed sequences.

[0078] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their native conformations and which usually determine the class to which the antibody belongs.

[0079] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their native conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0080] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0081] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response can involve either antibody production, or activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen.

[0082] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life span, a reduction in tumor cell proliferation, a reduction in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure to prevent the development of tumors in the first place, such as prophylactic therapy or treatment.

[0083] As used herein, the term "autologous" refers to any material derived from the same subject that is subsequently reintroduced into the subject.

[0084] 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 from individuals of the same species may be sufficiently genetically different to interact antigenically at certain genes, such as MHC alleles. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically identical to not interact antigenically at certain genes, such as MHC alleles.

[0085] The isolated nucleic acid molecules of the present disclosure include any nucleic acid molecule that encodes a polypeptide or fragment thereof of the present disclosure. Such nucleic acid molecules do not need to 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 can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to pairing to form a double-stranded molecule between a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof under various stringency conditions. For example, stringent salt concentrations can usually be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. In the absence of organic solvent, such as formamide, low stringency hybridization can be obtained, while in the presence of at least about 35% formamide or at least about 50% formamide, high stringency hybridization can be obtained.Stringent temperature conditions usually include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency can be achieved by combining these various conditions as needed.

[0086] "Substantially identical" or "substantially homologous" means that a polypeptide or nucleic acid molecule exhibits at least 50% homology or identity with a reference amino acid sequence (e.g., any 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 sequences are at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical at the amino acid level or at the nucleic acid level to the sequence used for comparison. Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. An exemplary method for measuring the degree of identity may use the BLAST program, where a probability score of e-3 to e-100 indicates closely related sequences.

[0087] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, or a biological property resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that the nucleotide sequence encoding the protein may include introns in some versions.

[0088] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, allowing for intercellular recognition and / or interaction.

[0089] 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.

[0090] 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.

[0091] As used herein, the term "immune effector response" or "immune effector function" refers to, for example, the function or response of an immunoresponsive cell that enhances or promotes the immune attack of a target cell. For example, immune effector function or response can refer to the property of T cells or NK cells that promotes the killing or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0092] As used herein, the term "flexible polypeptide linker" or "linker" refers to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n or (Gly-Gly-Gly-Ser)n, where n is a positive integer greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10. In some embodiments, the flexible polypeptide linker comprises, but is not limited to, Gly4Ser or (Gly4Ser)3. In other embodiments, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). In some embodiments, the flexible polypeptide linker comprises a Whitlow linker (e.g., GSTSGSGKPGSGEGSTKG [SEQ ID NO: 36]). Also included within the scope of this disclosure are linkers described, for example, in WO2012 / 138475.

[0093] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or alleviation of the progression, severity, and / or duration of a proliferative disorder (e.g., cancer), or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the present disclosure). In some embodiments, the reduction or amelioration refers to the improvement of at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to the inhibition of progression of the proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In some embodiments, the reduction or amelioration includes the reduction or stabilization of tumor size or cancer cell number.

[0094] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0095] Other aspects of the disclosure are described in the following sections and are within the scope of the claims.

[0096] Other Rules of Interpretation Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0097] Unless otherwise indicated, reference to a compound having one or more stereocenters contemplates each stereoisomer and all combinations of stereoisomers.

[0098] Endomucin specific antigen-binding domain The present disclosure provides an antigen-binding domain (e.g., single-chain variable fragment) that binds to endomucin (EMCN), a chimeric protein that includes an antigen-binding domain that binds to EMCN, and a nucleic acid encoding such an antigen-binding domain and chimeric protein. Without wishing to be bound by theory, EMCN is a sialoglycoprotein that prevents the assembly of focal adhesion complexes and inhibits the interaction between cells and the extracellular matrix. The EMCN-specific antigen-binding domain binds to human EMCN (e.g., Uniprot Q9ULC0, incorporated herein by reference for all purposes) or an epitope fragment thereof. EMCN may be expressed in hematopoietic stem and progenitor cells (HSPCs). EMCN may be expressed in cells that are generally considered healthy, such as healthy HSPCs. EMCN-specific antibodies have been previously described, including CBFYE-0213, V.7.C7.1, L4B1, L5F12, L10B5, L3F12, L6H3, L6H10 (also referred to herein as Ab1), L9H8, and L10F12, as described in Samulowitz U. et al., Am. J. Path., 2002 May, 160(5):1669-1681, which is incorporated herein by reference for all purposes.

[0099] The present disclosure provides an EMCN-specific antigen-binding domain comprising one or more of the amino acid sequences listed in Table A.

[0100] [Table 1] TIFF2025513284000003.tif146163

[0101] In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, the VH comprising a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 8). In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, the VH comprising a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence selected from the group consisting of SEQ ID NOs: 12-20. In some embodiments, the VH region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-15. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19. In some embodiments, the VH region has the amino acid sequence of SEQ ID NO: 16. In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, an EMCN-specific antigen-binding domain having the above VH sequence can have a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained in the VL region amino acid sequence of SEQ ID NO: 20. In some embodiments, an EMCN-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:9, a heavy chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:10, and a heavy chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:11.

[0102] In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 20. In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the EMCN-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) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in a VH region amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 20. In some embodiments, the EMCN-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 WGNGN SEQ ID NO: 7, and heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, an EMCN-specific antigen binding domain having the above VL sequence can have a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO:3, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of WGNGN SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO:8.In some embodiments, an EMCN-specific antigen binding domain having the above VL sequence can have a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8. In some embodiments, an EMCN-specific antigen binding domain having the above VL sequence can have a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8.

[0103] In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO:2, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO:8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:11. In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO:3, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO:8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO:9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:11. In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO:4, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO:8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO:9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:11.In some embodiments, an EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where (1) the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO:5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO:8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO:9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO:10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO:11.

[0104] In some embodiments, an EMCN-specific antigen-binding domain comprises a variable heavy chain (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:6, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:8; and a variable light chain (VL) region comprising a VL complementarity region L (CDRL1) having the amino acid sequence of SEQ ID NO:9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:11, wherein the antigen-binding domain is humanized.

[0105] In some embodiments, an EMCN-specific antigen-binding domain has a VH region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, an EMCN-specific antigen-binding domain has a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:12.

[0106] In some embodiments, an EMCN-specific antigen-binding domain has a VH region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an EMCN-specific antigen-binding domain has a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:16.

[0107] In some embodiments, an EMCN-specific antigen binding domain has a VL region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an EMCN-specific antigen binding domain has a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20.

[0108] In some embodiments, an EMCN-specific antigen-binding domain has (1) a VH region comprising the amino acid sequence of SEQ ID NO:12, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20, or a VL region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, an EMCN-specific antigen-binding domain has (1) a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:12, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20 or a VL region comprising the amino acid sequence of SEQ ID NO:20.

[0109] In some embodiments, an EMCN-specific antigen-binding domain has (1) a VH region comprising the amino acid sequence of SEQ ID NO:16, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20, or a VL region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, an EMCN-specific antigen-binding domain has (1) a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:16, and (2) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20 or a VL region comprising the amino acid sequence of SEQ ID NO:20.

[0110] In some embodiments, an EMCN-specific antigen-binding domain has (1) a VL region comprising the amino acid sequence of SEQ ID NO:20, and (2) a VH region comprising the amino acid sequence of SEQ ID NO:12, or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, an EMCN-specific antigen-binding domain has (1) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20, and (2) a VH region comprising an amino acid sequence of SEQ ID NO:12 or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:12.

[0111] In some embodiments, an EMCN-specific antigen-binding domain has (1) a VL region comprising the amino acid sequence of SEQ ID NO:20, and (2) a VH region comprising the amino acid sequence of SEQ ID NO:16, or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:16. In some embodiments, an EMCN-specific antigen-binding domain has (1) a VL region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:20, and (2) a VH region comprising an amino acid sequence of SEQ ID NO:16 or a VH region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:16.

[0112] EMCN-specific antigen-binding domains are Fab, Fab', and F(ab') 2 The EMCN-specific antigen-binding domain can be in any of the formats described herein, such as Fv, scFv, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH, and multispecific formats. In some embodiments, the EMCN-specific antigen-binding domain is in F(ab) format. In some embodiments, the EMCN-specific antigen-binding domain is in F(ab') format.

[0113] In some embodiments, the EMCN-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 EMCN-specific antigen-binding domain has the structure VH-L-VL or VL-L-VH, where L is a peptide linker.

[0114] In some embodiments, the scFV has an amino acid sequence selected from SEQ ID NOs: 68, 70, 72, and 74.

[0115] In some embodiments, the EMCN-specific antigen-binding domain is humanized.

[0116] The present disclosure also provides an EMCN-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, (1) the VH comprising a heavy chain complementarity determining region 1 (CDR-H1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8, and (2) the VL comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 11.

[0117] The present disclosure also provides an EMCN-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, (1) the VH comprising a heavy chain complementarity determining region 1 (CDR-H1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8, and (2) the VL comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 11.

[0118] In some embodiments, an EMCN-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a distinct human EMCN 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 11. In some embodiments, an EMCN-specific antigen-binding domain binds to the same or essentially the same epitope (e.g., a distinct human EMCN 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 8, and (2) the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 10, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the EMCN-specific antigen-binding domain binds to the same or substantially the same epitope (e.g., a distinct human EMCN 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: 12-19. In some embodiments, the EMCN-specific antigen-binding domain binds to the same or substantially the same epitope (e.g., a distinct human EMCN epitope) as a reference antibody, or antigen-binding fragment thereof, having a VL comprising the amino acid sequence of SEQ ID NO: 20.

[0119] The present disclosure also provides chimeric proteins and nucleic acids encoding such chimeric proteins, including an EMCN-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 EMCN-specific antigen-binding domains.

[0120] Chimeric antigen receptors (CARs) Certain aspects of the present disclosure relate to a chimeric receptor having any one of the EMCN-specific antigen binding domains described herein and capable of specifically binding to an EMCN protein, an EMCN-derived antigen, or an EMCN-derived epitope. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, a CAR is a chimeric protein that includes an antigen binding domain and a polypeptide molecule that is heterologous to the antigen binding domain, such as a peptide that is heterologous to the antibody from which the antigen binding domain can be derived. The polypeptide molecule that is heterologous to the antigen binding domain includes, but is not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or a combination thereof.

[0121] In some embodiments, CARs are engineered receptors that transfer or confer specificity of interest (e.g., EMCN) to immune effector cells. In certain embodiments, CARs can be used to transfer antibody specificity to immune responsive cells such as T cells. In some embodiments, the CARs of the present disclosure comprise an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0122] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, and also commonly referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of an immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of an immunoresponsive cell. In some embodiments, activation of the immunoresponsive cell results in killing of the target cell. In some embodiments, activation of the immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of the immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of the immunoresponsive cell can be a combination of the above responses.

[0123] The CAR of the present disclosure can be a first, second, or third generation CAR. A "first generation" CAR contains a single intracellular signaling domain, generally derived from a T cell receptor chain. A "first generation" CAR generally has an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the main transmitter of signals from endogenous TCR. A "first generation" CAR provides de novo antigen recognition and transduces CD4 T cells via the CD3ζ chain signaling domain in a single fusion molecule, independent of antigen presentation via HLA. + and CD8 +"Second generation" CARs add a second intracellular signaling domain from one of a variety of costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide an additional signal to the T cell. "Second generation" CARs provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3 zeta). In preclinical studies, it has been shown that "second generation" CARs can enhance the antitumor activity of immune responsive cells such as T cells. "Third generation" CARs have multiple intracellular costimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3 zeta).

