Chimeric receptor and method of use thereof

Abstract

Provided herein are EMCN-specific antigen-binding domains and chimeric proteins comprising the EMCN-specific antigen-binding domains. Also provided herein are cells, nucleic acids, vectors, compositions, and methods relating to proteins comprising the EMCN-specific antigen-binding domains.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 151,483, filed on 19 February 2021, and U.S. Provisional Application No. 63 / 092,736, filed on 16 October 2020, each of which is incorporated herein by reference in whole for all purposes.

[0002] Sequence List This application includes a sequence listing filed via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy thereof, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and has a size of X,XXX,XXX bytes. [Background technology]

[0003] Chimeric antigen receptor (CAR) 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 remission in patients with CD19-expressing malignancies that have resulted in drug resistance and tumor progression due to chemotherapy. The success of CD19 CAR therapy offers optimism for treating other hematological malignancies, such as acute myeloid leukemia (AML), the most common acute leukemia in adults. AML is a cancer of the myeloid blood cells, characterized by the rapid proliferation of abnormal cells that accumulate in the bone marrow and blood, interfering with normal blood cells. Sometimes AML can spread to the brain, skin, or gums. Standard chemotherapy for AML has remained virtually unchanged for the past 40 years (Pulte et al., 2008), and overall survival rates remain very low.

[0004] One challenge in developing CAR therapies for AML is the lack of suitable targets. The ability to identify appropriate CAR targets is crucial for effectively targeting and treating tumors without damaging normal cells that express the same target antigen. Therefore, the need for CAR-T cell-based AML therapies that target AML cells without targeting normal cells or tissues remains. [Overview of the project]

[0005] Chimeric proteins containing an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety are provided herein. The antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, (a) VH comprises heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and VL comprises KSSQSLVA It includes light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of SDENTYLN (SEQ ID NO: 10), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12), and the amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are K Defined based on the abat numbering scheme, (b) VH includes heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and VL has the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10). It includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12), and the amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia numbering scheme;Alternatively, (c) VH includes heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3) contained within the VH region amino acid sequence of SEQ ID NO: 1; VL includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3) contained within the VL region amino acid sequence of SEQ ID NO: 9; optionally, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat or Chothia numbering scheme.

[0006] In some embodiments, the antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region, where VH includes a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and VL includes KSSQSLVASDENTYLN ( It includes light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of sequence number 10), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). The amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Kabat numbering scheme.

[0007] In some embodiments, the antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region, where VH includes a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence RIKD (SEQ ID NO: 4), and VL includes KSSQSLVASDENTYLN (SEQ ID NO: 10) The light chain complementarity determination region includes light chain complementarity determination region 1 (CDR-L1) having an amino acid sequence, light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). The amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia numbering scheme.

[0008] In some embodiments, the antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region, where VH includes a heavy chain complementarity-determining region 1 (CDR-H1) contained within the VH region amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity-determining region 2 (CDR-H2) contained within the VH region amino acid sequence of SEQ ID NO: 1, and a heavy chain complementarity-determining region 3 (CDR-H3) contained within the VH region amino acid sequence of SEQ ID NO: 1, and VL is contained within the VL region amino acid sequence of SEQ ID NO: 9 It includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2) contained within the VL region amino acid sequence of SEQ ID NO: 9, and light chain complementarity determination region 3 (CDR-L3) contained within the VL region amino acid sequence of SEQ ID NO: 9. Optionally, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat or Chothia numbering scheme.

[0009] In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region contains the amino acid sequence of SEQ ID NO: 9.

[0010] In some aspects, the antigen binding domain comprises a single-chain variable fragment (scFv). In some aspects, the VH and VL of the scFv are separated by a peptide linker. In some aspects, the antigen binding domain comprises the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. In some aspects, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 22.

[0011] In some aspects, 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.

[0012] In some aspects, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some aspects, the intracellular inhibitory domain comprises an enzyme inhibitory domain or an intracellular inhibitory co-signaling domain.

[0013] Also provided herein are engineered nucleic acids encoding the chimeric proteins described above.

[0014] Also provided herein are expression vectors comprising the engineered nucleic acids described above.

[0015] Also provided herein are isolated cells comprising the engineered nucleic acids described above.

[0016] Also provided herein are populations of engineered cells expressing the engineered nucleic acids or expression vectors described above.

[0017] In some embodiments, isolated cells or populations of cells further comprise one or more tumor-targeted chimeric receptors expressed on the cell surface. In some embodiments, one or more tumor-targeted chimeric receptors are chimeric antigen receptors (CARs) or engineered T cell receptors.

[0018] In some embodiments, the cells or population of cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0019] Pharmaceutical compositions comprising an effective amount of a previously described population of cells or manipulated cells, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, are also provided herein.

[0020] Also provided herein is a method for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a subject having a tumor a therapeutically effective dose of either previously described cells or a previously described composition.

[0021] A method for treating a subject having a tumor, comprising administering either a previously described cell or a previously described composition in a therapeutically effective dose, is also provided herein.

[0022] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, and the VH region comprises a heavy-chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence RIKD (SEQ ID NO: 4).

[0023] Chimeric proteins containing an antigen-binding domain and heterologous molecules or moieties specific to endomucin (EMCN) are also provided herein, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region comprises a heavy chain complementarity-determining region 1 (CDR-H1), a heavy chain complementarity-determining region 2 (CDR-H2), and a heavy chain complementarity-determining region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the amino acid sequence of the VH region of SEQ ID NO: 1.

[0024] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH comprises (a) a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), or (b) a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4).

[0025] In some embodiments, the VL includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Chothia numbering scheme. In some embodiments, VL includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12).

[0026] Chimeric proteins containing an antigen-binding domain and heterologous molecules or moieties specific to endomucin (EMCN) are also provided herein, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VL comprising a light chain complementarity-determining region 1 (CDR-L1), a light chain complementarity-determining region 2 (CDR-L2), and a light chain complementarity-determining region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Chothia numbering scheme.

[0027] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VL comprising a light-chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), a light-chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence QVSKLDS (SEQ ID NO: 11), and a light-chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12).

[0028] In some embodiments, VH comprises heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, VH comprises heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4). In some embodiments, VH includes a heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4).

[0029] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety are also provided herein, wherein the antigen-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH is (a) a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), or (b) a heavy chain phase having the amino acid sequence of RYDMH (SEQ ID NO: 102). The complementarity-determining region 1 (CDR-H1), the heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and the heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) are included. VL includes the light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), the light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and the light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0030] In some embodiments, the VH region includes 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 with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH region includes the amino acid sequence of SEQ ID NO: 1.

[0031] In some embodiments, the VL region includes 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 with the amino acid sequence of SEQ ID NO: 9.

[0032] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, the 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 with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 9.

[0033] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment, and the antibody or antigen-binding fragment comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VL 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 with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL region comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 1.

[0034] Chimeric proteins comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain competes with a reference antibody or its antigen-binding fragment for binding to EMCN, and the reference antibody or its antigen-binding fragment comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein VH has a heavy-chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2) and a WGNGN (SEQ ID NO: 3) amino acid sequence The heavy chain complementarity determination region 2 (CDR-H2) and the heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) are included, and the VL includes the light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), the light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and the light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0035] Chimeric proteins comprising an endomucin (EMCN)-specific antigen-binding domain and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain is essentially bound to the same EMCN epitope as the reference antibody or its antigen-binding fragment, and the reference antibody or its antigen-binding fragment comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein VH has the amino acid sequence GFSLSRY (SEQ ID NO: 2) and WGNGN (SEQ ID NO: 3). The VL includes a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of RIKD (SEQ ID NO: 4), and a light chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0036] Chimeric proteins comprising an endomucin (EMCN)-specific antigen-binding domain and a heterologous molecule or portion are also provided herein, wherein the antigen-binding domain binds to the same human EMCN epitope as the EMCN epitope bound by the reference antibody or its antigen-binding fragment, and the reference antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, where VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), WGNGN (SEQ ID NO: 3). The VL region includes a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of , and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4). The VL region includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). In some embodiments, the VH region of the reference antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL region of the reference antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 9.

[0037] In some embodiments, the antigen-binding domain includes an F(ab) fragment, an F(ab') fragment, or a single-stranded variable fragment (scFv). In some embodiments, the antibody or antigen-binding fragment includes a single-stranded variable fragment (scFv).

[0038] In some embodiments, the VH and VL of 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 the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-39.

[0039] In some embodiments, scFv includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-22.

[0040] In some embodiments, the chimeric protein is an antibody-drug conjugate, in which the heterologous molecule or portion contains a therapeutic agent.

[0041] In some embodiments, the chimeric protein is a chimeric antigen receptor (CAR), and the heterogeneous molecule or 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. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR is an activated CAR comprising one or more intracellular signaling domains that stimulate an immune response. In some embodiments, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some embodiments, the intracellular inhibitory domain comprises an enzyme inhibitory domain. In some embodiments, the intracellular inhibitory domain comprises an intracellular inhibitory co-signaling domain. In some embodiments, the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-48.

[0042] Single-stranded variable fragments (scFv) specific to endomucin (EMCN), including heavy-chain variable (VH) and light-chain variable (VL) regions, are also provided herein. VH contains heavy chain complementarity-determining region 3 (CDR-H3) which has the amino acid sequence of RIKD (SEQ ID NO: 4).

[0043] Endomucin (EMCN)-specific single-strand variable fragments (scFv) are also provided herein, the antigen-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VH region comprising 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), the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the reference antibody are defined based on the Kabat numbering scheme. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the reference antibody are defined based on the Chothia numbering scheme.

[0044] Endomucin (EMCN)-specific single-chain variable fragments (scFv) are also provided herein, the antigen-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VH region comprising (a) a heavy-chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), a heavy-chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy-chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CD The amino acid sequences of R-L2 and CDR-L3 are defined based on the Kabat numbering scheme; or (b) the reference antibody comprises heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), wherein the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia numbering scheme.

[0045] In some embodiments, the VL of scFv includes a light chain complementarity determination region 1 (CDR-L1), a light chain complementarity determination region 2 (CDR-L2), and a light chain complementarity determination region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9.

[0046] In some embodiments, the VL of scFv includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12).

[0047] Endomucin (EMCN)-specific single-strand variable fragments (scFv) are also provided herein, the antigen-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VL comprising a light-chain complementarity-determining region 1 (CDR-L1), a light-chain complementarity-determining region 2 (CDR-L2), and a light-chain complementarity-determining region 3 (CDR-L3), the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 being contained within the VL region amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme. In some embodiments, the amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia numbering scheme.

[0048] A single-stranded variable fragment (scFv) specific to endomucin (EMCN) is also provided herein, the antigen-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VL comprising a light-chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), a light-chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence QVSKLDS (SEQ ID NO: 11), and a light-chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12).

[0049] In some embodiments, the VH region of scFv includes heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the reference antibody are defined based on the Kabat numbering scheme. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the reference antibody are defined based on the Chothia numbering scheme.

[0050] In some embodiments, the VH of scFv is a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), It includes heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4).

[0051] In some embodiments, the VH of scFv includes a heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4).

[0052] A single-stranded variable fragment (scFV) specific to endomucin (EMCN) is also provided herein, wherein the antigen-binding domain comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, and VH comprises (a) a heavy-chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy-chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy-chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), or (b) a heavy-chain complementarity-determining region 1 (C) having the amino acid sequence of RYDMH (SEQ ID NO: 102). The CDR-H1) includes a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4). The VL includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0053] In some embodiments, the VH of scFv includes 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 with the amino acid sequence of SEQ ID NO: 1.