[0124] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, 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, bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0125] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, for example, as a non-logical gate to control, regulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. For example, if a healthy cell expresses both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by an inhibitory chimeric receptor, the immunoresponsive cell expressing the tumor antigen may bind to the healthy cell. In such a case, the inhibitory chimeric antigen also binds to its cognate ligand on the healthy cell, and the inhibitory function of the inhibitory chimeric receptor reduces, reduces, prevents, or inhibits the activation of the immunoresponsive cell via the tumor-targeting chimeric receptor ("non-logical gating"). In some embodiments, the inhibitory chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, the immunoresponsive cells may contain one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors that target antigens that are not expressed or generally not considered to be expressed on tumors (e.g., EMCN). A combination of tumor-targeting chimeric receptors and inhibitory chimeric receptors on the same immunoresponsive cells can be used to reduce extratumoral toxicity on the target.

[0126] In some embodiments, the extracellular antigen binding domain of a CAR of the disclosure comprises about 2×10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 M or less, approximately 2×10 -9 M or less, or about 1 x 10 -9 The dissociation constant (K d ) binds to one or more antigens (e.g., EMCN). In some embodiments, Kd is about 2 x 10 -7 M ~ approx. 1×10 -9 The range is M.

[0127] Binding of the extracellular antigen-binding domain of the CAR of the present disclosure can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeling reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFvs can be radioactively labeled and used in RIA assays. Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific for the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., with a radioactive or fluorescent marker).

[0128] In some embodiments, a CAR of the present disclosure comprises an extracellular antigen binding domain that binds to EMCN (e.g., an EMCN protein, an EMCN-derived antigen, or an EMCN-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 can be crosslinked. In certain embodiments, the extracellular binding domain comprises an F(ab) 2 It is a fragment.

[0129] Extracellular antigen-binding domain The extracellular antigen binding domain of the CAR of the present disclosure specifically binds to EMCN (e.g., an EMCN protein, an EMCN-derived antigen, or an EMCN-derived epitope). In certain embodiments, the extracellular antigen binding domain binds to EMCN expressed on hematopoietic stem cells. In certain embodiments, the extracellular antigen binding domain binds to EMCN expressed on cells that are generally considered healthy, such as healthy HSCP. In some embodiments, the EMCN is human EMCN.

[0130] Antigen binding domains of the present disclosure can include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single domain antibodies (sdAbs), such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), as well as any domain associated with an alternative scaffold known in the art to function as an antigen binding domain, such as recombinant fibronectin domains, T cell receptors (TCR), recombinant affinity-improved TCRs, or fragments thereof, e.g., single chain TCRs. In some cases, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used.

[0131] 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.

[0132] 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.

[0133] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind 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).

[0134] 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 embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked to each other, each scFv can be linked to the next scFv to which a peptide is linked. In some embodiments, each of the one or more scFvs is separated by a peptide linker.

[0135] [Table 2]

[0136] 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 suitable antigen-binding domains described herein or known in the art. For example, the first or second antigen-binding domain can be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFvs), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first chimeric receptor can be specific for EMCN, and the chimeric receptor can be specific for a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on myeloid cells, such as AML cells).

[0137] 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.

[0138] In some embodiments, the 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 different epitopes of the same antigen. In some embodiments, each of the two or more antigen binding domains binds to different antigens.

[0139] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains can be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, an sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-improved recombinant TCR, and a single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0140] In certain embodiments, the bispecific CAR or tanCAR comprises an antigen-binding domain that comprises a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is located upstream of its VL (VL 1 ) upstream of its VH (VH 1 ), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH 2 ) upstream of that VL(VL 2 ) so that the overall bispecific antibody molecule has the arrangement VH 1 -VL 1 -VL 2 -VH 2In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VH (VH 1 ) upstream of that VL(VL 1 ) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL 2 ) upstream of its VH (VH 2 ) and the overall bispecific antibody molecule is arranged in the arrangement VL 1 VH 1 -VH 2 -VL 2 In some embodiments, a linker is provided between two antibodies or antibody fragments (e.g., scFv), for example, when the construct has a VH 1 -VL 1 -VL 2 -VH 2 If placed as VL 1 and V.L. 2 Between or between the construct VL 1 -VH 1 -VH 2 -VL 2 If placed as VH 1 and V.H. 2 The linker may be any of the linkers described herein, e.g., (Gly 4 -Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. In general, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is placed between the VL and VH of the first scFv. In some embodiments, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR comprises a VL, a VH, and may further comprise one or more linkers in the arrangement described herein.

[0141] In some embodiments, the chimeric receptor comprises a bivalent CAR. In some embodiments, the bivalent CAR is an EMCN bivalent CAR. In some embodiments, the bivalent EMCN CAR comprises one or more of the anti-EMCN sequences shown in Table 1. In some embodiments, each of the ABDs of the bivalent EMCN CAR comprises the same ABD.

[0142] In some embodiments, the chimeric receptor comprises a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises an EMCN CAR. In some embodiments, the bicistronic EMCN CAR comprises one or more of the anti-EMCN sequences shown in Table 1.

[0143] Transmembrane domain In some embodiments, the transmembrane domain of the CAR of the present disclosure (e.g., the EMCN-specific CAR described herein) comprises a hydrophobic alpha helix that spans at least a portion of the cell membrane. It has been shown that different transmembrane domains can result in different receptor stabilities. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR of the present disclosure can comprise a transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0144] In some embodiments, the transmembrane domain is from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide can be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is from an OX40 polypeptide. Any suitable OX40 polypeptide can be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is from an ICOS polypeptide. Any suitable ICOS polypeptide can be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508.In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Exemplary BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0145] 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_00500 9, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a polypeptide comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a fragment thereof. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can include one conservative amino acid substitution, no more than two conservative amino acid substitutions, or no more than three conservative amino acid substitutions.In some embodiments, the polypeptide is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length (NCBI reference numbers NP_001139345, AAA92533.1, NP_006130 ... 31668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0146] Further examples of suitable polypeptides from which a transmembrane domain may be derived include T cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and the transmembrane domains of the alpha, beta, or zeta chains of NG2C, but are not limited to these.

[0147] 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).

[0148] Spacer Region In some embodiments, the CAR of the present disclosure (e.g., the EMCN-specific CAR described herein) can also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region can be a hinge derived from a human protein. For example, the hinge can be a human Ig (immunoglobulin) hinge, including but not limited to an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region can include an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some aspects, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may comprise any of the amino acid sequences listed in Table 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.

[0149] [Table 3]

[0150] [Table 4]

[0151] 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.

[0152] In some embodiments, the CAR of the present disclosure may further comprise a short oligopeptide or polypeptide linker that is 2 to 10 amino acid residues in length and can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine-serine duplex. In some embodiments, the linker comprises the amino acid sequence of GGCKJSGGCKJS (SEQ ID NO: 62).

[0153] In some aspects, the transmembrane domain further comprises at least a portion of the extracellular domain of the same protein.

[0154] Intracellular signaling domains In some embodiments, a CAR of the present disclosure (e.g., an EMCN-specific CAR described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of a CAR may comprise an intracellular signaling domain.

[0155] Examples of suitable intracellular signaling domains that may be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act coordinately to regulate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0156] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of T cells, and thus secondary and / or costimulatory signals are typically also required for full activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). In addition, T cell signaling and function (e.g., activation signaling cascades) can be negatively controlled by inhibitory receptors present in T cells via intracellular inhibitory co-signaling domains.

[0157] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can include 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 includes one or more intracellular inhibitory co-signaling domains. In some embodiments, one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., transmembrane domains) via a peptide linker (see, e.g., Table 2) or a spacer or hinge sequence (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 via a peptide linker (see, e.g., Table 2) or a spacer or hinge sequence (see, e.g., Table 3). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, one or more intracellular inhibitory co-signaling domains of the chimeric protein comprise one or more ITIM-containing proteins, or fragments thereof. ITIMs are conserved amino acid sequences found in the cytoplasmic tail 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 scaffold proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, GITR, and PD-L1. The inhibitory intracellular signaling domain can further comprise an enzyme inhibitory domain. In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain. In some embodiments, the enzyme catalytic domain is derived from an enzyme 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.

[0158] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can include a primary signaling domain that controls the primary activation of the TCR complex, either in a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can include signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in the CAR of the present disclosure include, but are not limited to, those of CD3-zeta, FcR gamma, FcR beta beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0159] In some embodiments, a CAR of the present disclosure (e.g., an EMCN-specific CAR described herein) comprises an intracellular signaling domain, e.g., a primary signaling domain of a CD3-zeta polypeptide. A CD3-zeta polypeptide of the present disclosure may have an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI reference number NP_932170 or NP_001106864.2. In some embodiments, a CD3-zeta polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, a polypeptide can have an amino acid sequence that is a contiguous portion of NCBI reference number NP_932170 or NP_001106864.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids in length.

[0160] 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.

[0161] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure can comprise a CD3-zeta signaling domain by itself, or it can be combined with any other desired intracellular signaling domain useful in the context of the CAR of the present disclosure.For example, the intracellular signaling domain of the CAR can comprise a portion of the CD3-zeta chain and a costimulatory signaling domain.A costimulatory signaling domain can refer to a portion of the CAR that comprises the intracellular domain of a costimulatory molecule.A costimulatory molecule of the present disclosure is a cell surface molecule other than an antigen receptor or its ligand that can be required for the efficient response of lymphocytes to antigens.Examples of suitable costimulatory molecules include CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor. , CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and the like.

[0162] In some embodiments, the intracellular signaling sequences within the cytoplasmic portion of the CAR of the present disclosure can be linked together in a random or specific order. In some embodiments, a short oligopeptide or polypeptide linker, for example, 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form a bond between the intracellular signaling sequences. In one embodiment, a glycine-serine duplex can be used as a suitable linker. In one embodiment, a single amino acid, for example, alanine or glycine, can be used as a suitable linker.

[0163] In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains, for example, 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.

[0164] In some embodiments, a cell of the present disclosure expresses a CAR that comprises an antigen binding domain that binds EMCN, a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0165] In some embodiments, cells of the present disclosure express an iCAR that includes an antigen binding domain that binds EMCN (e.g., an EMCN-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, cells of the present disclosure express a CAR that includes an antigen binding domain that binds EMCN (e.g., an EMCN-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, cells of the present disclosure express a CAR that includes an antigen binding domain that binds EMCN (e.g., an EMCN-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 located between the antigen binding domain and the transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0166] In some embodiments, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR and comprises 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, respectively.

[0167] 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.

[0168] Natural killer cell receptor (NKR) CAR In some embodiments, a CAR of the present disclosure (e.g., an EMCN-specific CAR described herein) comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR components include KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS The NKR-CAR may be a transmembrane domain, hinge domain, or cytoplasmic domain from any suitable natural killer cell receptor, including, but not limited to, killer cell immunoglobulin-like receptors (KIRs) such as DPI; natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0169] Further chimeric receptor targets Certain aspects of the present disclosure relate to chimeric receptors that bind to antigens of interest in addition to EMCN and nucleic acids that code for such chimeric receptors.Certain aspects of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, that are genetically modified to express one or more of such chimeric receptors that bind to antigens of interest in addition to EMCN, and methods of using such receptors and cells to treat and / or prevent myeloid malignancies, such as AML, and other conditions where antigen-specific immune responses are desired.Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination.The present disclosure provides the immunogenicity within the tumor microenvironment to treat such malignant cells.