[0054] In some embodiments, the VH of scFv contains the amino acid sequence of SEQ ID NO: 1.

[0055] In some embodiments, the VL of scFv includes 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 with the amino acid sequence of SEQ ID NO: 9.

[0056] In some embodiments, the VL of scFv contains the amino acid sequence of SEQ ID NO: 9.

[0057] Endomucin (EMCN)-specific single-stranded variable fragments (scFVs) are also provided herein. The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO: 1.

[0058] In some embodiments, the VH region of scFv contains the amino acid sequence of SEQ ID NO: 1.

[0059] In some embodiments, the VL region of scFv includes 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 with the amino acid sequence of SEQ ID NO: 9.

[0060] In some embodiments, the VL region of scFv contains the amino acid sequence of SEQ ID NO: 9.

[0061] Also provided herein are single-stranded variable fragments (scFVs) containing an antigen-binding domain specific to endomucin (EMCN), wherein the antigen-binding domain comprises an antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the VL 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 with the amino acid sequence of SEQ ID NO: 9.

[0062] In some embodiments, the VH region of scFv contains the amino acid sequence of SEQ ID NO: 1.

[0063] A single-chain variable fragment (scFV) containing an antigen-binding domain specific to endomucin (EMCN) is also provided herein, wherein the antigen-binding domain competes with a reference antibody or its antigen-binding fragment for binding to EMCN, and the reference antibody or its antigen-binding fragment comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein VH comprises a heavy-chain complementarity determination region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), a heavy-chain complementarity determination region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), and a heavy-chain complementarity determination region having the amino acid sequence RIKD (SEQ ID NO: 4). Including region 3 (CDR-H3), VL includes light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on Chothia annotation and numbering schemes.

[0064] A single-stranded variable fragment (scFV) containing an antigen-binding domain specific to endomucin (EMCN) is also provided herein. The antigen-binding domain essentially binds to the same EMCN epitope as the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH includes heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence RIKD (SEQ ID NO: 4). VL includes light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10). It includes a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). The amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme.

[0065] In some embodiments, the previously described scFv includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-22.

[0066] A single-stranded variable fragment (scFV) containing an antigen-binding domain specific to endomucin (EMCN) is also provided herein. The antigen-binding domain binds to the same human EMCN epitope as the EMCN epitope bound by the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH includes heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence RIKD (SEQ ID NO: 4). VL includes light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10). It includes a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). The amino acid sequences of the reference antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme.

[0067] In some embodiments, the VH region of the reference antibody or its antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 1.

[0068] In some embodiments, the VL region of the reference antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 9.

[0069] Compositions comprising either a chimeric protein or scFv provided herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, are also provided herein.

[0070] Manipulated nucleic acids encoding either a chimeric protein or scFv, as provided herein, are also provided herein. Expression vectors containing any of the manipulated nucleic acids provided herein are also provided herein.

[0071] Compositions comprising any of the manipulated nucleic acids provided herein or any of the expression vectors provided herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, are also provided herein.

[0072] A method for producing engineered cells, comprising transducing isolated cells using any of the engineered nucleic acids or expression vectors provided herein, is also provided herein.

[0073] Manipulated cells produced by any of the methods provided herein are also provided herein.

[0074] Isolated cells containing any of the manipulated nucleic acids provided herein, any of the expression vectors provided herein, or any of the compositions provided herein are also provided herein.

[0075] A population of manipulated cells expressing any of the manipulated nucleic acids or any of the expression vectors provided herein is also provided herein.

[0076] Isolated cells or populations of cells containing either a chimeric protein or scFv, as provided herein, are also provided herein.

[0077] Populations of engineered cells expressing any of the chimeric proteins provided herein are also provided herein. In some embodiments, the chimeric proteins are recombinantly expressed. In some embodiments, the chimeric proteins are expressed from a vector or a locus selected from the cell genome. In some embodiments, the cells or populations of cells further include one or more tumor-targeted chimeric receptors expressed on the cell surface. In some embodiments, each of the one or more tumor-targeted chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor. In some embodiments, the cells or population of cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0078] Pharmaceutical compositions comprising an effective amount of any of the manipulated cells or populations provided herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, are also provided herein.

[0079] Pharmaceutical compositions comprising genetically modified cells expressing any of the chimeric proteins provided herein in an effective amount, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof, are also provided herein. In some embodiments, the pharmaceutical compositions are for the treatment and / or prevention of tumors.

[0080] A method for treating a subject in need thereof, comprising administering a therapeutically effective dose of any of the compositions or cells provided herein, is also provided herein.

[0081] Also provided herein is a method for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a subject having a tumor a therapeutically effective dose of any of the compositions provided herein or any of the cells provided herein.

[0082] A method for treating a subject having a tumor is also provided herein, comprising administering a therapeutically effective dose of any of the compositions provided herein or any of the cells provided herein.

[0083] In some embodiments, the method comprises administering a composition comprising immune checkpoint cells or a population of cells expressing an inhibitor described herein, wherein the cells or population of cells further express one or more tumor-targeted chimeric receptors. In some embodiments, the method comprises administering an equivalent composition comprising one or more tumor-targeted chimeric receptors but lacking inhibitory CARs, resulting in reduced off-target effects.

[0084] Kits for treating and / or preventing tumors, comprising one of the chimeric proteins provided herein, are also provided herein. In some embodiments, the kit further includes described instructions for using the chimeric protein to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0085] Kits for treating and / or preventing tumors, comprising either cells or populations of cells provided herein, are also provided herein. In some embodiments, the kit further includes a written instruction for using the cells to treat and / or prevent tumors in a subject.

[0086] Kits for treating and / or preventing tumors, comprising any of the isolated nucleic acids provided herein, are also provided herein. In some embodiments, the kit further includes described instructions for using the nucleic acids to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0087] Kits for treating and / or preventing tumors, comprising one of the vectors provided herein, are also provided herein. In some embodiments, the kit further includes described instructions for using the vectors to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject.

[0088] Kits for treating and / or preventing tumors, comprising any of the compositions provided herein, are also provided herein. In some embodiments, the kit further includes a set of instructions for using the compositions for treating and / or preventing tumors in a subject. [Invention 1001] A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. (a) The above VH is Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, The aforementioned VL is, Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Kabat numbering scheme, or (b) The above VH is Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4). Includes, The aforementioned VL is, Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). Includes, The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the aforementioned reference antibody are defined based on the Chothia numbering scheme, or (c) The above VH is Heavy chain complementarity determination region 1 (CDR-H1) contained within the VH region amino acid sequence of SEQ ID NO: 1, heavy chain complementarity determination region 2 (CDR-H2) contained within the VH region amino acid sequence of SEQ ID NO: 1, and heavy chain complementarity determination region 3 (CDR-H3) contained within the VH region amino acid sequence of SEQ ID NO: 1 Includes, The aforementioned VL is, Light chain complementarity determination region 1 (CDR-L1) contained within the VL region amino acid sequence of SEQ ID NO: 9, light chain complementarity determination region 2 (CDR-L2) contained within the VL region amino acid sequence of SEQ ID NO: 9, and light chain complementarity determination region 3 (CDR-L3) contained within the VL region amino acid sequence of SEQ ID NO: 9 Includes, Optionally, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat or Chothia numbering scheme. The aforementioned chimeric protein. [Invention 1002] The chimeric protein of the present invention 1001, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1. [Invention 1003] The VL region comprises the amino acid sequence of SEQ ID NO: 9, a chimeric protein according to the present invention 1001 or 1002. [Invention 1004] The aforementioned antigen-binding domain comprises a single-stranded variable fragment (scFv), and is a chimeric protein according to any of the present invention 1001 to 1003. [Invention 1005] The chimeric protein of the present invention 1004, wherein the VH and VL of the scFv are separated by a peptide linker. [Invention 1006] The chimeric protein of the present invention 1005, wherein the antigen-binding domain 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. [Invention 1007] The aforementioned scFv is a chimeric protein according to any of the present invention 1004 to 1006, wherein the scFv contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 17 to 22. [Invention 1008] The chimeric protein according to any one of the present invention 1001 to 1007, wherein the chimeric protein is a chimeric antigen receptor (CAR), and the heterogeneous molecule or 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. [Invention 1009] The chimeric protein of the present invention 1008, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. [Invention 1010] The chimeric protein of the present invention 1009, wherein the intracellular inhibitory domain includes an enzyme inhibitory domain or an intracellular inhibitory co-signaling domain. [Invention 1011] A modified nucleic acid encoding any of the chimeric proteins of the present invention 1001 to 1010. [Invention 1012] An expression vector comprising the manipulated nucleic acid of the present invention 1011. [Invention 1013] Isolated cells containing the manipulated nucleic acid of Invention 1011 or the expression vector of Invention 1012. [Invention 1014] A population of manipulated cells expressing the manipulated nucleic acid of Invention 1011 or the expression vector of Invention 1012. [Invention 1015] A cell or population of cells according to the present invention 1013 or 1014, further comprising one or more tumor-targeting chimeric receptors expressed on the cell surface. [Invention 1016] The cell or population of cells of the present invention 1015, wherein each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor. [Invention 1017] A cell or population of cells according to any of the present invention 1013 to 1016, selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. [Invention 1018] A pharmaceutical composition comprising an effective amount of any of the cells or manipulated cells according to invention 1013 to 1017, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. [Invention 1019] A method for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a subject having a tumor a therapeutically effective dose of any of the cells of any of Invention 1013 to 1017 or a composition of Invention 1018. [Invention 1020] A method for treating a subject having a tumor, comprising administering a therapeutically effective dose of any of the cells of any of Invention 1013 to 1017 or a composition of Invention 1018. [Brief explanation of the drawing]

[0089] This patent or application document includes at least one drawing made in color. Copies of this patent or patent application publication including the color drawing are available from the Patent Office upon request and payment of the necessary fees.

[0090] These and other features, aspects, and advantages of this disclosure will be better understood in relation to the following description and accompanying drawings.

[0091] [Figure 1] Figure 1 shows the sequencing results of the light chain variable region of antibody 1 (Ab1) using the Chothia naming scheme. Figure 1 discloses sequence number 9.

[0092] [Figure 2] Figure 2 shows the sequencing results of the heavy chain variable region of antibody 1 (Ab1) using the Chothia naming scheme. Figure 2 discloses sequence number 1.

[0093] [Figure 3A]Figure 3A shows the gating strategy for establishing an EMCN expression baseline.

[0094] [Figure 3B] Figure 3B shows a control cell line that has not been transduced by the virus and exhibits baseline EMCN expression.

[0095] [Figure 3C] Figure 3C shows the transduced cell line exhibiting EMCN expression 3 days after transduction.

[0096] [Figure 4A] Figure 4A shows transduced cell lines exhibiting EMCN expression 24 days after transduction following 21 days of drug selection with 1 μg / mL puromycin.

[0097] [Figure 4B] Figure 4B shows transduced cell lines exhibiting EMCN expression 24 days after transduction following 21 days of drug selection with 0.5 μg / mL puromycin.

[0098] [Figure 5] Figure 5 shows a gating strategy for establishing an EMCN expression baseline for the analysis of cells manipulated to express EMCN.

[0099] [Figure 6A] Figure 6A shows the gating strategy established for establishing a CAR expression baseline, using a control that has not undergone viral transduction.

[0100] [Figure 6B] Figure 6B shows the CAR expression in transduced cells for CAR constructs SB00819, SB01052, SB02405, and SB02406.

[0101] [Figure 6C]Figure 6C shows the CAR expression in transduced cells for CAR constructs SB02407, SB02408, SB02409, and SB02410.

[0102] [Figure 7A] Figure 7A shows the euthanasia percentage (standardized against non-transduced T cell controls) after co-culture of FLT3-specific or CD33-specific CAR T cells with parental Molm13 (upper panel) or SEM (lower panel).

[0103] [Figure 7B] Figure 7B shows the euthanasia percentage (standardized against non-transfected T cell controls) after co-culture with Molm13 (upper panel) or SEM (lower panel) target cells manipulated to express EMCN in FLT3-specific or CD33-specific CAR T cells.

[0104] [Figure 8] Figure 8 shows the expression profiles of various iCAR forms with anti-FLT3 aCAR and anti-EMCN binding domains, including co-expression after transduction in NK cells, as evaluated by flow cytometry. 1–3 biological copies (shown as separate points) are shown for each condition.

[0105] [Figure 9] Figure 9 shows NK cell-mediated killing (upper panel) and cytokine secretion (lower panel). Shown are various NK cells manipulated to co-express anti-FLT3 aCAR and anti-EMCN iCAR. “Separate” = each type of SEM cell presented separately (upper left panel). “Mixed” = both types of SEM cells mixed together in the same culture (upper right panel). 1-3 biological replicas per condition (shown as separate points). 3 technical replicas per measurement, with X and Y SEMs plotted where applicable. KLRG1, which is negative for iCAR protection, is not shown.

[0106] [Figure 10] Figure 10 shows the euthanasia rates of hematopoietic stem cells and progenitor cells (HSPCs) co-cultured with NK cells expressing various chimeric antigen receptors. [Modes for carrying out the invention]

[0107] Detailed explanation The practices described herein, unless otherwise indicated, employ conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the scope of the art. Such methods are fully described in the literature, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006); Fundamental Virology, 3 rd Edition,vol.I & II(BNFields and DMKnipe,eds.);Handbook of Experimental Immunology,Vols.I-IV(DMWeir and CCBlackwell eds.,Blackwell Scientific Publications);ALLehninger,Biochemistry(Worth Publishers,Inc.,current addition);Sambrook,et al.,Molecular Cloning:A Laboratory Manual(3 rd See Edition, 2001; Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0108] definition Unless otherwise defined, all terms, notations, and other scientific terms used herein are intended to have meanings generally understood by those skilled in the art. In some cases, terms having generally understood meanings are defined herein for clarity and / or immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from what is generally 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, for example, 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 manufacturers, unless otherwise stated.

[0109] As used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise. Terms such as "includes" and "etc." are intended to convey inclusion without limitation unless otherwise indicated.

[0110] As used herein, the term “including” also specifically includes embodiments that “consist of” and “essentially consist of” the listed elements, unless otherwise indicated.

[0111] The term "approximately" indicates and encompasses both the value and the range above and below that value. In certain embodiments, the term "approximately" indicates a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "approximately" indicates a specified value ± one standard deviation of that value.

[0112] As used herein, the terms “stimulating a cell-mediated immune response” or “stimulating an immune response” refer to the generation of signals by one or more cell types or cell populations that result in an immune response. Immunostimulatory activity may include pro-inflammatory activity. In various embodiments, the immune response occurs after the activation of immune cells (e.g., T cells or NK cells) or, non-limited, simultaneously via receptors, including 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 having ligands stimulate receptors of pro-inflammatory polypeptides and / or their ligands may enhance the immune response of immune-responsive cells. While not bound by any particular theory, receiving multiple stimulatory signals (e.g., co-stimulation) is crucial for carrying out robust and long-lasting cell-mediated immune responses, such as T-cell-mediated immune responses, in which T cells may be inhibited and unresponsive to antigens (also called "T-cell anergy") in the absence of co-stimulatory signals. The various effects of co-stimulatory signals are not fully understood, especially when combined with each other, but co-stimulation generally leads to increased gene expression to generate long-lived, proliferative, and apoptosis-resistant cells, such as T cells or NK cells, that respond strongly to antigens, for example, when mediating the complete and / or sustained elimination of target cells expressing congenital antigens.

[0113] As used herein, the terms “chimeric antigen receptor” or, alternatively, “CAR” refer to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an “intracellular signaling domain”) which includes a functional signaling domain.

[0114] As used herein, the terms “activated CAR” or “aCAR” refer to a CAR construct / structure that has the ability to induce changes in signaling or protein expression in activated CAR-expressing cells that initiate, activate, stimulate, or increase an immune response upon binding to a congeneral aCAR ligand.

[0115] As used herein, the terms “inhibitory CAR” or “iCAR” refer to a CAR construct / structure that has the ability to induce alterations in signaling or protein expression in inhibitory CAR-expressing cells that inhibit, attenuate, reduce, decrease, suppress, or repress the immune response in response to a congeneral iCAR ligand, including a reduction in the activation of one or more stimulating signals, including co-stimulatory signals, or immune-responsive cells that have received or are receiving such signals.

[0116] As used herein, the term “intracellular signaling domain” refers to a functional portion of a protein that acts by transmitting information within a cell to modulate cellular activity via a defined signaling pathway, either by generating a second messenger or by acting as an effector in response to such a messenger.

[0117] As used herein, the terms “extracellular antigen-binding domain” or “antigen-binding domain” (ABD) refer to a polypeptide sequence or polypeptide complex that specifically recognizes or binds to a given antigen or epitope, such as a polypeptide sequence or polypeptide complex portion of a chimeric protein described herein that results in EMCN-specific binding. An ABD (or antibody, antigen-binding fragment, and / or a chimeric protein containing it) is said to “recognize” the epitope (or more commonly, antigen) to which the ABD specifically binds, and the epitope is said to be the “recognition specificity” or “binding specificity” of the ABD. An ABD is said to bind to its specific antigen or epitope with a particular affinity. As described herein, “affinity” refers to the strength of the non-covalent intermolecular force interaction between one molecule and another. Affinity, i.e., the strength of the interaction, can be expressed as the dissociation equilibrium constant (KD), where a lower KD value indicates a stronger intermolecular interaction. The KD value of an antibody construct is 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, evaluated by affinity, may refer to binding molecules having affinity between ABD and its congener antigen or epitope, and the KD value is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M is less than M. Specific binding can also include the recognition and binding of a biological molecule of interest (e.g., polypeptide), while not specifically recognizing or binding to other molecules in a sample, e.g., a biological sample naturally containing the polypeptide of this disclosure. In certain embodiments, specific binding refers to the binding of an epitope or antigen or antigenic determinant of an ABD, antibody, or antigen-binding fragment in such a manner that the binding may be substituted for or compete with a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0118] ABD can be an antibody. As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be of natural or recombinant origin. Antibodies can be tetramers of immunoglobulin molecules.

[0119] An ABD can be an antigen-binding fragment of an antibody. As used herein, the term “antigen-binding fragment” refers to at least one portion of an intact antibody or 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, single-domain antibodies such as Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, sdAb(VL or VH), a camel VHH domain, and a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at a hinge region, as well as isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1 136, 2005). Antigen-binding fragments can also be transplanted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies).

[0120] The number of antigen-binding domains (ABDs) in a binding molecule such as the chimeric protein described herein defines the "valency" of the binding molecule. A binding molecule having a single ABD is "monovalent". A binding molecule having a plurality of ABDs is referred to as "multivalent". A multivalent binding molecule having two ABDs is "bivalent". A multivalent binding molecule having three ABDs is "trivalent". A multivalent binding molecule having four ABDs is "tetravalent". In various multivalent embodiments, all of the plurality of ABDs have the same recognition specificity, and can be referred to as a "monospecific multivalent" binding molecule. In other multivalent embodiments, at least two of the plurality of ABDs have different recognition specificities. Such binding molecules are multivalent and "multispecific". In multivalent embodiments 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 a plurality of recognition specificities for different epitopes present on the same antigen, the binding molecule is "multiparatopic". A multivalent embodiment in which ABDs collectively recognize two epitopes on the same antigen is "biparatopic".

[0121] In various multivalent embodiments, multivalent binding molecules improve the binding avidity of the binding molecule for a particular target. As described herein, "binding avidity" refers to the overall strength of interaction between two or more molecules, for example, between a multivalent binding molecule and a particular target, and binding avidity is the cumulative strength of interaction provided by the affinities of a plurality of ABDs. Binding avidity can be measured by the same methods used to determine affinity as described above. In certain embodiments, the binding avidity of a binding molecule for a specific target is such that the interaction is a specific binding interaction, and the binding avidity between two molecules is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10It has a KD value less than M. In certain embodiments, the binding activity of a binding molecule to a specific target has a KD value such that the interaction is a specific binding interaction, and one or more affinities of individual ABDs do not have a KD value that recognizes them as specific binding to their respective antigens or epitopes. In certain embodiments, binding activity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to a shared specific target or distinct antigens on a complex, such as distinct antigens found on individual cells. In certain embodiments, binding activity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to distinct epitopes on shared individual antigens.

[0122] As used herein, the terms “single-chain variable fragment” or “scFv” refer to a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are linked via a short, mobile polypeptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless otherwise specified, an scFv may have VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0123] As used herein, “variable region” refers to a variable region arising from recombination, for example, in an immunoglobulin gene in a B cell or a T cell receptor (TCR) gene in a T cell, after V, J, and / or D segment recombination. In immunoglobulin genes, variable regions are typically defined from the antibody chain from which they are induced; for example, VH refers to the variable region of the antibody heavy chain, and VL refers to the variable region of the antibody light chain. Selective VH and VL can bind together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0124] As used herein, the terms “complementarity-determining region” or “CDR” refer to sequences within the antibody variable regions VH and VL that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using one 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 acids of VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of VH, e.g., mammalian VH, e.g., human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of VL, e.g., mammalian VL, e.g., human VL. In various embodiments, the CDR is a mammalian sequence, non-limitingly including mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CDR is a human sequence. In various embodiments, the CDR is a naturally occurring sequence.

[0125] As used herein, the term “framework region” or “FR” typically refers to a generally conserved sequence within the antibody variable regions VH and VL, which functions as a scaffold for scattered CDRs in the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (N-terminus to C-terminus). In various embodiments, FR is a mammalian sequence, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In certain embodiments, FR is a human sequence. In various embodiments, FR is a naturally occurring sequence. In various embodiments, FR is a synthetic sequence, including, but not limited to, a reasonably designed sequence.

[0126] As used herein, the term “antibody heavy chain” refers to the larger of two types of polypeptide chains present in antibody molecules in their naturally occurring three-dimensional structure, which typically determine the class to which the antibody belongs.

[0127] As used herein, the term “antibody light chain” refers to the smaller of two types of polypeptide chains present in antibody molecules in their naturally occurring three-dimensional structures. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0128] As used herein, the term “recombinant antibody” refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as meaning an antibody produced by the synthesis of a DNA molecule encoding the antibody, in which the DNA molecule expresses an antibody protein, or an amino acid sequence that identifies the antibody, and the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technologies that are available and well known in the art.

[0129] As used herein, the terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may involve antibody production, activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen.

[0130] As used herein, the terms “antitumor effect” or “antitumor activity” refer to a biological effect that can be manifested by a variety of means, including, but not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in lifespan, a decrease in tumor cell proliferation, a decrease in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. The “antitumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of this disclosure to prevent tumor development in the first place, such as through prophylactic therapy or treatment.

[0131] As used herein, the term “autologous” refers to any substance derived from the same subject that is later reintroduced into the subject.