[0170] In some embodiments, the first chimeric receptor comprises an antigen binding domain that binds EMCN (e.g., an EMCN-specific antigen binding domain having one or more of the amino acid sequences listed in Table 1), and the second chimeric receptor comprises an additional antigen binding domain that binds a second antigen, such as a tumor-associated antigen (e.g., an AML-associated antigen). In some embodiments, the cell can express a first chimeric receptor specific for EMCN (e.g., a CAR comprising an EMCN-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., an AML-associated antigen). In some embodiments, the cell can express a first inhibitory chimeric receptor specific for EMCN (e.g., an inhibitory CAR comprising an EMCN-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., an AML-associated antigen). For example, a cell (e.g., an immunoresponsive cell) can be engineered to co-express or be capable of co-expressing an iCAR that includes an antigen-binding domain that binds EMCN (e.g., an EMCN-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., an AML-associated antigen). In addition to EMCN, suitable antibodies that bind antigens include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to a second antigen, such as a tumor-associated antigen (e.g., an AML-associated antigen). Examples of AML-associated antigens include FLT3, CD33, CD123, CLEC12A, CXCR4, and EphA3. In some embodiments, a commercially available antibody that binds to a second antigen, such as a tumor-associated antigen (e.g., an AML-associated antigen), may be used. The CDRs of commercially available antibodies are readily accessible by those skilled in the art using conventional sequencing techniques. Furthermore, one 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.

[0171] T cell receptor (TCR) Certain aspects of the present disclosure relate to a chimeric receptor that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., an AML-associated antigen), where the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). The TCR of the present disclosure is a disulfide-linked heterodimeric protein that includes two variable chains expressed as part of a complex with an invariant CD3 chain molecule. The TCR is found on the surface of T cells and is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR of the present disclosure includes an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR includes a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0172] Each chain of the TCR consists of two extracellular domains, the variable (V) region and the constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable regions bind to the peptide / MHC complex. Each of the variable regions has three complementarity determining regions (CDRs).

[0173] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. Upon complexing of the TCR complex with its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0174] In some embodiments, the TCR of the present disclosure is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from the naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from the naturally occurring TCR by at least 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residues. In certain embodiments, the TCR is a naturally occurring TCR modified by at least one amino acid residue. In some embodiments, the TCR is a naturally occurring TCR modified by at least 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residues.

[0175] Chimeric TCR In some embodiments, the TCR of the present disclosure comprises one or more antigen binding domains that can be grafted onto one or more constant domains of a TCR chain, e.g., a TCR alpha chain or a TCR beta chain, to create a chimeric TCR that specifically binds to a second antigen of interest, such as a tumor-associated antigen (e.g., an AML-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 at least a portion of the constant domains, e.g., the extracellular constant domain, the transmembrane domain, and the cytoplasmic domain, of a TCR chain, such as a TCR alpha chain and / or a TCR beta chain. As another example, the CDRs of an antibody or antibody fragment can be grafted onto a TCR alpha chain and / or a beta chain to create a chimeric TCR that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., an AML-associated antigen). Such chimeric TCRs can be produced by methods well known in the art (see, e.g., Willemsen RA et al., Gene Therapy 2000; 7:1369-1377; Zhang T et al., Cancer Gene Ther 2004 11: 487-496; and Aggen et al., Gene Ther. 2012 Apr; 19(4): 365-74).

[0176] Immunoresponsive cells Certain aspects of the present disclosure relate to cells, e.g., 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 to treat myeloid malignancies (e.g., AML).

[0177] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primary cell. In some embodiments, the mammalian cell is a cell line. In some embodiments, the mammalian cell is a bone marrow cell, a blood cell, a skin cell, a bone cell, a muscle cell, a neuronal cell, an adipocyte, a liver cell, or a cardiac cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell derived from or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. The immune cells of the present disclosure can be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or an iPSC). Exemplary immune cells include, but are not limited to, T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary neuronal cells include, but are not limited to, neural progenitor cells, neurons (e.g., sensory neurons, motor neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, or serotonergic neurons), astrocytes, oligodendrocytes, and microglia.

[0178] 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, provide for the production of antibodies, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, and the like. Examples of immunoresponsive cells of 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 lymphocytes can be derived or differentiated). T cells mature in the thymus and can be lymphocytes primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present disclosure include T helper cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells, e.g., T EM Cells and T EMRA The T cells can be any type of T cell, including but not limited to T cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as chimeric TCRs or CARs.

[0179] 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.

[0180] In some embodiments, the immunoresponsive cells of the present disclosure are T cells. The T cells of the present disclosure can be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

[0181] In some embodiments, the immunoresponsive cell of the present disclosure is a universal T cell with defective TCR-αβ. Methods for generating universal T cells are described in the art, for example, in Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0182] In some embodiments, the immunoresponsive cells of the present disclosure are isolated immunoresponsive cells that comprise one or more chimeric receptors of the present disclosure. In 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.

[0183] In some embodiments, the immunoresponsive cell is a T cell. In some embodiments, the immunoresponsive cell is a natural killer (NK) cell.

[0184] In some embodiments, the immunoresponsive cell expresses or is capable of expressing an immunoreceptor. An immunoreceptor is generally capable of inducing signal transduction or changes in protein expression in an immunoreceptor-expressing cell, thereby resulting in the regulation of an immune response upon binding to a cognate ligand (e.g., modulating, activating, initiating, stimulating, increasing, preventing, attenuating, inhibiting, reducing, decreasing, inhibiting, or suppressing an immune response). For example, when the CD3 chains present on a TCR / CAR cluster in response to ligand binding, a signal transduction cascade occurs via an immunoreceptor tyrosine-based activation motif (ITAM). Specifically, in certain embodiments, when an endogenous TCR, an exogenous TCR, a chimeric TCR, or a CAR (specifically an activated CAR) binds to their respective antigens, the formation of an immunological synapse occurs, which includes the clustering of many molecules (e.g., CD4 or CD8, CD3 / / / , etc.) in the vicinity of the bound receptor. Such clustering of membrane-bound signaling molecules results in phosphorylation of ITAM motifs contained within the CD3 chains, which initiates the T cell activation pathway and ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors can induce global gene expression in T cells to initiate T cell-mediated immune responses such as cytokine production and / or T cell-mediated killing by increasing IL-2 production for proliferation and expression of master regulator T cell proteins.

[0185] Cells expressing multiple chimeric receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the disclosure comprises two or more chimeric receptors of the disclosure. In some embodiments, the cell comprises two or more chimeric receptors, 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.

[0186] In some embodiments, each of the two or more chimeric receptors comprises a different antigen binding domain, e.g., an antigen binding domain that binds to the same antigen or different antigens. In some embodiments, each antigen bound by the two or more chimeric receptors is expressed on the same cell, such as a myeloid cell type (e.g., the same AML cell type). In some embodiments, each antigen bound by the two or more chimeric receptors is an AML-associated antigen (e.g., FLT3, CD33, CD123, CLEC12A, CXCR4, EphA3, etc.).

[0187] 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., an EMCN-specific chimeric receptor) and a second chimeric receptor can include a first chimeric receptor that includes an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor can include an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can include a VHH.

[0188] Without wishing to be bound by theory, it is believed that in cells having multiple chimeric membrane-embedded receptors, each comprising an antigen-binding domain, interactions between the antigen-binding domains of each receptor may be undesirable as such interactions may inhibit the ability of one or more antigen-binding domains to bind their cognate antigen. Thus, in embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more chimeric receptors, the chimeric receptors 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.

[0189] In some embodiments, when present on the surface of a cell, binding of the antigen binding domain of the first chimeric receptor to its cognate antigen (e.g., an EMCN-specific chimeric receptor that binds EMCN) is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, binding of the antigen binding domain of the first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen binding domain of the first chimeric receptor to its cognate antigen in the absence of the second chimeric receptor. In some embodiments, when present on the surface of a cell, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other less than when both are scFv antigen binding domains. In some embodiments, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other less than when both are scFv antigen binding domains.

[0190] 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 typically expressed on normal cells (e.g., cells typically considered to be healthy) but not expressed on tumor cells, such as AML cells. In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain that binds to EMCN (e.g., an EMCN-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 1).

[0191] 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, bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0192] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, e.g., as a non-logic gate to control, regulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. In some embodiments, an inhibitory chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure.

[0193] In some embodiments, the cells of the present disclosure comprise one or more of the inhibitory chimeric 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 an AML-associated antigen. In some embodiments, the one or more tumor-associated antigens comprise CD33. In some embodiments, the one or more tumor-associated antigens comprise FLT3. In some embodiments, the one or more tumor-associated antigens comprise CD33 and FLT3.

[0194] Costimulatory Ligands In some embodiments, the cells (e.g., immunoresponsive cells) of the present disclosure can further include one or more recombinant or exogenous costimulatory ligands. For example, the cells can be further transduced with one or more costimulatory ligands such that the cells co-express or are induced to co-express one or more chimeric receptors (e.g., EMCN-specific CARs described herein) of the present disclosure and one or more costimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between one or more chimeric receptors and one or more costimulatory ligands may provide a non-antigen-specific signal that is important for the full activation of the cells. Examples of suitable costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its main role is in regulating immune cells. Members of the TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) that contain a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas Ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell activating factor (B AFF) / Bly s / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and contain immunoglobulin domains (folds).Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, and PD-L1 / (B7-H1), a ligand for PD-1. In certain embodiments, the one or more costimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0195] Chemokine Receptors In some embodiments, the cells (e.g., immunoresponsive cells) of the present disclosure comprise one or more chimeric receptors (e.g., EMCN-specific CARs described herein) and may further comprise one or more chemokine receptors. For example, transgenic expression of chemokine receptors CCR2b or CXCR2 in cells such as T cells enhances trafficking to CCL2- or CXCL1-secreting solid tumors (Craddock et al, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw et al. Hum Gene Ther. 2002 Nov 1; 13(16): 1971 -80). Without wishing to be bound by theory, it is believed that the chemokine receptors expressed on the chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by the tumor and improve targeting of the cells to the tumor, which may promote invasion of the cells into the tumor and enhance the antitumor effect of the cells. The chemokine receptor of the present disclosure may comprise a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment thereof.The suitable chemokine receptor examples that can 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 cells are selected based on the chemokines secreted by tumors.

[0196] Chimeric receptor regulation Some embodiments of the present disclosure relate to controlling one or more chimeric receptor activities of the chimeric receptor expressing cells of the present disclosure (e.g., EMCN-specific CARs described herein). There are several ways in which chimeric receptor activity can be regulated. In some embodiments, a regulatable chimeric receptor that can control one or more chimeric receptor activities would 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 apoptosis of the cells. In some embodiments, iCaspase-9 contains a binding domain that contains a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, resulting in inducible and selective depletion of chimeric receptor-expressing cells.

[0197] Alternatively, in some embodiments, the chimeric receptor of the present disclosure may be modulated by utilizing small molecules or antibodies that inactivate or inhibit chimeric receptor activity. For example, an antibody may delete chimeric receptor-expressing cells by inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the chimeric receptor-expressing cells of the present disclosure may further express an antigen that is recognized by a molecule capable of inducing ADCC cell death or complement-induced cell death. For example, the chimeric receptor-expressing cells of the present disclosure may further express a receptor that can be targeted by an antibody or antibody fragment. Examples of suitable receptors that can be targeted by an antibody or antibody fragment include, but are not limited to, EpCAM, VEGFR, integrins (e.g., ανβ3, α4, αΙ 3 / 4 β3, 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, CD5, CD11, CD11a / LFA-1, CD15, CD 18 / 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, as well as truncated versions thereof.