[0132] As used herein, the term “homogeneous” refers to any material originating from different animals of the same species as the subject into which the material is introduced. Two or more subjects are said to be homogeneous if their genes at one or more loci are not identical. In some embodiments, homogeneous materials from individuals of the same species may be sufficiently genetically different to interact antigenically at certain genes, such as MHC alleles. In some embodiments, homogeneous materials from individuals of the same species may be sufficiently genetically identical to not interact antigenically at certain genes, such as MHC alleles.

[0133] The isolated nucleic acid molecules of this disclosure include any nucleic acid molecules encoding a polypeptide or fragment thereof of this disclosure. Such nucleic acid molecules do not need to be 100% homologous or identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. Nucleic acids having “substantial identity” or “substantial homology” to the 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 with a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof, under conditions of varying strictness. For example, the exact salt concentrations may typically be about 750 mM NaCl and less than 75 mM trisodium citrate, about 500 mM NaCl and less than 50 mM trisodium citrate, or about 250 mM NaCl and less than 25 mM trisodium citrate. Low-tight hybridization can be obtained in the absence of organic solvents, such as formamide, while high-tight hybridization can be obtained in the presence of at least about 35% or at least about 50% formamide. Tight temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergent, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various levels of tightness can be achieved.

[0134] "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 of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical with the sequence used for comparison at the amino acid level or at the nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program). 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 and tyrosine. An exemplary method for measuring the degree of identity may involve using the BLAST program, where probability scores from e-3 to e-100 indicate closely related sequences.

[0135] As used herein, the term “encodes” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, or the biological properties derived therefrom, that serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a prescribed sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a prescribed sequence of amino acids. Thus, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, where the nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, used as a template for the transcription of a gene or cDNA, may be said to code for a protein or other product of that gene or cDNA. Unless otherwise indicated, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. The phrase “nucleotide sequence encoding a protein or RNA” may include introns to the extent that a nucleotide sequence encoding a protein may include introns in some versions.

[0136] 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, enabling intercellular recognition and / or interaction.

[0137] The terms “effective dose” and “therapeutic dose” are used interchangeably herein and refer to the amount of a compound, formulation, substance, or composition described herein that is effective in achieving a particular biological outcome. In some embodiments, the “effective dose” or “therapeutic dose” is an amount sufficient to block, improve, or inhibit the continued proliferation, growth, or metastasis of a disease or disorder of interest, such as a bone marrow disorder.

[0138] As used herein, the term “immunely responsive cell” refers to a cell that functions in an immune response (e.g., an immune effector response), its precursor, or its offspring. 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 phagocytic cells.

[0139] As used herein, the terms “immune effector response” or “immune effector function” refer to the function or response of an immune-responsive cell, for example, that enhances or promotes the immune attack of a target cell. For example, an immune effector function or response may refer to the properties of a T cell or NK cell that promote the death or inhibition of growth or proliferation of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0140] As used herein, the terms “mobile polypeptide linker” or “linker” refer to peptide linkers consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link variable heavy chain and variable light chain regions together. In one embodiment, the mobile polypeptide linker is a Gly / Ser linker with the amino acid sequence (Gly-Gly-Gly-Gly-Ser) n (Sequence ID 107) Or (Gly-Gly-Gly-Ser) n (Sequence ID 108) The formula includes, 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 mobile polypeptide linker is Gly4Ser (Sequence ID 33) Or (Gly4Ser)3 (Sequence ID 35) This includes, but is not limited to, the linker (Gly2Ser) (Sequence No. 23) , (GlySer) or (Gly3Ser) (Sequence No. 28)This includes multiple iterations of the above. In some embodiments, the mobile polypeptide linker includes a Whitlow linker (e.g., GSTGSSGKPGSGEGSTKG [SEQ ID NO: 38]). The scope of this disclosure also includes, for example, the linkers described in WO2012 / 138475.

[0141] As used herein, the terms “treat,” “treatment,” and “treating” refer to the reduction or mitigation of the progression, severity, and / or duration of a proliferative disorder (e.g., cancer), or the mitigation of one or more symptoms (preferably one or more identifiable symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as CARs of this disclosure). In some embodiments, reduction or improvement refers to the improvement of at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms “treat,” “treatment,” and “treating” refer to the inhibition of the progression of a proliferative disorder, physically, for example, by stabilization of an identifiable symptom; physiologically, for example, by stabilization of a physical parameter; or both. In some embodiments, reduction or improvement includes a reduction or stabilization of tumor size or cancer cell count.

[0142] As used herein, the term “subject” is intended to include living organisms (e.g., mammals, humans) from which an immune response may be induced.

[0143] Other aspects of this disclosure are described in the following sections and are within the claims.

[0144] Other Interpretation Rules The ranges listed herein are understood to be all abbreviations of values ​​within a range that include the listed endpoints. For example, the range 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.

[0145] Unless otherwise specified, references to compounds having one or more stereocenters refer to each stereoisomer and all combinations of those stereoisomers.

[0146] Endomucin-specific chimeric protein and antigen-binding domain This disclosure provides an antigen-binding domain (e.g., a single-stranded variable fragment) that binds to endomucin (EMCN), a chimeric protein comprising the antigen-binding domain that binds to EMCN, and nucleic acids encoding such antigen-binding domains and chimeric proteins. While not wishing to be bound by theory, EMCN is a sialocyoglycoprotein that interferes with the assembly of local adhesion complexes and inhibits interactions between cells and the extracellular matrix. In some embodiments, the EMCN-specific chimeric protein binds to human EMCN (e.g., Uniprot Q9ULC0, incorporated herein by reference for all purposes) or its epitope fragment. EMCN may be expressed in hematopoietic stem cells and progenitor cells (HSPCs). EMCN may be expressed in cells generally considered healthy, such as healthy HSPCs. EMCN-specific antibodies, including CBFYE-0213, V.7.C7.1, L4B1, L5F12, L10B5, L3F12, L6H3, L6H10 (also referred to herein as Ab1), L9H8, and L10F12, have already been described, as described in Samulowitz U. et al., Am.J. Path., 2002 May, 160(5):1669-1681, which are incorporated herein by reference for all purposes.

[0147] This disclosure provides an EMCN-specific antigen-binding domain having one or more of the amino acid sequences listed in Table A.

[0148] In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, and VH includes a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4). In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, and VH includes a heavy chain complementarity-determining region 1 (CDR-H1), a heavy chain complementarity-determining region 2 (CDR-H2), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where VH includes a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence RIKD (SEQ ID NO: 4). In some embodiments, the EMCN-specific antigen-binding domain having the above VH sequence may 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), where the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. In some embodiments, the EMCN-specific antigen-binding domain having the above-mentioned VH sequence may have a heavy chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a heavy chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a heavy chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0149] In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, and the VL region includes 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), which have the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 9. In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, where the VL includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). In some embodiments, the EMCN-specific antigen-binding domain having the above VL sequence may have a heavy chain complementarity determination region 1 (CDR-H1), a heavy chain complementarity determination region 2 (CDR-H2), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 1. In some embodiments, the EMCN-specific antigen-binding domain having the above-described VL sequence may have a heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4).

[0150] In some embodiments, the EMCN-specific antigen-binding domain has a heavy chain variable (VH) region and a light chain variable (VL) region, (1) VH includes a heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and (2) VL includes a light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0151] In some embodiments, the EMCN-specific antigen-binding domain has a VH region containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the EMCN-specific antigen-binding domain has a VH region containing 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 with the amino acid sequence of SEQ ID NO: 1.

[0152] In some embodiments, the EMCN-specific antigen-binding domain has a VL region containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the EMCN-specific antigen-binding domain has a VL region containing 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 with the amino acid sequence of SEQ ID NO: 9.

[0153] In some embodiments, the EMCN-specific antigen-binding domain has (1) a VH region containing the amino acid sequence of SEQ ID NO: 1, and (2) a VL region containing 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 with the amino acid sequence of SEQ ID NO: 9, or a VL region containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the EMCN-specific antigen-binding domain includes (1) a VH region containing 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%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, and (2) a VL region containing 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%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, or a VL region containing the amino acid sequence of SEQ ID NO: 9.

[0154] In some embodiments, the EMCN-specific antigen-binding domain has (1) a VL region containing the amino acid sequence of SEQ ID NO: 9, and (2) a VH region containing the amino acid sequence of SEQ ID NO: 1 or a VH region containing 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 with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the EMCN-specific antigen-binding domain has a VL region containing 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%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, and (2) a VH region containing an amino acid sequence of SEQ ID NO: 1 or a VH region containing 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%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0155] In some embodiments, the EMCN-specific antigen-binding domain competes with a reference antibody or its antigen-binding fragment having a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (1) VH includes a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and (2) VL includes a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12).

[0156] In some embodiments, the EMCN-specific antigen-binding domain binds to the same or substantially the same epitope (e.g., a separate human EMCN epitope) as the reference antibody or its antigen-binding fragment having a heavy chain variable (VH) region and a light chain variable (VL) region, (1) VH has a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence WGNGN (SEQ ID NO: 3), (1) The VL includes a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), and (2) the VL includes a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12). In some embodiments, the EMCN-specific antigen-binding domain binds to the same or substantially the same epitope (e.g., a separate human EMCN epitope) as the reference antibody or its antigen-binding fragment having a VH containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the EMCN-specific antigen-binding domain binds to the same or substantially the same epitope (e.g., a separate human EMCN epitope) as the reference antibody or its antigen-binding fragment having a VL containing the amino acid sequence of SEQ ID NO: 9.

[0157] The EMCN-specific antigen-binding domain can be any of the formats described herein, such as Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camel 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.

[0158] In some embodiments, the EMCN-specific antigen-binding domain is a single-stranded variable fragment (scFv) format comprising an scFv format having one of the peptide linkers described herein (see, for example, Table 1). 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. The disclosure also provides chimeric proteins and nucleic acids encoding such chimeric proteins and comprising an EMCN-specific antigen-binding domain having one or more of the amino acid sequences listed in Table A. The chimeric protein may comprise one of the EMCN-specific antigen-binding domains already described.

[0159] Chimeric antigen receptor (CAR) Certain aspects of this disclosure relate to a chimeric receptor having one of the EMCN-specific antigen-binding domains described herein and having the ability to specifically bind 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). Generally, a CAR is a chimeric protein comprising an antigen-binding domain and a polypeptide molecule heterogeneous to the antigen-binding domain, such as a peptide heterogeneous to an antibody in which the antigen-binding domain can be induced. Polypeptide molecules heterogeneous to the antigen-binding domain include, but are not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or a combination thereof.

[0160] In some embodiments, a CAR is an engineered receptor that implants or confers a desired specificity (e.g., EMCN) to immune effector cells. In certain embodiments, a CAR can be used to implant antibody specificity into immune-responsive cells such as T cells. In some embodiments, the CARs of this disclosure include an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0161] In some embodiments, the chimeric antigen receptor is an activated 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 homologous ligand is sufficient to induce activation of immune-responsive cells. In some embodiments, binding of the chimeric antigen receptor to its homologous ligand is sufficient to induce stimulation of immune-responsive cells. In some embodiments, activation of immune-responsive cells results in the death of target cells. In some embodiments, activation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces differentiation of immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces proliferation of immune-responsive cells. In some embodiments, activation and / or stimulation of immune-responsive cells can be a combination of the above responses.

[0162] The CARs of this disclosure may be first, second, or third-generation CARs. "First-generation" CARs generally contain a single intracellular signaling domain derived from the T cell receptor chain. "First-generation" CARs generally have an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary signaling factor from the endogenous TCR. "First-generation" CARs give de novo antigen recognition and, independently of HLA-mediated antigen presentation, transmit signals via the CD3ζ chain signaling domain within a single fusion molecule to CD4 + and CD8 +It induces activation of both T cells. Second-generation CARs provide additional signaling to T cells by adding a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR. Second-generation CARs provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that second-generation CARs can enhance the antitumor activity of immune-responsive cells such as T cells. Third-generation CARs have multiple intracellular co-stimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0163] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, one or more chimeric inhibitory receptors bind to antigens expressed on non-tumor cells derived from tissues selected from the group consisting of brain, nerve tissue, endocrine system, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

[0164] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used in conjunction with one or more activated chimeric receptors (e.g., activated chimeric TCRs or CARs) expressed on cells of the Disclosure (e.g., immune-responsive cells) as, for example, not a logic gate to control, modulate, or otherwise inhibit the activity of one or more activated chimeric receptors. For example, if healthy cells express both antigens recognized by tumor-targeted chimeric receptors and antigens recognized by inhibitory chimeric receptors, immune-responsive cells expressing tumor-associated antigens may bind to healthy cells. In such cases, the inhibitory chimeric antigen also binds to its homologous ligand on healthy cells, and the inhibitory function of the inhibitory chimeric receptor reduces, mitigates, prevents, or inhibits the activation of immune-responsive cells via tumor-targeted chimeric receptors ("not a logic gate"). In some embodiments, the inhibitory chimeric receptors of the Disclosure may inhibit the activity of one or more cells of the Disclosure (e.g., immune-responsive cells). In some embodiments, immune-responsive cells may comprise one or more tumor-targeted chimeric receptors and one or more inhibitory chimeric receptors targeting antigens (e.g., EMCNs) that are not expressed on tumors or are not generally considered to be expressed on tumors. Combinations of tumor-targeted chimeric receptors and inhibitory chimeric receptors in the same immune-responsive cells can be used to reduce extratumor toxicity on the target.

[0165] In some embodiments, the extracellular antigen-binding domain of the CAR disclosed herein is approximately 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 approximately 1 × 10 -9 Dissociation constants (K) less than or equal to M d ) binds to one or more antigens (e.g., EMCN). In some embodiments, Kd It is approximately 2 x 10 -7 M ~ approx. 1×10 -9 It is within the range of M.

[0166] The binding of the extracellular antigen-binding domain of the CARs of this 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 the complex of interest and a specific labeling reagent (e.g., antibody or scFv). For example, scFv may be radiolabeled and used in RIA assays. Radioisotopes can be detected by means such as the use of a γ 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 proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of a CAR is labeled with a secondary antibody specific to the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., with a radioactive or fluorescent marker).

[0167] In some embodiments, the CAR of this 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 is an F(ab)2 fragment.

[0168] Extracellular antigen-binding domain The extracellular antigen-binding domain of the CARs of this disclosure specifically binds to EMCN (e.g., EMCN protein, EMCN-derived antigen, or EMCN-derived epitope). In certain embodiments, the extracellular antigen-binding domain binds to EMCN expressed in hematopoietic stem cells. In certain embodiments, the extracellular antigen-binding domain binds to EMCN expressed in cells generally considered healthy, such as healthy HSPCs. In some embodiments, the EMCN is human EMCN.

[0169] The antigen-binding domains of this disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, complex antibodies, human antibodies, humanized antibodies, and functional fragments thereof, as well as any domain that functions as an antigen-binding domain and binds to an alternative scaffold known in the art, such as recombinant fibronectin domains, T cell receptors (TCRs), recombinant TCRs with enhanced affinity, or fragments thereof, such as single-chain TCRs. In some cases, it is beneficial that the antigen-binding domain originates from the same species in which the CAR is ultimately used.

[0170] In some embodiments, the extracellular antigen-binding domain includes 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 includes an antigen-binding fragment of the antibody.

[0171] In some embodiments, the extracellular antigen-binding domain includes the F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain includes the F(ab') fragment.

[0172] In some embodiments, the extracellular antigen-binding domain includes scFv. In some embodiments, the extracellular antigen-binding domain includes two single-stranded variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain includes a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to distinct epitopes on the same antigen. In certain embodiments, the scFv is a mammalian scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

[0173] In certain embodiments, VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker contains one of the amino acid sequences shown in Table 1. In certain embodiments, the scFv contains 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 one or more scFvs contains the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked together, each scFv can be linked to the next scFv to which a peptide is linked. In some embodiments, each of one or more scFvs is separated by a peptide linker.

[0174] (Table 1) Peptide linkers TIFF0007913758000001.tif72128

[0175] In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domains of the first and second chimeric receptors may be suitable antigen double-binding domains described herein or known in the art. For example, the first or second antigen-binding domain may be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-stranded variable fragments (scFv), or single-domain antibodies (sdAb). In some embodiments, the antigen-binding domains of the first and / or second chimeric receptors comprise two single-stranded variable fragments (scFv). In some embodiments, each of the two scFv binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first chimeric receptor may be specific to EMCN, and the chimeric receptor may be specific to a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed in bone marrow cells, such as AML cells).

[0176] In some embodiments, the extracellular antigen-binding domain includes a single-domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0177] In some embodiments, the CAR of this disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds to the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different antigen.

[0178] 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 mobile linker. In some embodiments, each of the two antigen-binding domains may be independently selected from an antibody, an antigen-binding fragment of an antibody, scFv, sdAb, recombinant fibronectin domain, T cell receptor (TCR), a recombinant TCR with enhanced affinity, and a single-stranded TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0179] In certain embodiments, a bispecific CAR or tanCAR includes an antigen-binding domain containing 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, VH may be upstream or downstream of VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VL (VL1) with its VH (VH1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VH (VH2) with its VL (VL2), resulting in the overall bispecific antibody molecule having the configuration VH1-VL1-VL2-VH2. In other embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VH(VH1) with its VL(VL1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned upstream of its VL(VL2) with its VH(VH2), so that the overall bispecific antibody molecule has the configuration VL1VH1-VH2-VL2. In some embodiments, a linker is positioned between two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2 when the construct is positioned as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is positioned as VL1-VH1-VH2-VL2. The linker may be a linker as described herein, for example, a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. (Sequence ID 109) Generally, the linker between two scFvs must be long enough to avoid mismatching between the domains of the two scFvs. In some embodiments, the linker is located between the VL and VH of the first scFv. In some embodiments, the linker is located between the VL and VH of the second scFv. In a construct having multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR includes VL, VH and may further include one or more linkers in the arrangements described herein.

[0180] In some embodiments, the chimeric receptor includes a bivalent CAR. In some embodiments, the bivalent CAR is an EMCN bivalent CAR. In some embodiments, the bivalent EMCN CAR includes one or more of the anti-EMCN sequences shown in Table A. In some embodiments, the ABDs of the bivalent EMCN CAR each include the same ABD.

[0181] 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 A.

[0182] transmembrane domain In some embodiments, the transmembrane domain of the CARs of this disclosure (e.g., EMCN-specific CARs as described herein) includes a hydrophobic alpha-helix spanning at least a portion of the cell membrane. It has been shown that different transmembrane domains can contribute to the stability of different receptors. After antigen recognition, the receptors cluster, and the signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CARs of this disclosure may include the transmembrane domains of CD8 polypeptide, CD28 polypeptide, CD3-zeta polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, LIR-1 (LILRB1) polypeptide, or synthetic peptides, or any combination thereof.

[0183] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide 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 derived 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 derived 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 the 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.

[0184] In some embodiments, the transmembrane domains are NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009 The polypeptide comprises an amino acid sequence or fragment thereof 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. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may contain one conservative amino acid substitution, two or fewer conservative amino acid substitutions, or three or fewer conservative amino acid substitutions.In some embodiments, the polypeptide has an amino acid length of 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, as indicated by NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_03 It may have an amino acid sequence that is a contiguous portion of 1668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0185] Further examples of suitable polypeptides from which the 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, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD10 3. This includes, but is not limited to, the transmembrane regions of the alpha, beta, or zeta strands of 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 NG2C.

[0186] In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 82). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 83). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 84).

[0187] In some embodiments, the transmembrane domain further comprises at least a portion of the extracellular domain of the same protein.

[0188] Spacer area In some embodiments, the CARs of this disclosure (e.g., EMCN-specific CARs as described herein) may also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge derived from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including but not limited to the IgG4 hinge, IgG2 hinge, CD8a hinge, or IgD hinge. In some embodiments, the spacer region may include the IgG4 hinge, IgG2 hinge, IgD hinge, CD28 hinge, KIR2DS2 hinge, LNGFR hinge, or PDGFR-beta extracellular linker. In some embodiments, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may include any of the amino acid sequences listed in Table 2, or any amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table 2. In some embodiments, the nucleic acid encoding any of the spacer regions of this disclosure may include any of the nucleic acid sequences listed in Table 3, or any nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 3.

[0189] (Table 2) Spacer amino acid sequence TIFF0007913758000002.tif80156

[0190] (Table 3) Spacer nucleic acid sequences TIFF0007913758000003.tif140156

[0191] In some embodiments, the spacer region includes the sequence shown in sequence number 40. In some embodiments, the spacer region includes the sequence shown in sequence number 41. In some embodiments, the spacer region includes the sequence shown in sequence number 42. In some embodiments, the spacer region includes the sequence shown in sequence number 43. In some embodiments, the spacer region includes the sequence shown in sequence number 44. In some embodiments, the spacer region includes the sequence shown in sequence number 45. In some embodiments, the spacer region includes the sequence shown in sequence number 46. In some embodiments, the spacer region includes the sequence shown in sequence number 47. In some embodiments, the spacer region includes the sequence shown in sequence number 48. In some embodiments, the spacer region includes the sequence shown in sequence number 49. In some embodiments, the spacer region includes the sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (sequence number 85). In some embodiments, the spacer region includes the sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 86). In some embodiments, the spacer region includes the sequence FVPVFLPAKPTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 87).

[0192] In some embodiments, the CAR of the present disclosure may further include a short oligopeptide or polypeptide linker having a length of 2 to 10 amino acid residues that can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a preferred linker is a glycine-serine double chain. In some embodiments, the linker includes the amino acid sequence GGCKJSGGCKJS (SEQ ID NO: 88).

[0193] Intracellular signal transduction domains In some embodiments, the CARs of this disclosure (e.g., the EMCN-specific CARs described herein) include one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of the CAR may include intracellular signaling domains.

[0194] Examples of suitable intracellular signaling domains that may be used in the CARs of this disclosure include, but are not limited to, cytoplasmic sequences of T cell receptors (TCRs), co-receptors that act cooperatively to modulate signaling after antigen receptor engagement, and any derivatives or variants of these sequences, and any recombinant sequences having the same functional capacity.

[0195] While we do not wish to be bound by theory, the signals generated through the TCR alone are insufficient for complete T cell activation, and therefore, typically, secondary and / or co-stimulatory signals are also considered necessary for complete 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 co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains). Furthermore, T cell signaling and function (e.g., activation signaling cascades) can be negatively regulated by inhibitory receptors present on T cells via intracellular inhibitory co-signaling domains.

[0196] In some embodiments, the intracellular signaling domain of the CARs of this disclosure may include an inhibitory intracellular signaling domain. Examples of inhibitory intracellular domains that may 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 may include 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 peptide linkers (e.g., see Table 2) or spacer or hinge sequences (e.g., see Table 3). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, they can be linked via peptide linkers (e.g., see Table 2) or spacer or hinge sequences (e.g., see Table 3). In some embodiments, the intracellular inhibitory co-signaling domains are inhibitory domains. In some embodiments, one or more intracellular inhibitory co-signaling domains in a chimeric protein include one or more ITIM-containing proteins or fragments thereof. ITIM is a conserved amino acid sequence found at the cytoplasmic terminals of many inhibitory immune receptors. 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, one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins or fragments thereof. In some embodiments, 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 may 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.