[0198] In some embodiments, a chimeric receptor-expressing cell of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capability but retains an epitope recognized by a molecule capable of inducing ADCC (e.g., WO2011 / 056894).

[0199] In some embodiments, the chimeric receptor expressing cells of the present disclosure further comprise a highly expressed compact marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor expressing cells that binds with an anti-CD20 antibody (e.g., rituximab) and results in selective depletion of the chimeric receptor expressing cells by ADCC. Other methods for depleting the chimeric receptor expressing cells of the present disclosure include, but are not limited to, administration of a monoclonal anti-CD52 antibody that selectively binds and targets the chimeric receptor expressing cells for destruction by inducing ADCC. In some embodiments, the chimeric receptor expressing cells can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can initiate effector cell activity, such as ADCC activity or ADC activity. In some embodiments, the chimeric receptor ligand can further bind to an agent that induces cell death, such as a toxin. In some embodiments, the chimeric receptor expressing cells of the present disclosure can further express a target protein that is recognized by the cell depletion agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depletion agent is an anti-CD20 antibody. In such embodiments, the cell depletion agent is administered when it is desired to reduce or eliminate chimeric receptor expressing cells. In some embodiments, the cell depletion agent is an anti-CD52 antibody.

[0200] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides in which the components of the chimeric receptor of the present disclosure are distributed on separate polypeptides or members. For example, the set of polypeptides can include a dimerization switch that, in the presence of a dimerization molecule, can bind the polypeptides to each other to form a functional chimeric receptor.

[0201] EMCN-specific protein-encoding nucleic acid constructs Certain aspects of the present disclosure relate to nucleic acids (e.g., isolated nucleic acids) encoding one or more EMCN-specific proteins of the present disclosure (e.g., an EMCN-specific CAR 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.

[0202] In some embodiments, the 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., EMCN), 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.

[0203] 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, inducing the gene of interest from a vector known to contain the gene, or isolating the gene of interest directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0204] In some embodiments, the nucleic acid of the disclosure is contained within a vector. In some embodiments, the nucleic acid of the disclosure is expressed in a cell via a transposon, a CRISPR / Cas9 system, a TALEN, or a zinc finger nuclease.

[0205] 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 regulating expression of the desired nucleic acid.

[0206] 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.

[0207] The nucleic acid of the present disclosure can be cloned into several types of vectors.For example, the nucleic acid 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.

[0208] In some embodiments, the plasmid vector comprises a transposon / transposase system for integrating the 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 herein by reference in its entirety. In some embodiments, the transposon system is Sleeping Beauty transposon / transposase or piggyBac transposon / transposase.

[0209] 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, since 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 an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and US6326193). Numerous virus-based systems for gene transfer into mammalian cells have been developed. 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. Numerous retroviral systems are known in the art.

[0210] In some embodiments, vectors of the present disclosure include additional promoter elements, such as enhancers that regulate the frequency of transcription initiation. Enhancers are typically located in the region 30 bp to 110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements can be flexible so that promoter function is maintained when elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decrease. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Exemplary promoters can include, but are not limited to, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EF1a promoter, the ubiquitin C promoter, and the phosphoglycerokinase (PGK) promoter.

[0211] In some embodiments, the promoter that can express the nucleic acid of the present disclosure in mammalian cells, such as the immunoresponsive cells of the present disclosure, is EF1a promoter.Natural EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes.EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in promoting chimeric receptor expression from nucleic acid cloned into lentiviral vector.

[0212] In some embodiments, the promoter capable of expressing the nucleic acid of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is a constitutive promoter. For example, a suitable constitutive promoter is the immediate early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter that can drive high levels of 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.

[0213] 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 an inducible promoter.The use of an inducible promoter can provide a molecular switch that can induce or suppress the expression of the 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 promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0214] In some embodiments, vectors of the present disclosure may further include a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator, elements allowing episomal replication, and / or elements allowing selection.

[0215] In some embodiments, the vectors of the present disclosure may further comprise a selectable marker gene and / or a reporter gene to facilitate identification and selection of chimeric receptor expressing cells from a population of cells transduced with the vector. In some embodiments, the selectable marker may be encoded by a nucleic acid separated from the vector and used in the co-transfection procedure. Either the selectable marker or the reporter gene may be flanked by appropriate control sequences to allow expression in the host cell. Examples of selectable markers include, but are not limited to, antibiotic resistance genes, such as neo and the like.

[0216] In some embodiments, reporter genes can be used to identify transduced cells and to evaluate the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene that encodes a polypeptide that is not present or expressed in the recipient organism or tissue, and whose expression results in an easily detectable property, such as an enzymatic activity. Expression of the reporter gene can be assayed at a suitable time after the nucleic acid is introduced into the recipient cell. Examples of reporter genes include, but are not limited to, genes encoding luciferase, genes encoding beta-galactosidase, genes encoding chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, and genes encoding green fluorescent protein. Suitable expression systems are well known in the art and can be prepared using known techniques or obtained commercially. In some embodiments, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0217] In some embodiments, a vector comprising a nucleic acid sequence encoding an EMCN-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.

[0218] In embodiments in which the EMCN-specific protein-expressing cell comprises two or more heterologous proteins (e.g., two or more chimeric receptors), a single nucleic acid may encode the two or more proteins under a single regulatory control element (e.g., promoter) or under separate regulatory control elements for the nucleotide sequence encoding each protein contained in the nucleic acid. In some embodiments in which the EMCN-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., 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, such as an autocleavage site or a substrate for an intracellular protease. 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, hi some embodiments, two or more chimeric receptors comprise a T2A and an E2A peptide cleavage site.

[0219] 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 acid 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 acid 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 acid into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0220] In some embodiments, liposomes may be used as a non-viral delivery system for introducing the nucleic acid or vector of the present disclosure into a host cell in vitro, ex vivo, or in vivo. In some embodiments, the nucleic acid may be associated with a lipid, for example, by being encapsulated in the aqueous interior of the liposome, by being interspersed within the lipid bilayer of the liposome, by being attached to the liposome via a linking molecule that is associated with both the liposome and the nucleic acid, by being entrapped in the liposome, by being complexed with the liposome, by being dispersed in a solution containing lipids, by being mixed with lipids, by being combined with lipids, by being contained as a suspension in lipids, by being contained or complexed in micelles, or by being otherwise associated with lipids. As disclosed herein, lipid-associated nucleic acid or vector compositions are not limited to any particular structure in solution. In some embodiments, such compositions may exist as micelles or in bilayer structures with a "collapsed" structure. Such compositions may also be interspersed in the solution to form aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that can be naturally occurring or synthetic. In some embodiments, lipids can include lipid droplets naturally occurring in the cytoplasm, or classes of compounds that include long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Suitable lipids may be obtained from commercial sources, including, but 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 single and multi-layer lipid vesicles formed by the production of enclosed lipid bilayers or aggregates.In some embodiments, liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. In some embodiments, multilamellar liposomes can have multiple lipid layers separated by aqueous medium. Multilamellar liposomes can form spontaneously when phospholipids are suspended in an excess of aqueous solution. In some embodiments, the lipid components may undergo self-rearrangement before the formation of a closed structure, and can trap water and dissolved solutes between the lipid bilayers. In some embodiments, the lipids can assume a micellar structure or exist only as heterogeneous aggregates of lipid molecules.

[0221] In some embodiments, the nucleic acid or vector of the disclosure is introduced into a mammalian host cell, such as an immunoresponsive cell of the disclosure. In some embodiments, the presence of the nucleic acid or vector of the 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.

[0222] In some embodiments, the nucleic acid or vector of the present disclosure is stably transduced into the immunoresponsive cell of the present disclosure. In some embodiments, the cell exhibiting stable expression of the nucleic acid or vector expresses the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months after transduction.

[0223] In embodiments in which an EMCN-specific protein (e.g., a chimeric receptor) of the present disclosure is transiently expressed in a cell, a nucleic acid or vector encoding an EMCN-specific protein of the present disclosure is transfected into an immunoresponsive cell of the present disclosure. In some embodiments, the immunoresponsive cell expresses the EMCN-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.

[0224] In some embodiments, the nucleic acid construct encodes a bicistronic encoded chimeric antigen receptor. In some embodiments, the encoded bicistronic chimeric antigen receptor comprises an EMCN CAR (e.g., an EMCN inhibitory CAR) and a CAR specific for a second antigen (e.g., a tumor-targeting chimeric receptor).

[0225] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor. In some embodiments, the encoded bivalent chimeric antigen receptor comprises an EMCN antigen binding domain and a second antigen binding domain.

[0226] Pharmaceutical Compositions and Administration Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising one or more EMCN-specific proteins (e.g., chimeric receptors) of the present disclosure or immunoresponsive cells of the present disclosure expressing such one or more EMCN-specific proteins. In some embodiments, compositions comprising EMCN-specific proteins (e.g., chimeric receptors) or genetically modified immunoresponsive cells expressing such EMCN-specific proteins can be provided systemically or directly to a subject for treatment of a proliferative disorder, such as a bone marrow disorder. In certain embodiments, the composition is directly injected into an organ of interest (e.g., an organ affected by the disorder). Alternatively, the composition can be provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). Proliferative and differentiation agents can be provided before, during, or after administration of the composition to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

[0227] Compositions containing the genetically modified cells of the present disclosure can be administered in any physiologically acceptable vehicle, for example, intravascularly, but they can also be introduced into bone or other convenient sites where the genetically modified cells can find a suitable site for regeneration and differentiation (e.g., the thymus). In some embodiments, at least 1×10 5 cells may be administered, ultimately resulting in a total of 1 x 10 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, including, but not limited to, fluorescence-activated cell sorting (FACS). In some embodiments, the purity of genetically modified cells in a population of cells can be about 50%, about 55%, about 60%, or about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more of the cells in the population of cells. The dosage can be easily adjusted by one skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc. In some embodiments, factors can also include, for example, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, G-CSF, MCSF, GM-CSF, gamma interferon, and erythropoietin.

[0228] In certain embodiments, the composition is a pharmaceutical composition comprising genetically modified cells, such as immunoresponsive cells or their precursor cells, and a pharma- ceutically acceptable carrier. Administration can be autologous or xenogeneic. For example, immunoresponsive cells or precursors can be obtained from one subject and administered to the same subject or to a different compatible subject. In some embodiments, the immunoresponsive cells or their progeny of the present disclosure can be derived from peripheral blood cells (e.g., from in vivo, ex vivo, or in vitro) and can be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the therapeutic composition of the present disclosure (e.g., pharmaceutical composition comprising genetically modified cells of the present disclosure), it is generally formulated in a unit dose injectable form (solution, suspension, emulsion).