[0197] In some embodiments, the intracellular signaling domain of the CARs of this disclosure may include a primary signaling domain that controls primary activation of the TCR complex either stimulatingly or inhibitorily. A primary intracellular signaling domain acting stimulatingly may include a known signaling motif as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of suitable ITAM-containing primary intracellular signaling domains that may be used in the CARs of this disclosure include, but are not limited to, those of CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0198] In some embodiments, the CARs of this disclosure (e.g., EMCN-specific CARs as described herein) include an intracellular signaling domain, such as the primary signaling domain of a CD3-zetapolypeptide. The CD3-zetapolypeptides of this 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, the CD3-zetapolypeptide may contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide may have an amino acid sequence that is a contiguous portion of NCBI reference number NP_932170 or NP_001106864.2, having a length of 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.

[0199] In other embodiments, the primary signaling domain includes a modified ITAM domain, such as a mutant ITAM domain, whose activity has been altered (e.g., increased or decreased) compared to a native ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four, or more ITAM motifs.

[0200] In some embodiments, the intracellular signaling domain of the CAR of the Disclosure may comprise a CD3-zeta signaling domain by itself, or it may be combined with any other desired intracellular signaling domain useful in the context of the CAR of the Disclosure. For example, the intracellular signaling domain of the CAR may comprise a portion of the CD3 zeta chain and a costimulatory signaling domain. The costimulatory signaling domain may refer to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. The costimulatory molecule of the Disclosure is a cell surface molecule other than an antigen receptor or its ligand that may be required for an efficient lymphocyte response to an antigen.Examples of suitable co-stimulatory molecules include ligands that specifically bind to 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, CD83, MHC class I molecules, TNF receptor proteins, and immunoglobulins. Robulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4 , VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGA X, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, C This includes, but is not limited to, D96 (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, etc.

[0201] In some embodiments, intracellular signaling sequences within a portion of the cytoplasm of the CARs of this disclosure may be linked to one another in a random or specific order. In some embodiments, for example, short oligopeptides or polypeptide linkers of length from 2 to 10 amino acids (e.g., 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids) may form links with the intracellular signaling sequences. In one embodiment, a glycine-serine double chain may be used as a suitable linker. In one embodiment, a single amino acid, such as alanine or glycine, may be used as a suitable linker.

[0202] In some embodiments, the intracellular signaling domain includes two or more co-stimulatory signaling domains, for example, two co-stimulatory signaling domains, three co-stimulatory signaling domains, four co-stimulatory signaling domains, five co-stimulatory signaling domains, six co-stimulatory signaling domains, seven co-stimulatory signaling domains, eight co-stimulatory signaling domains, nine co-stimulatory signaling domains, ten co-stimulatory signaling domains, or more co-stimulatory signaling domains. In one embodiment, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, two or more co-stimulatory signaling domains are separated by a linker of the Disclosure (for example, any of the linkers listed in Table 1). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0203] In some embodiments, the cells of the Disclosure express a CAR comprising an antigen-binding domain that binds to EMCN, a transmembrane domain of the Disclosure, a primary signaling domain, and one or more co-stimulatory signaling domains.

[0204] In some embodiments, the cells of the Disclosure express an iCAR comprising 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 A), the transmembrane domain of the Disclosure, and one or more intracellular inhibitory co-signaling domains. In some embodiments, the cells of the Disclosure express a CAR comprising (1) 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 A), the transmembrane domain of the Disclosure, a primary signaling domain, and one or more co-stimulatory signaling domains.

[0205] In some embodiments, the transmembrane domain is derived from the same protein as 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 proteins selected from 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, respectively.

[0206] In some embodiments, the transmembrane domain originates from a first protein, and one or more intracellular signaling domains originate from a second protein distinct from the first protein.

[0207] Natural killer cell receptor (NKR) CAR In some embodiments, the CARs of this disclosure (e.g., EMCN-specific CARs as described herein) comprise one or more components of natural killer cell receptors (NKRs) to form 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, the following: killer cell immunoglobulin-like receptors (KIRs) such as DPI; innate cytotoxic receptors (NCRs) such as NKp30, NKp44, and NKp46; the immune cell receptor signaling lymphocyte activating molecule (SLAM) family such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published on September 18, 2014.

[0208] Further targeting of chimeric receptors Certain aspects of this disclosure relate to chimeric receptors that bind to an antigen of interest in addition to EMCNs, and nucleic acids encoding such chimeric receptors. Furthermore, certain aspects of this disclosure relate to cells such as immune-responsive cells and genetically modified chimeric receptors expressing one or more of the chimeric receptors that bind to an antigen of interest in addition to EMCNs, as well as methods for treating and / or preventing myeloid malignancies such as AML and other conditions for which an antigen-specific immune response is desired using such receptors and cells. Malignant cells have developed a set of mechanisms to protect themselves from immune recognition and elimination. This disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0209] In some embodiments, the first 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 A), and the second chimeric receptor comprises a further antigen-binding domain that binds to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen). In some embodiments, cells can express a first chimeric receptor specific to EMCN (e.g., a 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 to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen). In some embodiments, cells can express a first inhibitory chimeric receptor specific to 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 to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen). For example, cells (e.g., immune-responsive cells) can co-express, or be engineered to be capable of co-expressing, an iCAR containing 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 A) and an aCAR that targets a tumor-associated antigen (e.g., AML-associated antigen). In addition to EMCN, suitable antigen-binding antibodies include any antibody that binds sufficiently strongly and specifically to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen), whether natural or synthetic, full-length or fragments, monoclonal or polyclonal. In some embodiments, commercially available antibodies that bind to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen), may be used. CDRs of commercially available antibodies are readily accessible to those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art can construct nucleic acids encoding scFv and chimeric receptors (e.g., CAR and TCR) based on the CDRs of such commercially available antibodies.

[0210] T cell receptor (TCR) Certain embodiments of this disclosure relate to a chimeric receptor that specifically binds to a second antigen, such as a tumor-associated antigen (e.g., AML-associated antigen), where the chimeric receptor for the second antigen is an engineered T cell receptor (TCR). The TCR of this disclosure is a disulfide-bonded heterodimer protein comprising 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 plays a role in recognizing the antigen as a peptide bound to a major histocompatibility complex (MHC) molecule. In certain embodiments, the TCR of this disclosure comprises an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0211] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to peptide / MHC complexes. Each variable region has three complementarity-determining regions (CDRs).

[0212] In certain embodiments, the TCR can form receptor complexes with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. When the TCR complex conjugates with its antigen and MHC (peptide / MHC), the T cells expressing the TCR complex are activated.

[0213] In some embodiments, the TCRs of this disclosure are recombinant TCRs. In certain embodiments, the TCRs are non-naturally derived TCRs. In certain embodiments, the TCRs differ from naturally derived TCRs by at least one amino acid residue. In some embodiments, the TCRs differ from naturally derived TCRs by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least eleven amino acid residues, at least twelve amino acid residues, at least thirteen amino acid residues, at least fourteen amino acid residues, at least fifteen amino acid residues, at least twenty amino acid residues, at least twenty-five amino acid residues, at least thirty amino acid residues, at least forty amino acid residues, at least fifty amino acid residues, at least sixty amino acid residues, at least seventy amino acid residues, at least eighty amino acid residues, at least ninety amino acid residues, at least one hundred amino acid residues, or more amino acid residues. In certain embodiments, the TCR is a naturally occurring TCR modified by at least one amino acid residue. In some embodiments, the TCR is a naturally occurring TCR modified by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least eleven amino acid residues, at least twelve amino acid residues, at least thirteen amino acid residues, at least fourteen amino acid residues, at least fifteen amino acid residues, at least twenty amino acid residues, at least twenty-five amino acid residues, at least thirty amino acid residues, at least forty amino acid residues, at least fifty amino acid residues, at least sixty amino acid residues, at least seventy amino acid residues, at least eighty amino acid residues, at least ninety amino acid residues, at least one hundred amino acid residues, or more amino acid residues.

[0214] Chimeric TCR In some embodiments, a 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 generate a chimeric TCR that specifically binds 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 chimeric TCRs 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 domain of a TCR chain, such as a TCR alpha chain and / or a TCR beta chain, e.g., an extracellular constant domain, a transmembrane domain, and a cytoplasmic domain. As another example, the CDRs of an antibody or antibody fragment can be grafted into a TCR alpha chain and / or beta chain to generate 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 generated 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).

[0215] Immune responsive cells Certain aspects of the present disclosure relate to cells, e.g., immune responsive 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 to methods of using such cells to treat myeloid malignancies (e.g., AML).

[0216] In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are primary cells. In some embodiments, the mammalian cells are cell lines. In some embodiments, the mammalian cells are bone marrow cells, blood cells, skin cells, osteocytes, muscle cells, nerve cells, adipocytes, hepatocytes, or cardiac cells. In some embodiments, the cells are stem cells. 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 cells are cells derived from or differentiated from the stem cells of this disclosure. In some embodiments, the cells are immune cells. The immune cells of this disclosure may be isolated or differentiated from the stem cells of this disclosure (e.g., from ESCs or iPSCs). Exemplary immune cells include, but are not limited to, T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha-beta T cells, and gamma-delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cells are nerve cells. The nerve cells of this disclosure may be isolated or differentiated from the stem cells of this 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.

[0217] In some embodiments, the cell is an immunoresponsive cell. The immunoresponsive cells of the present disclosure can be isolated or differentiated from the stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary immunoresponsive cells of the present disclosure include, but are not limited to, cells of the lymphoid lineage. Lymphoid lineages, including B cells, T cells, and natural killer (NK) cells, provide for antibody production, regulation of the cellular immune system, detection of foreign substances in 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., those derived from or capable of differentiating into lymphocytes). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity, and 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 cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells, can be any type of T cell, including but not limited to. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce death of infected somatic cells or tumor cells. A patient's own T cells can be genetically modified to target a specific antigen through introduction of one or more chimeric receptors such as chimeric TCR or CAR.

[0218] Natural killer (NK) cells are part of cell-mediated immunity and can be lymphocytes that act during the innate immune response. NK cells do not require prior activation to exert their cytotoxic effect on target cells.

[0219] In some embodiments, the immunoresponsive cell of the present disclosure is a T cell. The T cells of the present disclosure can be induced in vitro from autologous, allogeneic, or engineered progenitor or stem cells.

[0220] In some embodiments, the immune-responsive cells of this disclosure are universal T cells having a TCR-αβ defect. Methods for developing universal T cells are described in the Art, for example, Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119: 5697-5705.

[0221] In some embodiments, the immune-responsive cells of the Disclosure are isolated immune-responsive cells comprising one or more chimeric receptors of the Disclosure. In some embodiments, the immune-responsive 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 Disclosure.

[0222] In some embodiments, the immune-responsive cells are T cells. In some embodiments, the immune-responsive cells are natural killer (NK) cells.

[0223] In some embodiments, immune-responsive cells express or are capable of expressing immune receptors. Immune receptors can generally induce changes in signaling or protein expression in immune receptor-expressing cells, thereby modulating the immune response upon binding to homogeneous ligands (e.g., modulating, activating, initiating, stimulating, increasing, preventing, attenuating, inhibiting, reducing, decreasing, inhibiting, or suppressing the immune response). For example, when CD3 chains present in TCRs / CARs cluster in response to ligand binding, a signaling cascade occurs via an immune receptor tyrosine-based activation motif (ITAM). Specifically, in certain embodiments, when an endogenous TCR, exogenous TCR, chimeric TCR, or CAR (specifically an activated CAR) binds to its respective antigen, immunosynaptic formation occurs, involving the clustering of many molecules near the binding receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). Such clustering of membrane-bound signaling molecules phosphorylates the ITAM motif within the CD3 chain, initiating the T cell activation pathway and ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors can induce global gene expression in T cells, increasing IL-2 production for the proliferation and expression of master regulator T cell proteins, thereby initiating T cell-mediated immune responses such as cytokine production and / or T cell-mediated injury.

[0224] Cells that express multiple chimeric receptors In some embodiments, the cells of the Disclosure (e.g., immune-responsive cells) comprise two or more chimeric receptors of the Disclosure. In some embodiments, the cells comprise two or more chimeric receptors, one of which is a chimeric inhibitory receptor. In some embodiments, the cells comprise three or more chimeric receptors, at least one of which is a chimeric inhibitory receptor. In some embodiments, the cells comprise four or more chimeric receptors, at least one of which is a chimeric inhibitory receptor. In some embodiments, the cells comprise five or more chimeric receptors, at least one of which is a chimeric inhibitory receptor.

[0225] In some embodiments, each of the two or more chimeric receptors includes a different antigen-binding domain, for example, an antigen-binding domain that binds to the same antigen or a different antigen. In some embodiments, each antigen bound by the two or more chimeric receptors is expressed in the same cells, such as myeloid cells (e.g., the same AML cell type).

[0226] In embodiments of the present disclosure in which cells (e.g., immune-responsive cells) express two or more distinct chimeric receptors, the antigen-binding domains of each of the different chimeric receptors may be designed so that the antigen-binding domains do not interact with each other. For example, cells (e.g., immune-responsive cells) of the present disclosure expressing a first chimeric receptor (e.g., an EMCN-specific chimeric receptor) and a second chimeric receptor may include a first chimeric receptor having an antigen-binding domain that does not form association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor may contain an antibody fragment such as scFv, while the antigen-binding domain of the second chimeric receptor may contain VHH.

[0227] While we do not wish to be bound by theory, in cells having multiple chimeric membrane-embedded receptors, each containing an antigen-binding domain, interactions between the antigen-binding domains of each receptor may be undesirable, as such interactions could inhibit the ability of one or more antigen-binding domains to bind to their congener antigens. Therefore, in embodiments of the present disclosure in which cells (e.g., immune-responsive cells) express two or more chimeric receptors, the chimeric receptors include antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor includes an scFv, and the antigen-binding domain of a second chimeric receptor includes 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. In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of the first chimeric receptor to its congener antigen (e.g., an EMCN-specific chimeric receptor that binds to EMCN) is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, the binding of the antigen-binding domain of the first chimeric receptor to its congener 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 congener antigen in the absence of the second chimeric receptor. In some embodiments, when present on the cell surface, the antigen-binding domains of the first and second chimeric receptors associate with each other less than when both are scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first and second chimeric receptors associate with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than when both are scFv antigen-binding domains.

[0228] Chimeric inhibitory receptors In some embodiments, the cells of the Disclosure (e.g., immune-responsive cells) include one or more chimeric inhibitory receptors of the Disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors includes an antigen-binding domain that binds to an antigen that is commonly expressed on normal cells (e.g., cells generally considered healthy) but not expressed on tumor cells such as AML cells. In some embodiments, the inhibitory chimeric receptor includes 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 A).

[0229] In some embodiments, one or more chimeric inhibitory receptors bind to antigens expressed on non-tumor cells derived from tissues selected from the group consisting of brain, nerve tissue, endocrine system, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

[0230] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used in conjunction with one or more activated chimeric receptors (e.g., activated chimeric TCRs or CARs) expressed on the cells of the Disclosure (e.g., immune-responsive cells) as, for example, not a logic gate for controlling, modulating, or otherwise inhibiting the activity of one or more activated chimeric receptors. In some embodiments, the inhibitory chimeric receptors of the Disclosure may inhibit the activity of one or more cells of the Disclosure (e.g., immune-responsive cells).

[0231] In some embodiments, the cells of the Disclosure comprise one or more inhibitory chimeric receptors of the Disclosure, further comprising tumor-targeted chimeric receptors that bind to one or more tumor-associated antigens. In some embodiments, one or more tumor-associated antigens comprise AML-associated antigens. In some embodiments, one or more tumor-associated antigens comprise CD33. In some embodiments, one or more tumor-associated antigens comprise FLT3. In some embodiments, one or more tumor-associated antigens comprise CD33 and FLT3. While not intended to be theoretically bound, when immune checkpoint cells expressing tumor-targeted chimeric receptors that bind to tumor-associated antigens are administered, the off-target effects of the tumor-targeted chimeric receptors can be reduced if the tumor-associated antigens are also expressed by healthy cells, such as healthy HSPCs, which further express EMCN-specific inhibitory chimeric receptors as described herein. As used herein, “off-target effects” refer to the killing of off-target cells (i.e., non-tumor cells that also express tumor-associated antigens) by immune cells expressing tumor-targeted chimeric receptors. In some embodiments, the reduction of off-target effects is a reduction in the killing of healthy HSPCs. In some embodiments, the reduction of off-target effects is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% less killing of off-target cells compared to equivalent immune checkpoint cells that express tumor-targeting antigens but do not express the inhibitory chimeric receptors disclosed herein.

[0232] Co-stimulatory ligand In some embodiments, the cells of the present disclosure (e.g., immune-responsive cells) may further comprise one or more recombinant or exogenous costimulatory ligands. For example, cells may be further transduced with one or more costimulatory ligands so that they co-express or are induced to co-express one or more chimeric receptors of the present disclosure (e.g., EMCN-specific CARs described herein) and one or more costimulatory ligands. While we do not wish to be constrained by theory, it is thought that interactions between one or more chimeric receptors and one or more costimulatory ligands may provide non-antigen-specific signals important for the full activation of cells. Examples of suitable costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates acute-phase responses. Its primary role is the regulation of immune cells. Members of the TNF superfamily share several common characteristics. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Suitable examples of TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell activator (B AFF) / Bly s / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducible ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess an immunoglobulin domain (folding).Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, ligands for both CD28, and PD-L1 / (B7-H1), which is a ligand for PD-1. In certain embodiments, one or more co-stimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0233] Chemokine receptors In some embodiments, the cells of this disclosure (e.g., immune-responsive cells) comprise one or more chimeric receptors (e.g., EMCN-specific CARs as described herein) and may further comprise one or more chemokine receptors. For example, transgenic expression of the chemokine receptor CCR2b or CXCR2 in cells such as T cells enhances transport to CCL2-secreting or CXCL1-secreting solid tumors (Craddock et al, J Immunother. 2010 Oct;33(8):780-8 and Kershaw et al. Hum Gene Ther. 2002 Nov 1;13(16):1971-80). While we do not wish to be constrained by theory, it is conceivable that chemokine receptors expressed on chimeric receptor-expressing cells of this disclosure may recognize chemokines secreted by tumors and improve the targeting of cells to tumors, thereby promoting cell invasion into tumors and enhancing the antitumor effect of cells. The chemokine receptors of this disclosure may include naturally occurring chemokine receptors, recombinant chemokine receptors, or chemokine-binding fragments thereof. Preferred chemokine receptors that may be expressed on cells of this 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 chemokines secreted by tumors.

[0234] Chimeric receptor regulation Some embodiments of this disclosure relate to controlling the activity of one or more chimeric receptors in chimeric receptor-expressing cells of this disclosure (e.g., EMCN-specific CARs as described herein). Several methods exist for controlling chimeric receptor activity. In some embodiments, controllable chimeric receptors capable of controlling the activity of one or more chimeric receptors may be desirable to optimize the safety and / or efficacy of chimeric receptor therapy. For example, inducing apoptosis using a caspase fused to a dimerizing 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, the chimeric receptor-expressing cells of the present disclosure can also express inducible caspase-9 (i-caspase-9), which induces caspase-9 activation and leads to cellular apoptosis upon administration of a dimerizing agent such as limiducide (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). In some embodiments, i-caspase-9 includes a binding domain containing a chemical inducer of dimerization (CID) that mediates dimerization in the presence of CID, resulting in inducible and selective depletion of chimeric receptor-expressing cells.

[0235] Alternatively, in some embodiments, the chimeric receptor of the present disclosure can be controlled by using a small molecule or antibody that inactivates or inhibits chimeric receptor activity. For example, an antibody can deplete chimeric receptor-expressing cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the chimeric receptor-expressing cells of the present disclosure can further express an antigen that is recognized by a molecule capable of inducing cell death by ADCC or complement-induced cell death. For example, the chimeric receptor-expressing cells of the present disclosure can further express a receptor that can be targeted by an antibody or an 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, αΙΙb / αΙΙbβ3, α4β7, α5β1, ανβ3, αν), members of the TNF receptor superfamily (e.g., TRAIL-R1 and TRAIL-R2), PDGF receptors, interferon receptors, folate receptors, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptors, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE receptors, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, and EGFR, and truncated versions thereof.

[0236] In some embodiments, the chimeric receptor-expressing cells of the present disclosure can also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling ability but retains an epitope recognized by a molecule capable of inducing ADCC (see, e.g., WO2011 / 056894).

[0237] In some embodiments, the chimeric receptor-expressing cells of this disclosure further include a highly expressed small marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor-expressing cells, which binds to an anti-CD20 antibody (e.g., rituximab) and results in selective depletion of the chimeric receptor-expressing cells by ADCC. Other methods for depleting the chimeric receptor-expressing cells of this disclosure include, but are not limited to, the administration of a monoclonal anti-CD52 antibody that selectively binds to and targets the chimeric receptor-expressing cells for destruction by inducing ADCC. In some embodiments, the chimeric receptor-expressing cells may be selectively targeted using a chimeric receptor ligand, such as an anti-idiotype antibody. In some embodiments, the anti-idiotype antibody can induce effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can further bind to a cell death-inducing agent, such as a toxin. In some embodiments, the chimeric receptor-expressing cells of this disclosure may further express a target protein recognized by the cell depletion agent of this 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 desirable to reduce or eliminate chimeric receptor-expressing cells. In some embodiments, the cell depletion agent is an anti-CD52 antibody.

[0238] In some embodiments, a controlled chimeric receptor comprises a set of polypeptides in which the components of the chimeric receptor of this disclosure are distributed onto separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, in the presence of a dimerizing molecule, can conjugate the polypeptides together to form a functional chimeric receptor.

[0239] EMCN-specific protein-coding nucleic acid constructs Certain aspects of this disclosure relate to nucleic acids (e.g., isolated nucleic acids) encoding one or more EMCN-specific proteins (e.g., EMCN-specific CARs as described herein). In some embodiments, the nucleic acid is an RNA construct such as a messenger RNA (mRNA) transcript or modified RNA. In some embodiments, the nucleic acid is a DNA construct.

[0240] In some embodiments, the nucleic acids of this disclosure encode 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 acids encode 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 acids further comprise a nucleotide sequence encoding a spacer region. In some embodiments, the antigen-binding domain is connected to the transmembrane domain by the spacer region. In some embodiments, the spacer region comprises a nucleic acid sequence selected from any of the nucleic acid sequences listed in Table 3. In some embodiments, the nucleic acids further comprise a nucleotide sequence encoding a leader sequence.

[0241] The nucleic acids of this disclosure can be obtained using any suitable recombinant methods known in the art, including, but not limited to, screening a library from cells expressing the gene of interest, inducing the gene of interest from a vector known to contain the gene, or directly isolating the gene of interest from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

[0242] In some embodiments, the nucleic acids of the Disclosure are contained within a vector. In some embodiments, the nucleic acids of the Disclosure are expressed intracellularly via transposons, CRISPR / Cas9 systems, TALENs, or zinc finger nucleases.