[0229] formulation Certain aspects of the present disclosure relate to formulations of compositions comprising the disclosed EMCN-specific proteins (e.g., chimeric receptors) or genetically modified cells expressing such proteins (e.g., immunoresponsive cells of the present disclosure). In some embodiments, the disclosed compositions comprising genetically modified cells may be provided as sterile liquid preparations, including, but not limited to, isotonic aqueous solutions, suspensions, emulsions, dispersions, and viscous compositions that may be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions may be more conveniently administered, particularly by injection. In some embodiments, viscous compositions may be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0230] In some embodiments, a sterile injectable solution can be prepared by incorporating the genetically modified cells of the present disclosure in a sufficient amount of a suitable solvent with various amounts of any other components as desired. Such compositions can be mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, dextrose, etc. In some embodiments, the compositions can also be lyophilized. The compositions can include auxiliary substances such as wetting agents, dispersants, pH buffers, and antibacterial agents, depending on the route of administration and the desired preparation.

[0231] In some embodiments, the composition of the present disclosure may further comprise various additives that can enhance the stability and sterility of the composition. Examples of such additives include, but are not limited to, antibacterial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination can be prevented by including any of various antibacterial and antifungal agents, including, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable preparation of the present disclosure can be achieved by using a suitable agent that delays absorption, such as aluminum monostearate and gelatin.

[0232] In some embodiments, the compositions of the present disclosure can be isotonic, i.e., have the same osmotic pressure as blood and tears. In some embodiments, the desired isotonicity can be achieved using, for example, sodium chloride, dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.

[0233] 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.

[0234] One consideration regarding 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 varies depending on the subject being treated. In certain embodiments, the amount of genetically modified cells administered to a subject in need thereof is 1×10 4 cells ~ 1×10 10 The amount of cells that is considered an effective dose may range from 10 to 100 cells. In some embodiments, the exact amount of cells that is considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage amount can be easily ascertained by one of ordinary skill in the art based on this disclosure and technical knowledge.

[0235] Heterologous Moieties and Modifications In a further set of embodiments, the EMCN-specific chimeric proteins herein (e.g., EMCN-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.

[0236] Drug Conjugates In various embodiments, a protein comprising an EMCN-specific antigen-binding domain described herein is conjugated to a therapeutic agent (i.e., a drug) to form an antibody-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In certain embodiments, the therapeutic agent is attached to the antigen-binding domain via a linker peptide, as discussed in more detail herein.

[0237] Methods for preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate a drug to an antigen-binding domain disclosed herein (e.g., having one or more of the amino acid sequences listed in Table 1) are described, for example, in U.S. Pat. No. 8,624,003 (the Pott method), U.S. Pat. No. 8,163,888 (one-step), U.S. Pat. No. 5,208,020 (two-step method), U.S. Pat. No. 8,337,626 (a method for preparing antibody-drug conjugates), and U.S. Pat. ,856, U.S. Patent No. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, U.S. Patent No. 7,745,394, WO 2017 / 136623, WO 2017 / 015502, WO 2017 / 015496, WO 2017 / 015495, WO 2004 / 010957, WO 2005 / 077090, WO 2005 / 082023, WO 2006 / 065533, WO 2007 / 030642, WO 2007 / 103288, WO 2013 / 173337, WO 2015 / 057699, WO 2015 / 095755, WO 2015 / 123679, WO 2015 / 157286, WO 2017 / 165851, WO 2009 / 073445 No. 2010 / 068759, WO 2010 / 138719, WO 2012 / 171020, WO 2014 / 008375, WO 2014 / 093394, WO 2014 / 093640, WO 2014 / 160360, WO 2015 / 054659, WO 2015 / 195925, WO 2017 / 160754, Storz (MAbs. 2015 November-December; 7(6): 989-1009), Lambert et al. (Adv Ther, 2017 34: 1015). Diamantis et al. (British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007. 3: 321-2), We et al. (Proc Natl Acad Sci USA, 2009.106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7:1052-67), Agarwal et al. (Proc Natl Acad Sci USA., 2013, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. (Drug Dev. and D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2014, 25:1331-41), Liang et al. (J Am Chem Soc., 2014, 136:10850-3), Drake et al. (Curr Opin Chem Biol., 2015, 28:174-80), and York et al. (BMC Biotechnology, 2016, 16(1):23), each of which is incorporated herein by reference in its entirety for all of its teachings.

[0238] Further binding moieties In various embodiments, the EMCN-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 moieties are antibody fragments or antibody formats, including, but not limited to, full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, diabodies, sc diabodies, DARTs, tandAbs, minibodies, camelid VHHs, and other antibody fragments or formats known to those of skill in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), the entire teachings of which are incorporated herein by reference.

[0239] In certain embodiments, the one or more additional binding moieties are attached to the C-terminus of one or more peptides of an EMCN-specific antigen-binding domain, such as a VH and / or VL, a Fab heavy and / or light chain fragment, or an scFv. In certain embodiments, the one or more additional binding moieties are attached to the N-terminus of one or more peptides of an EMCN-specific antigen-binding domain, such as a VH and / or VL, a Fab heavy and / or light chain fragment, or an scFv.

[0240] In certain embodiments, the one or more additional binding moieties are specific for a different antigen or epitope than EMCN. In certain embodiments, the one or more additional binding moieties are specific for EMCN.

[0241] 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) via Fc-mediated attachment (e.g., protein A / G). In certain embodiments, one or more additional binding moieties are attached to an antigen binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table 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 in the same expression vector (e.g., plasmid).

[0242] 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 functional or chemically reactive groups that can be used in downstream processes, such as binding to additional moieties (e.g., drug conjugates and additional binding moieties), as well as downstream purification processes.

[0243] In certain embodiments, the modification is a chemically reactive group, including, but not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkyne groups), and aldehydes resulting in formylglycine (FGly). In certain embodiments, the modification is a functional group, including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and streptosystems, etc.). In certain embodiments, the functional group or chemically reactive group has a cleavable peptide sequence. In certain embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In certain embodiments, the protease cleavage is performed by an intracellular protease. In certain embodiments, the protease cleavage is performed by an extracellular or membrane-bound protease. ADC therapy using protease cleavage is described in detail by Choi et al. (Theranostics, 2012; 2(2): 156-178.), the entire teachings of which are incorporated herein by reference.

[0244] Treatment method Certain aspects of the present disclosure relate to methods of using the disclosed EMCN-specific proteins (e.g., chimeric receptors) and genetically modified cells (e.g., immunoresponsive cells) expressing such proteins to treat a subject in need thereof. In some embodiments, the disclosed methods are useful for treating a cancer in a subject, such as a bone marrow disorder. In some embodiments, the bone marrow disorder is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myelogenous leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, or polycythemia vera. In some embodiments, the bone marrow disorder is AML. Other aspects of the present disclosure relate to the use of the disclosed chimeric receptors and genetically modified cells (e.g., immunoresponsive cells) expressing an EMCN-specific chimeric receptor in methods for treating a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. In some embodiments, the methods of the present disclosure may 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, the 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, the effective amount may be provided by bolus or continuous perfusion.

[0245] As disclosed herein, an "effective amount" or a "therapeutically effective amount" is an amount sufficient to affect beneficial or desired clinical results during treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to palliate, improve, stabilize, reverse, or delay the progression of a disease, or reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person of ordinary skill in the art. In determining the appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the immunoresponsive cells being administered.

[0246] For adoptive immunotherapy using antigen-specific cells (e.g., immune-responsive cells such as T cells), approximately 1 × 10 6 ~1×10 10 cells (e.g., about 1 x 10 9 A cell dose ranging from 1000 to 10000 cells is usually injected. Upon administration of the cells to a subject and subsequent differentiation, immunoresponsive cells are induced to be specifically directed against a particular antigen. In some embodiments, induction of immunoresponsive cells can include, but is not limited to, inactivation of antigen-specific cells, such as by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance in autoimmune disorders and the like. Genetically modified cells can be administered by any method known in the art, including, but not limited to, intravenously, subcutaneously, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

[0247] In some embodiments, the method of use includes a method of inhibiting an immune response. Inhibiting an immune response can refer to, for example, preventing, attenuating, or inhibiting a cell-mediated immune response induced by a chimeric receptor expressed on the surface of an immunomodulatory cell. In embodiments, the method includes preventing, attenuating, or inhibiting the activation of an activating chimeric receptor expressed on the surface of an immunomodulatory cell.

[0248] In some embodiments, the inhibitory chimeric receptors of the present disclosure are used to prevent, attenuate, inhibit, or suppress immune responses initiated by tumor-targeting chimeric receptors (e.g., activating CARs). For example, an immunomodulatory cell expresses an inhibitory chimeric receptor that recognizes antigen target 1 (e.g., a non-tumor antigen) and a tumor-targeting chimeric receptor that recognizes antigen target 2 (e.g., a tumor target). In this example, when an exemplary immunomodulatory cell contacts a 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, where the target cell is a non-tumor cell expressing 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 prevention, attenuation, or inhibition of signaling of the tumor-targeting chimeric receptor, and the immunomodulatory cell is not activated. Similarly, in an exemplary example, where the target cell is a non-tumor cell expressing only antigen target 1, only the inhibitory chimeric receptor can be activated. In contrast, in an illustrative example where the target cell is a tumor cell expressing only antigen target 2, the inhibitory chimeric receptor cannot be activated, while the tumor-targeting chimeric receptor can be activated, resulting in signaling that leads to activation of immune regulatory cells.

[0249] The inhibition of the immune response initiated by the tumor-targeting chimeric receptor can be the inhibition or reduction of the activation of the tumor-targeting chimeric receptor, the inhibition or reduction of the signal transduction of the tumor-targeting chimeric receptor, or the inhibition or reduction of the activation of immunoregulatory cells. The inhibitory chimeric receptor can inhibit the activation of the tumor-targeting chimeric receptor, the signal transduction by the tumor-targeting chimeric receptor, or the 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 the activation of the tumor-targeting chimeric receptor, the signal transduction or activation of immunoregulatory cells compared to the activation of the tumor-targeting chimeric receptor, the signal transduction or activation of immunoregulatory cells that lack the inhibitory chimeric receptor. In some embodiments, the inhibition refers to the decrease or reduction of the activity of the tumor-targeting chimeric receptor before or after its activation.

[0250] The immune response can be the production and secretion of cytokines or chemokines from activated immunoregulatory cells. The immune response can be a cell-mediated immune response against target cells.

[0251] 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, where the immune response is induced by activation of the immunomodulatory cells.

[0252] Treatment In some embodiments, the methods of the disclosure increase an immune response in a subject in need thereof. In some embodiments, the methods of the disclosure include methods for treating and / or preventing bone marrow disorders in a subject. In some embodiments, the subject is a human. In some embodiments, human subjects suitable for treatment may include two treatment groups that may be distinguished by clinical criteria. Subjects with "progressive disease" or "high tumor burden" are subjects with clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., based on percentage of leukemic cells by palpation, CAT scan, ultrasound, mammogram, or X-ray; positive biochemical or histopathological markers are insufficient to identify this population by themselves). In some embodiments, the pharmaceutical compositions of the disclosure are administered to these subjects to induce an anti-tumor response with the aim of alleviating their condition. In some embodiments, a reduction in tumor burden occurs as a result of administration of the pharmaceutical composition, but any clinical improvement would constitute a benefit. In some embodiments, clinical improvement includes a reduced risk or reduced rate of progression in pathological outcomes of the tumor. In some embodiments, a second group of suitable human subjects are "adjuvant group" subjects. These subjects are individuals who have a history of bone marrow disorders but have responded to another treatment. Previous therapy may include, but is not limited to, surgical resection, radiation therapy, and / or conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk for disease progression near the original tumor site or by metastasis. In some embodiments, this group can be further subdivided into high-risk and low-risk individuals. Subdivision can be based on characteristics observed before or after initial treatment. These characteristics are known in the clinical arts and are suitably defined for different bone marrow diseases. Typical characteristics of the high-risk subgroup are those in which the tumor has invaded adjacent tissues or shows lymph node involvement.

[0253] In any and all aspects of the increased immune response described herein, any increase or decrease or change in an aspect of a characteristic or function is compared to a cell that has not been contacted with an immunoresponsive cell as described herein.

[0254] Increasing an immune response can be both an enhancement of an immune response or an induction of an immune response. For example, increasing an immune response encompasses both initiating or initiating an immune response or increasing or amplifying an ongoing or existing immune response. In some embodiments, the treatment induces an immune response. In some embodiments, the induced immune response is an adaptive immune response. In some embodiments, the induced immune response is an innate immune response. In some embodiments, the treatment enhances an immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the treatment increases an immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response.

[0255] 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 detected positive for genetic mutations associated with AML, but are still of childbearing age, can benefit from receiving one or more cells (e.g., immunoresponsive cells) of the present disclosure in a prophylactic treatment to prevent the development of AML until suitable for performing prophylactic surgery.In some embodiments, subjects may have a progressive form of disease, in which case treatment goals may include alleviating or reversing disease progression and / or improving side effects.In some embodiments, subjects may have a history of a condition that they have already been treated for, in which case treatment goals may typically include reducing or delaying the risk of recurrence.

[0256] Combination therapy In some embodiments, the genetically modified cells (e.g., immune response cells) of the present disclosure expressing one or more proteins comprising the antigen binding domains (e.g., scFvs) of the present disclosure, such as the chimeric receptors of the present disclosure, may be used in combination with other known drugs and therapies. In some embodiments, the combination therapy of the present disclosure includes the genetically modified cells of the present disclosure, which may be administered in combination with one or more additional therapeutic agents. In some embodiments, the genetically modified cells and one or more additional therapeutic agents may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the genetically modified may be administered first and the one or more additional agents may be administered a second time, or the administration order may be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[0257] In some embodiments, the genetically modified cells of the present disclosure may be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunosuppressants (e.g., CAMPATH or anti-CD3 antibodies), cytotoxin, fludarabme, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, and peptide vaccines.

[0258] In some embodiments, the genetically modified cells of the present disclosure can be used in combination with lymphodepleting agents.Suitable lymphodepleting agents reduce or reduce lymphocytes, for example, B-cell lymphocytes and / or T-cell lymphocytes, before immunotherapy.Examples of suitable lymphodepleting agents include, but are not limited to, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[0259] In some embodiments, the genetically modified cells of the present disclosure may be used in combination with a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, anthracyclines (e.g., doxorubicin), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclarubicin A, gliotoxin, bortezomib), immunoderivatives such as thalidomide or thalidomide derivatives (e.g., lenalidomide), and the like.

[0260] Examples of common chemotherapeutic agents suitable for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Bleomycin®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chloramphenicol (Citrosil), and rivaroxaban (Ricin®). Bucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosal®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyte®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubicin®), daunorubicin citrate liposome Infusion (Daunozom®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Bepcid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adolsil®, Efudex®), flutamide (Eurexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idahivir®), Syn (registered trademark), ifosfamide (IFEX (registered trademark), irinotecan (Camptosar (registered trademark)), L-asparaginase (ELSPAR (registered trademark)), leucovorin calcium, melphalan (Alkeran (registered trademark)), 6-mercaptopurine (Purintor (registered trademark)), methotrexate (Forex (registered trademark)), mitoxantrone (Novantron (registered trademark)), Mylotarg, paclitaxel (Taxol (registered trademark), Phoenix (Yttrium 90 / MX-DTPA), pentostatin,These include, but are not limited to, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Bumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velbon®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0261] Examples of suitable alkylating agents include nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Rev), immune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional illustrative examples of alkylating agents include oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin D, Cosmegen®), melphalan (L-PAM, Also known as L-sarcolysin, and phenylalanine masterbate, Alkeran®), altretamine (also known as hexamethylmelamine (HMM), Hexalen®), carmustine (BiCNU®), bendamustine (Treanda®), busulfan (Busulfex® and Myleran®), carboplatin (Paraplatin®), lomustine (also known as CCNU, CeeNU®), cisplatin (also known as CDDP, Platinol® and Platinol®-AQ), chlorambucil (Leukeran®), cyclophosphamide (Cytoxan® and Neosar®), dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®), amphetamine (also known as hexamethylmelamine (HMM), Hexalen®), rifabutin (Rifabutin ... These include, but are not limited to, aitretamine (also known as hexamethylmelamine (HMM), Hexalen®), ifosfamide (Ifex®), Prednumustine, procarbazine (Matulane®), mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, Mustargen®), streptozocin (Zanosar®), thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine HC1 (Treanda®).

[0262] Examples of suitable mTOR inhibitors include, but are not limited to, temsirolimus, ridaforolimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RAD001), rapamycin (AY22989, Sirolimus®), and XL765.

[0263] 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.

[0264] Examples of suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin® and Rubex®), bleomycin (lenoxane®), daunorubicin (dauorubicin hydrochloride, daunomyem, and rubidomycin hydrochloride, Cerubidine®), daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®), mitoxantrone (DHAD, Novantrone®), epirubicin (Ellence™), idarubicin (idamycin®, idamycin PES®), mitomycin C (Mutamycin®), geldanamycin, herbimycin, rabidomycin, and desacetramycin.

[0265] Examples of suitable vinca alkaloids include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Erdisine®), vinblastine (also known as vinblastine sulfate, vincalucoblastine, and VLB, Alkaban-AQ®, and Velban®).

[0266] 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.

[0267] In some embodiments, the genetically modified cells of the present disclosure are administered in combination with a CD20 inhibitor, such as an anti-CD20 antibody, or a fragment thereof.Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), okaratuzumab, and Prol31921.

[0268] In some embodiments, the genetically modified cells of the present disclosure are administered in combination with an oncolytic virus. In some embodiments, the oncolytic virus can selectively replicate in cancer cells and induce the death or delay the growth of cancer cells. In some cases, the oncolytic virus has no effect or minimal effect on non-cancer cells. Suitable oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)). In some embodiments, the oncolytic virus is a recombinant oncolytic virus.

[0269] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, for example, an SHP-1 inhibitor or an SHP-2 inhibitor.In one embodiment, the genetically modified cells of the present disclosure can be used in combination with a kinase inhibitor.Examples of suitable kinase inhibitors include, but are not limited to, CDK4 inhibitor, CDK4 / 6 inhibitor, BTK inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, mTOR inhibitor, MNK inhibitor, and anaplastic lymphoma kinase (ALK) inhibitor.

[0270] In some embodiments, the genetically modified cells of the present disclosure are administered to subjects in combination with a regulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the effectiveness of chimeric receptor-expressing cell therapy. Without being bound by theory, it is believed that administration of MDSC regulators enhances the effectiveness of the genetically modified cells of the present disclosure. Examples of suitable regulators of MDSCs include, but are not limited to, MCS110 and BLZ945.

[0271] In some embodiments, the genetically modified cells of the present disclosure are administered to a subject in combination with an agent that inhibits or reduces the activity of immunosuppressive plasma cells. Immunosuppressive plasma cells have been shown to inhibit T cell-dependent immunogenic chemotherapy, such as oxaliplatin (Shalapour et al., Nature 2015, 521:94-101). In one embodiment, the immunosuppressive plasma cells can express one or more of IgA, interleukin (IL)-10, and PD-L1.

[0272] In some embodiments, the genetically modified cells of the disclosure are administered to a subject in combination with an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide. In some embodiments, the genetically modified cells of the disclosure are further modified to express an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide.

[0273] In some embodiments, a subject with a bone marrow disorder (e.g., AML) 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), cytarabme, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is a liposomal formulation containing cytarabine and daunorubicin in a molar ratio of 5:1. 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, the subject is administered the genetically modified cells of the present disclosure in combination with a biological therapy, e.g., an antibody or cell therapy, e.g., 225Ac-lintuzumab (Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma / Bristol Myers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer). In some embodiments, the subject is administered the genetically modified cells of the present disclosure in combination with an FLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo), crenolanib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), ASP2215 (Astelias).In some embodiments, the subject is administered the genetically modified cells of the present disclosure in combination with an isocitrate dehydrogenase (IDH) inhibitor, such as AG-221 (Celgene / Agios) or AG-120 (Agios / Celgene). In some embodiments, the subject is administered the genetically modified cells of the present disclosure in combination with an inhibitor of a cell cycle regulator, such as an inhibitor of polo-like kinase 1 (Plkl), such as volasertib (Boehringer Ingelheim), or an inhibitor of cyclin-dependent kinase 9 (Cdk9), such as 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, such as an inhibitor of B cell lymphoma 2 (Bcl-2), such as venetoclax (Abbvie / Roche), or an inhibitor of Bruton's tyrosine kinase (Btk), such as ibrutinib (Pharmacyclics / Johnson & Johnson Janssen Pharmaceutical). In some embodiments, the subject is administered the genetically modified cells of the present disclosure in combination with an inhibitor of M1 aminopeptidase, an inhibitor of histone deacetylase (HDAC), such as pracinostat (MEI Pharma), a multikinase inhibitor, such as rigosertib (Onconova Therapeutics / Baxter / SymBio), or a peptidic CXCR4 inverse agonist, such as BL-8040 (BioLineRx).

[0274] In some embodiments, the subject may be administered an agent that enhances the activity or compatibility of the genetically modified cells of the present disclosure. For example, the agent may inhibit a molecule that regulates or controls, 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 initiate immune effector responses. Examples of suitable inhibitory molecules include, but are not limited to, PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIGIT, 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, agents such as inhibitory nucleic acids, for example inhibitory nucleic acids, for example inhibitory nucleic acids, for example dsRNA, for example siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN), can be used to inhibit the expression of inhibitory molecules in genetically modified cells. In one embodiment, the inhibitor is an shRNA. In some embodiments, the genetically modified cells of the present disclosure can be further modified to express inhibitory nucleic acids, for example inhibitory nucleic acids, for example inhibitory nucleic acids, for example dsRNA, for example siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN), and can be used to inhibit the expression of inhibitory molecules in genetically modified cells.

[0275] In one embodiment, the agent that modulates or controls, e.g., inhibits, T cell function is inhibited in the genetically modified cell of the present disclosure. In such an embodiment, the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, e.g., inhibits, T cell function is linked to a nucleic acid encoding a component, e.g., all components, of the chimeric receptor of the present disclosure. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, T cell function is operably linked to a promoter, e.g., a promoter derived from HI or U6, such that the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, T cell function is expressed, e.g., in the genetically modified cell. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, e.g., inhibits, T cell function is present on the same vector, e.g., lentiviral vector, that contains the nucleic acid molecule encoding the component, e.g., all components, of the chimeric receptor. In such an embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits the expression of the molecule that modulates or controls, e.g., inhibits, T cell function is located on a vector, e.g., lentiviral vector, 5'- or 3'- of the nucleic acid encoding the component, e.g., all components, of the chimeric receptor. The nucleic acid molecule that encodes the dsRNA molecule that inhibits the expression of the molecule that regulates or controls, e.g., inhibits T cell function, can be transcribed in the same or different direction as the nucleic acid that encodes the components, e.g., all components, of the chimeric receptor.In one embodiment, the nucleic acid molecule that encodes the dsRNA molecule that inhibits the expression of the molecule that regulates or controls, e.g., inhibits T cell function, is present on a vector other than the vector that contains the nucleic acid molecule that encodes the components, e.g., all components, of the chimeric receptor.In one embodiment, the nucleic acid molecule that encodes the dsRNA molecule that inhibits the expression of the 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 that encodes the dsRNA molecule that inhibits the expression of the 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.

[0276] In one embodiment, the agent that regulates or controls, 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.

[0277] 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.

[0278] In one embodiment, the agent that enhances the activity of the genetically modified cells of the present disclosure is a cytokine. Cytokines have important functions related to immune responsive cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to a subject receiving the genetically modified cells of the present disclosure include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-12, L-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.

[0279] In some embodiments, the cytokine can be administered simultaneously or contemporaneously with the genetically modified cells, for example, on the same day. The cytokine can be prepared in the same pharmaceutical composition as the genetically modified cells, or in a separate pharmaceutical composition. The cytokine can be administered immediately after the administration of the genetically modified cells, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the administration of the genetically modified cells. In some embodiments, the cytokine is administered in a dosing regimen that occurs over one day, the first day of the cytokine dosing regimen can be the same day as the administration with the genetically modified cells, or the first day of the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the administration of the genetically modified cells. In one embodiment, on the first day, the genetically modified cells are administered to the subject, and on the second day, the cytokine is administered once a day for the next 7 days. In some embodiments, the cytokine is administered for a period of time 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 evaluation of the subject's response to the genetically modified cells.

[0280] kit Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of cancer (e.g., AML) or other diseases (e.g., immune-related or autoimmune disorders). In certain embodiments, the kit comprises 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 kit comprises a sterile container. In some embodiments, such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a drug.

[0281] In some embodiments, the therapeutic or prophylactic composition is provided with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., AML). 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, dosing schedules, dosing schedules for the treatment or prevention of a disease or its symptoms, precautions, warnings, indications, contraindications, overdosing information, adverse reactions, animal pharmacology, clinical trials, and / or bibliographic references. In some embodiments, the instructions may be printed directly on the container (if present), or may be printed as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0282] Enumeration of embodiments Embodiment 1: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), 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:6, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:8; b. the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:11; c. the antibody or antigen-binding fragment thereof is humanized; An antibody or an antigen-binding fragment thereof.

[0283] Embodiment 2: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a. VH comprises a VH complementarity region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 8; b. the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:11; c. the antibody or antigen-binding fragment thereof is humanized; An antibody or an antigen-binding fragment thereof.

[0284] Embodiment 3: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a. VH comprises a VH complementarity region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, a VH complementarity region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementarity region 3 (CDRH3) having the amino acid sequence of SEQ ID NO: 8; b. the VL comprises a VL complementarity region 1 (CDRL1) having the amino acid sequence of SEQ ID NO:9, a VL complementarity region 2 (CDRL2) having the amino acid sequence of SEQ ID NO:10, and a VL complementarity region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:11; An antibody or an antigen-binding fragment thereof.

[0285] Embodiment 4: The antibody or antigen-binding fragment thereof of embodiment 2 or embodiment 3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO:2.

[0286] Embodiment 5: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0287] Embodiment 6: The antibody or antigen-binding fragment thereof of embodiment 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 12.

[0288] Embodiment 7: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0289] Embodiment 8: The antibody or antigen-binding fragment thereof of embodiment 7, wherein the VH has the amino acid sequence set forth in SEQ ID NO: 16.

[0290] Embodiment 9: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0291] Embodiment 10: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, VH comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3) contained within VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 19; 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) contained within the VL having the amino acid sequence of SEQ ID NO: 20; An antibody or an antigen-binding fragment thereof.

[0292] Embodiment 11: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VL has the amino acid sequence set forth in SEQ ID NO: 20.

[0293] Embodiment 12: An isolated antibody or antigen-binding fragment thereof that specifically binds to human Endomucin (EMCN), comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0294] Embodiment 13: The antibody or antigen-binding fragment thereof according to any one of embodiments 10 to 12, wherein VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0295] Embodiment 14: The antibody or antigen-binding fragment thereof of embodiment 13, wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:12.

[0296] Embodiment 15: The antibody or antigen-binding fragment thereof according to any one of embodiments 10 to 12, wherein VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0297] Embodiment 16: The antibody or antigen-binding fragment thereof of embodiment 15, wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0298] Embodiment 17: An antibody or antigen-binding fragment thereof according to any one of embodiments 10 and 12 to 16, wherein the VL has the amino acid sequence set forth in SEQ ID NO: 20.

[0299] Embodiment 18: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0300] Embodiment 19: The antibody or antigen-binding fragment thereof of embodiment 18, wherein the antigen-binding fragment comprises a F(ab) fragment, a F(ab') fragment, or a single-chain variable fragment (scFV).

[0301] Embodiment 20: The antibody or antigen-binding fragment thereof of embodiment 19, wherein the antigen-binding fragment comprises a single-chain variable fragment (scFv).

[0302] Embodiment 21: The antibody or antigen-binding fragment thereof of embodiment 20, wherein the VH and VL of the scFv are separated by a peptide linker.

[0303] Embodiment 22: The antibody or antigen-binding fragment thereof of embodiment 21, 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.

[0304] Embodiment 23: The antibody or antigen-binding fragment thereof of embodiment 21 or embodiment 22, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 37.

[0305] Embodiment 24: The antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 23, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 67, 69, and 70.

[0306] Embodiment 25: A chimeric protein comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 24 and a heterologous molecule or moiety.

[0307] Embodiment 26: The chimeric protein of embodiment 25, wherein the chimeric protein is an antibody-drug conjugate and the heterologous molecule or moiety comprises a therapeutic agent.

[0308] Embodiment 27: The chimeric protein of embodiment 25, which is a chimeric antigen receptor (CAR), and wherein the heterologous molecule or moiety comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0309] Embodiment 28: The chimeric protein of embodiment 27, wherein the CAR comprises a transmembrane domain.

[0310] Embodiment 29: The chimeric protein of embodiment 27 or embodiment 28, wherein the CAR comprises one or more intracellular signaling domains.

[0311] Embodiment 30: The chimeric protein of any one of embodiments 27 to 29, wherein the CAR is an activated CAR comprising one or more intracellular signaling domains that stimulate an immune response.

[0312] Embodiment 31: The chimeric protein of any one of embodiments 27 to 29, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response.

[0313] Embodiment 32: The chimeric protein of embodiment 31, wherein the intracellular inhibitory domain comprises an enzyme inhibitory domain.

[0314] Embodiment 33: The chimeric protein of embodiment 31 or embodiment 32, wherein the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain.

[0315] Embodiment 34: The chimeric protein of any one of embodiments 27 to 33, wherein the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain.

[0316] Embodiment 35: The chimeric protein of embodiment 34, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52.

[0317] Embodiment 36: A composition comprising an antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 24, or a chimeric protein described in any one of embodiments 25 to 35, and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0318] Embodiment 37: An engineered nucleic acid encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 24, or the chimeric protein of any one of embodiments 25 to 35.

[0319] Embodiment 38: An expression vector comprising the engineered nucleic acid of embodiment 37.

[0320] Embodiment 39: A composition comprising the engineered nucleic acid of embodiment 37 or the expression vector of embodiment 38 and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, or a combination thereof.

[0321] Embodiment 40: A method of making an engineered cell, comprising transducing an isolated cell with the engineered nucleic acid of embodiment 37 or the expression vector of embodiment 38.

[0322] Embodiment 41: An isolated cell comprising the engineered nucleic acid of embodiment 37, the expression vector of embodiment 38, or the composition of embodiment 39.

[0323] Embodiment 42: A population of engineered cells expressing the engineered nucleic acid of embodiment 37 or the expression vector of embodiment 38.

[0324] Embodiment 43: An isolated cell comprising an antigen-binding fragment of any one of embodiments 1 to 24 or a chimeric protein of any one of embodiments 25 to 35.

[0325] Embodiment 44: A population of engineered cells expressing an antigen-binding fragment of any one of embodiments 1 to 24, or a chimeric protein of any one of embodiments 25 to 35.

[0326] Embodiment 45: The cell or population of cells according to any one of embodiments 41 to 44, wherein the chimeric protein is recombinantly expressed.

[0327] Embodiment 46: The cell or population of cells according to any one of embodiments 41 to 45, wherein the chimeric protein is expressed from a vector or from a selected locus derived from the genome of the cell.

[0328] Embodiment 47: The cell or population of cells according to any one of embodiments 41 to 46, wherein the cell or population of cells further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface.

[0329] Embodiment 48: The cell or population of cells of embodiment 47, wherein the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0330] Embodiment 49: The cell or population of cells of any one of embodiments 41-48, wherein the cell or population of cells is selected from the group consisting of: T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0331] Embodiment 50: The cell or population of cells according to any one of embodiments 41 to 49, wherein the cells are autologous.

[0332] Embodiment 51: A cell or population of cells according to any one of embodiments 41 to 49, wherein the cells are homogenous.

[0333] Embodiment 52: A pharmaceutical composition comprising an effective amount of the cell or population of engineered cells of any one of embodiments 41-51 and a pharma- ceutical acceptable carrier, a pharma-ceutical acceptable excipient, or a combination thereof.

[0334] Embodiment 53: A pharmaceutical composition comprising an effective amount of a genetically modified cell expressing an antigen-binding fragment of any one of embodiments 1 to 24, or a chimeric protein of any one of embodiments 25 to 35, and a pharma- ceutical acceptable carrier, a pharma-ceutical acceptable excipient, or a combination thereof.

[0335] Embodiment 54: The pharmaceutical composition of embodiment 52 or embodiment 53, for treating and / or preventing a tumor.

[0336] Embodiment 55: A method of treating a subject in need thereof, comprising administering a therapeutically effective dose of the composition of embodiment 36 or embodiment 39, or any of the cells of any one of embodiments 41-51, or the composition of embodiment 52 or embodiment 53.

[0337] Embodiment 56: A method of stimulating a cell-mediated immune response to tumor cells in a subject, comprising administering to a tumor-bearing subject a therapeutically effective dose of the composition of embodiment 36 or embodiment 39, or any of the cells of any one of embodiments 41 to 51, or the composition of embodiment 52 or embodiment 53.

[0338] Embodiment 57: The method of embodiment 56, comprising administering to a subject any of the cells of any one of embodiments 41 to 51, wherein the isolated cell or population of cells expresses a chimeric protein comprising an activating CAR of embodiment 31.

[0339] Embodiment 58: A method for inhibiting a cell-mediated immune response against tumor cells in a subject, comprising administering to a tumor-bearing subject a therapeutically effective dose of the composition of embodiment 36 or embodiment 39, or any of the cells of any one of embodiments 41 to 51, or the composition of embodiment 52 or embodiment 53.

[0340] Embodiment 59: The method of embodiment 58, comprising administering to a subject any of the cells of any one of embodiments 41 to 51, wherein the isolated cell or population of cells expresses a chimeric protein comprising an inhibitory CAR of embodiment 31.

[0341] Embodiment 60: A method of treating a subject having a tumor, comprising administering a therapeutically effective dose of the composition of embodiment 36 or embodiment 39, or any of the cells of any one of embodiments 41 to 51, or the composition of embodiment 52 or embodiment 53.

[0342] Embodiment 61: A kit for treating and / or preventing tumors, comprising a chimeric protein according to any one of embodiments 25 to 35.

[0343] Embodiment 62: The kit of embodiment 61, wherein the kit further comprises written instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0344] Embodiment 63: A kit for treating and / or preventing tumors comprising a cell or a population of cells according to any one of embodiments 41 to 51.

[0345] Embodiment 64: The kit of embodiment 63, wherein the kit further comprises written instructions for using the cells to treat and / or prevent a tumor in a subject.

[0346] Embodiment 65: A kit for treating and / or preventing tumors comprising an engineered nucleic acid according to embodiment 41.

[0347] Embodiment 66: The kit of embodiment 65, wherein the kit further comprises written instructions for using the nucleic acid to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0348] Embodiment 67: A kit for treating and / or preventing tumors comprising the vector of embodiment 38.

[0349] Embodiment 68: The kit of embodiment 67, wherein the kit further comprises written instructions for using the vector to produce one or more antigen-specific cells for treating and / or preventing a tumor in a subject.

[0350] Embodiment 69: A kit for treating and / or preventing tumors, comprising a composition according to embodiment 36, embodiment 39, embodiment 52, or embodiment 53.

[0351] Embodiment 70: The kit of embodiment 69, wherein the kit further comprises written instructions for using the composition to treat and / or prevent a tumor in a subject. EXAMPLES

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

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

[0354] Example 1: Construction of a humanized anti-EMCN antigen-binding domain To generate a humanized anti-EMCN antigen binding domain (e.g., scFv), the first, VH, and VL sequences from a human germline antibody were aligned to the murine-derived anti-EMCN VH and VL sequences of SEQ ID NO: 63 and SEQ ID NO: 64, respectively. The murine-derived anti-EMCN VH and VL sequences are shown in Table 5.

[0355] [Table 5]

[0356] Next, the framework regions surrounding the CDRs (as designated by the Kabat annotation and numbering scheme) were replaced with human germline antibody sequences. For the heavy chain human sequences, variable heavy chain 3-23 and 3-33 were selected for the framework regions. For the light chain, variable kappa chain 2-30 was selected for the framework regions. The germline sequences of VH 3-23, VH 3-33, and VK 2-30 are shown in Table 6.

[0357] [Table 6]

[0358] For the VH 3-33 based humanized VH, additional mutations were incorporated to backmutate residues present in mouse anti-EMCN at positions 50 and 52. Additionally, for both the 2-23 and 2-33 based humanized VH, CDR-H1 was engineered to match the human germline sequence of CDR positions 3 and 4 as specified by the Chothia annotation and numbering scheme.

[0359] Single chain variable fragments (scFv) were then engineered using each of the two humanized VH regions ("VH 3-23" and "VH 3-33") paired with a humanized VL region ("VL 2-30"). For each VH / VL combination, both orientations (N-terminus to C-terminus), VH-peptide linker-VL, and VL-peptide linker-VH were constructed. For all scFvs, the G4S3 peptide linker was used. The sequences are shown in Table 7.

[0360] [Table 7] TIFF2025513284000010.tif226161

[0361] Activating chimeric antigen receptors (aCARs) were engineered to contain humanized anti-EMCN antigen binding domains. The structures and sequences of the generated humanized anti-EMCN aCARs are shown in Table 8. Each aCAR contained a CD8 signal sequence and a FLAG tag at the N-terminus of the scFV, a CD28 transmembrane (TM) domain, a CD28 intracellular domain (ICD), and a CD3zeta activation domain.

[0362] [Table 8] TIFF2025513284000012.tif219162TIFF2025513284000013.tif202162TIFF2025513284000014.tif226161

[0363] Example 2: Humanized anti-EMCN antigen-binding domains retain binding function in T cell killing assays A CAR cell killing assay was performed to evaluate whether the humanized anti-EMCN antigen-binding domain allows the CAR to effectively target EMCN-expressing cells. The function of the humanized anti-EMCN scFv was compared to that of an activated CAR with the parent mouse anti-EMCN antigen-binding domain.

[0364] The CAR construct shown in Example 1 was cloned into a retroviral vector. In addition, as a control, the equivalent aCAR (SB02405) containing the antigen-binding domain of parental mouse anti-EMCN was also cloned into a retroviral vector. Then, the retrovirus was produced, and the primary T cells previously frozen and isolated from human donor PBMC were transduced with the retrovirus to express the CAR. On the 9th day after transduction, T cells and endomucin-expressing target cells were mixed and co-cultured (ET ratio: 1:1, 96-well plate, total medium volume of 200 μl). For the target cells, SEM and Molm13, two cell lines known to express potential cancer targets of interest for CAR-mediated killing (e.g., FLT3 (CD135) and CD33 (SIGLEC3)), were transduced to stably express EMCN, respectively. As controls, non-transduced SEM cells and non-transduced Molm13 were also used. Cells were harvested after 18 hours of co-incubation and T cell cytotoxicity against target cells was assessed by flow cytometry (analysis performed using FlowJo software) and presented as percent killing. The results of the killing assay are shown in Figure 1.

[0365] As shown in Figure 1, CARs containing each of the humanized anti-EMCN antigen-binding domains induced at least the same degree of killing or more killing of EMCN-expressing cell lines compared to CARs containing the parental murine anti-EMCN antigen-binding domain.

[0366] Example 3: Humanized anti-EMCN antigen-binding domain function is confirmed in an NK cell killing assay The functionality of the humanized anti-EMCN scFv was assessed by the T cell killing assay described in Example 2, and in this example was confirmed in a killing assay using NK cells expressing an activating CAR with a humanized anti-EMCN antigen-binding domain.

[0367] Retroviruses were produced using each of the retroviral vectors described in Example 2. Primary NK cells from donors were transduced with retroviruses to express CARs. Three days after transduction, CAR expression was evaluated by flow cytometry based on the FLAG epitope. The expression of each CAR on day 3 is shown in Figure 2. As shown in Figure 2, the expression of each of the CARs containing the humanized anti-EMCN antigen-binding domain was comparable to that of the CAR containing the parent antigen-binding domain.

[0368] Next, the killing of target cells by CAR NK cells was evaluated. Seven days after transduction, CAR NK cells and Endomucin-expressing target cells (EMCN-transduced SEM cells as described in Example 2) were mixed together and co-cultured (ET ratio: 1:1, 96-well plate, total medium volume 200 μl). As a control, the NK cell killing of non-transduced SEM cells was also evaluated. Cells were harvested after 18 hours of co-incubation, and T cell cytotoxicity against target cells was evaluated by flow cytometry (analysis performed using FlowJo software) and presented as killing percentage. The results of the killing assay are shown in Figure 3.

[0369] As shown in Figure 3, NK cells expressing the humanized anti-EMCN aCAR induced at least the same degree of killing of EMCN-expressing cell lines compared to a CAR containing the parental murine anti-EMCN antigen-binding domain.

[0370] Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0371] Equivalent While various specific embodiments have been shown and described, the above specification is not limiting. It will be understood that various changes can be made without departing from the spirit and scope of the disclosure. Many variations will be apparent to those skilled in the art in light of the scope of this specification.

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to human endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH comprises a VH complementary region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 6, and a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:

8. The VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:

11. The antibody or its antigen-binding fragment is humanized. The antibody or its antigen-binding fragment.

2. An isolated antibody or its antigen-binding fragment that specifically binds to human endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH comprises a VH complementary region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:

8. The VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:

11. The antibody or its antigen-binding fragment is optionally humanized. The antibody or its antigen-binding fragment.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the VH has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the VL has an amino acid sequence described in SEQ ID NO:

20.

4. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment is an antigen-binding fragment, and optionally the antigen-binding fragment comprises an F(ab) fragment, an F(ab') fragment, or a single-stranded variable fragment (scFV), and optionally the antigen-binding fragment comprises a single-stranded variable fragment (scFv).

5. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the VH and VL of the scFv are separated by a peptide linker, and optionally the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain, and optionally the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 37.

6. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 67, 69, and 70.

7. A chimeric protein comprising an antibody or its antigen-binding fragment and a heterologous molecule or heterologous portion, The antibody or its antigen-binding fragment (i) An isolated antibody or antigen-binding fragment thereof that specifically binds to human endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH comprises a VH complementary region 1 (CDRH1) having the amino acid sequence of SEQ ID NO: 1, a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 6, and a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:

8. The VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:

11. The antibody or its antigen-binding fragment is humanized. The antibody or its antigen-binding fragment, or (ii) An isolated antibody or antigen-binding fragment thereof that specifically binds to human endomucin (EMCN), comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH comprises a VH complementary region 1 (CDRH1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, a VH complementary region 2 (CDRH2) having the amino acid sequence of SEQ ID NO: 7, and a VH complementary region 3 (CDRH3) having the amino acid sequence of SEQ ID NO:

8. The VL includes a VL complementary region 1 (CDRL1) having the amino acid sequence of SEQ ID NO: 9, a VL complementary region 2 (CDRL2) having the amino acid sequence of SEQ ID NO: 10, and a VL complementary region 3 (CDRL3) having the amino acid sequence of SEQ ID NO:

11. The antibody or its antigen-binding fragment is optionally humanized. The antibody or its antigen-binding fragment; Selectively, the chimeric protein is an antibody-drug conjugate, and the heterologous molecule or heterologous portion contains a therapeutic agent. Optionally, the chimeric protein is a chimeric antigen receptor (CAR), and the heterologous molecule or heterologous portion comprises a polypeptide selected from the group consisting of a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof, and optionally, the CAR comprises a transmembrane domain. Optionally, the CAR comprises one or more intracellular signaling domains; 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. Selectively, the intracellular inhibitory domain includes an intracellular inhibitory co-signaling domain. Optionally, the CAR includes a spacer region between the antigen-binding domain and the transmembrane domain. The spacer region optionally has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52. The aforementioned chimeric protein.

8. An engineered nucleic acid encoding an antibody or antigen-binding fragment thereof according to claim 1 or 2, or a chimeric protein according to claim 7.

9. An expression vector comprising the manipulated nucleic acid described in claim 8.

10. A composition comprising an antibody or antigen-binding fragment thereof as described in claim 1 or 2, or a chimeric protein as described in claim 7, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

11. A method for producing engineered cells, comprising transducing isolated cells with the engineered nucleic acid described in claim 8.

12. Isolated cells comprising the antigen-binding fragment described in claim 1 or 2 or the chimeric protein described in claim 7.

13. A population of manipulated cells expressing the manipulated nucleic acid described in claim 8.

14. The aforementioned chimeric protein is expressed by recombination, The chimeric protein is optionally expressed from a vector or from a locus selected from the cell's genome. Optionally, the cells further comprise one or more tumor-targeting chimeric receptors expressed on the cell surface. Selectively, each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or a modified T cell receptor. The cells are optionally selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, 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 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, myelocytes, 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 selected from the group consisting of autologous cells, optionally selected from the group consisting of autologous cells, and optionally selected from the group consisting of iPSCs. The cell according to claim 12.

15. A pharmaceutical composition comprising an effective amount of the isolated cells described in claim 12, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, for use optionally to treat and / or prevent a tumor.

16. The composition according to claim 10 for treating a subject that requires such treatment.

17. The composition according to claim 10 for inhibiting a cell-mediated immune response against tumor cells in a subject having a tumor.

18. The composition according to claim 10 for treating a subject having a tumor.

19. A kit for treating and / or preventing tumors, comprising the engineered nucleic acid described in Claim 8, Optionally, the kit further includes written instructions for using the nucleic acid to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject. The aforementioned kit.

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