[0243] In some embodiments, the expression of nucleic acids encoding the chimeric receptors of this disclosure can be achieved by operably ligating the nucleic acid with a promoter and incorporating the construct into an expression vector. Suitable vectors can be replicated and incorporated in eukaryotic cells. Typical cloning vectors include transcription and translation terminators, start sequences, and promoters useful for controlling the expression of the desired nucleic acid.

[0244] In some embodiments, the expression constructs of the Disclosure may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols (e.g., US5399346, US5580859, and US5589466). In some embodiments, the vectors of the Disclosure are gene therapy vectors.

[0245] The nucleic acids of this disclosure may be cloned into several types of vectors. For example, the nucleic acids may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, or cosmids. In some embodiments, the vector may be an expression vector, a replication vector, a probe-generating vector, or a sequencing vector.

[0246] In some embodiments, the plasmid vector includes a transposon / transposase system for incorporating the nucleic acid of this disclosure into the 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 a Sleeping Beauty transposon / transposase or a piggyBac transposon / transposase.

[0247] In some embodiments, the expression vectors of the Disclosure may be delivered to cells in the form of viral vectors. Suitable viral vector systems are well known in the Art. For example, viral vectors may be derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In some embodiments, the vectors of the Disclosure are lentiviral vectors. Lentiviral vectors are suitable for long-term gene transfer because such vectors enable long-term and stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors are also advantageous over vectors derived from oncoretroviruses (e.g., mouse leukemia virus) in that lentiviral vectors can transduce non-proliferating cells. In some embodiments, the vectors of the Disclosure are adenovirus vectors (A5 / 35). In some embodiments, the vectors of the Disclosure include a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and US6326193). Numerous virus-based systems have been developed for gene transfer into mammalian cells. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to mammalian cells either in vivo or ex vivo. Several retroviral systems are known in the art.

[0248] In some embodiments, the vectors of this disclosure include additional promoter elements, such as enhancers, which control the frequency of transcription initiation. Enhancers are typically located 30 bp to 110 bp upstream of the initiation site, although some promoters have been shown to also include functional elements downstream of the initiation site. 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 before activity begins to decrease. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Exemplary promoters may 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.

[0249] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells such as the immune-responsive cells of the Disclosure is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in promoting chimeric receptor expression from nucleic acids cloned into lentiviral vectors.

[0250] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells such as immune-responsive cells of the Disclosure is a constitutive promoter. For example, a preferred constitutive promoter is the pre-early cytomegalovirus (CMV) promoter. The CMV promoter is a potent constitutive promoter capable of driving high levels of expression of any polynucleotide sequence operably linked to the promoter. Other preferred constitutive promoters include, but are not limited to, the ubiquitin C (UbiC) promoter, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) promoter, the human immunodeficiency virus (HIV) terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus pre-early promoter, the Roussarcoma virus promoter, the actin promoter, the myosin promoter, the elongation factor 1a promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0251] In some embodiments, the promoter capable of expressing the nucleic acids of the Disclosure in mammalian cells, such as immune-responsive cells of the Disclosure, is an inducible promoter. The use of an inducible promoter may provide a molecular switch that can induce or suppress the expression of the nucleic acids of the Disclosure when the promoter is operably linked to the nucleic acid. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0252] In some embodiments, the vectors of the present disclosure may further include signal sequences for promoting secretion, polyadenylation signals and transcription terminators, factors enabling episome replication, and / or factors enabling selection.

[0253] In some embodiments, the vectors of this disclosure may further include selectable marker genes and / or reporter genes to facilitate the identification and selection of chimeric receptor-expressing cells from a population of cells transduced with the vector. In some embodiments, the selectable markers may be encoded by nucleic acids that are isolated from the vector and used in a simultaneous translocation procedure. Either the selectable marker or reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Examples of selectable markers include, but are not limited to, antibiotic resistance genes such as neo.

[0254] In some embodiments, a reporter gene may be used to identify transduced cells and to evaluate the functionality of a regulatory sequence. As disclosed herein, a reporter gene is a gene that is not present or expressed in a recipient organism or tissue and encodes a polypeptide whose expression imposes easily detectable properties, such as enzymatic activity. The expression of a reporter gene can be assayed at a suitable time after the nucleic acid has been introduced into recipient cells. Examples of reporter genes include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloranphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein. Suitable expression systems are well known in the art and can be prepared using known techniques or are commercially available. In some embodiments, a construct having the smallest 5' adjacent region exhibiting the highest level of expression of the reporter gene is identified as a promoter. Such a promoter region may be ligated to a reporter gene and used to evaluate a drug for its ability to regulate promoter-driven transcription.

[0255] In some embodiments, a vector comprising a nucleic acid sequence encoding the chimeric receptor of the present disclosure further comprises a second nucleic acid encoding a polypeptide that increases the activity of the chimeric receptor.

[0256] In embodiments where EMCN-specific protein-expressing cells include two or more heterogeneous proteins (e.g., two or more chimeric receptors), a single nucleic acid may encode two or more proteins under a single regulatory element (e.g., a promoter) or under separate regulatory elements for each nucleotide sequence encoding each protein contained in the nucleic acid. In some embodiments where EMCN-specific protein-expressing cells include two or more heterogeneous proteins, each heterogeneous protein may be encoded by a separate nucleic acid. In some embodiments, each separate nucleic acid includes its own regulatory element (e.g., a promoter). In some embodiments, a single nucleic acid encodes two or more chimeric receptors, and the nucleotide sequences encoding the chimeric receptors are in the same reading frame and expressed as a single polypeptide chain. In such embodiments, two or more chimeric receptors may be separated by one or more peptide cleavage sites, e.g., autocleavage sites or substrates of intracellular proteases. Preferred peptide cleavage sites may include, but are not limited to, T2A peptide cleavage sites, P2A peptide cleavage sites, E2A peptide cleavage sites, and F2A peptide cleavage sites. In some embodiments, two or more chimeric receptors include a T2A peptide cleavage site. In some embodiments, two or more chimeric receptors include an E2A peptide cleavage site. In some embodiments, two or more chimeric receptors include a T2A and an E2A peptide cleavage site.

[0257] Methods for introducing and expressing genes in cells are well known in the art. For example, in some embodiments, expression vectors can be transferred to host cells by physical, chemical, or biological means. Examples of physical means for introducing nucleic acids into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Examples of chemical means for introducing nucleic acids into host cells include, but are not limited to, colloidal dispersions, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of biological means for introducing nucleic acids into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0258] In some embodiments, liposomes may be used as a nonviral delivery system for introducing nucleic acids or vectors of this disclosure into host cells in vitro, ex vivo, or in vivo. In some embodiments, nucleic acids may associate with lipids by, for example, being encapsulated within an aqueous liposome, being dispersed within the lipid bilayer of a liposome, being attached to a liposome via a linking molecule that associates with both the liposome and the nucleic acid, being confined within a liposome, being complexed with a liposome, being dispersed in a lipid-containing solution, being mixed with lipids, being combined with lipids, being contained as a suspension in lipids, being contained in or complexed in micelles, or being associated with lipids in other ways. 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 having a “broken-down” structure. Such compositions may also be dispersed in solution to form aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that may occur naturally or be synthesized. In some embodiments, lipids may include naturally occurring lipid droplets in the cytoplasm, or a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids may be obtained from commercial sources and include, but are not limited to, dimyristylphosphatidylcholine ("DMPC"), dicetyl phosphate ("DCP"), cholesterol, and dimyristylphosphatidylglycerol ("DMPG"). Storage solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the solvent because it evaporates more readily than methanol. As used herein, "liposomes" may encompass a variety of single and multilayer lipid vehicles formed by the formation of encapsulated 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, multilayer liposomes may have multiple lipid layers separated by an aqueous medium. Multilayer liposomes can be spontaneously formed by suspending phospholipids in an excess aqueous solution. In some embodiments, the lipid components may undergo self-rearrangement before the formation of a closed structure, allowing water and dissolved solutes to be trapped between the lipid bilayers. In some embodiments, lipids may be envisioned as having a micelle structure or may exist only as heterogeneous aggregates of lipid molecules.

[0259] In some embodiments, the nucleic acids or vectors of the Disclosure are introduced into mammalian host cells, such as immune-responsive cells of the Disclosure. In some embodiments, the presence of the nucleic acids or vectors of the Disclosure in host cells can be confirmed by any suitable assay known in the Art, including but not limited to Southern blot assays, Northern blot assays, RT-PCR, PCR, ELISA assays, and Western blot assays.

[0260] In some embodiments, the nucleic acids or vectors of the Disclosure are stably transduced into immune-responsive cells of the Disclosure. In some embodiments, cells exhibiting stable expression of the nucleic acids or vectors express the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months after transduction.

[0261] In embodiments in which the EMCN-specific protein of the Disclosure (e.g., a chimeric receptor) is transiently expressed in cells, the nucleic acid or vector encoding the EMCN-specific protein of the Disclosure is transfected into immune-responsive cells of the Disclosure. In some embodiments, the immune-responsive cells express the EMCN-specific protein for about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after transfection.

[0262] In some embodiments, the nucleic acid construct encodes a bicistronically encoded chimeric antigen receptor. In some embodiments, the encoded bicistronic chimeric antigen receptor includes an EMCN CAR (e.g., an EMCN inhibitory CAR) and a CAR specific to a second antigen (e.g., a tumor-targeting chimeric receptor).

[0263] 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.

[0264] Pharmaceutical composition and administration Certain embodiments of this disclosure relate to compositions (e.g., pharmaceutical compositions) comprising one or more EMCN-specific proteins (e.g., chimeric receptors) or immune-responsive cells expressing such one or more EMCN-specific proteins. In some embodiments, compositions comprising EMCN-specific proteins (e.g., chimeric receptors) or genetically modified immune-responsive cells expressing such EMCN-specific proteins can be delivered systemically or directly to a subject for the treatment of proliferative disorders, such as bone marrow abnormalities. In certain embodiments, the composition is injected directly into the organ of interest (e.g., the organ affected by the disorder). Alternatively, the composition may be delivered indirectly to the organ of interest, for example, by administration to the circulatory system (e.g., the tumor vascular system). Proliferative and differentiation agents can be delivered 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.

[0265] Compositions comprising the gene-modified cells of this disclosure can be administered intravascularly by any physiologically acceptable vehicle, for example, but they can also be introduced into bone or other convenient sites where the gene-modified cells can find suitable sites for regeneration and differentiation (e.g., the thymus). In some embodiments, at least 1 × 10⁻⁶ cells are present. 5 A number of cells may be administered, ultimately resulting in 1 × 10⁶ cells. 10It reaches one or more cells. The compositions comprising the gene-modified cells of this disclosure may include a purified cell population. Methods for determining the proportion of gene-modified cells in a cell population are well known in the art and include, but are not limited to, fluorescence-activated cell sorting (FACS). In some embodiments, the purity of gene-modified cells in a cell population may be about 50%, about 55%, about 60%, or about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more. The dosage can be easily adjusted by those skilled in the art (for example, a decrease in purity may require an increase in dosage). The cells may be introduced by injection, catheter, etc. In some embodiments, the factors may 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.

[0266] In certain embodiments, the composition is a pharmaceutical composition comprising genetically modified cells, such as immune-responsive cells or their progenitor cells, and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, immune-responsive cells or precursors may be obtained from one subject and administered to the same subject or subjects of different suitability. In some embodiments, the immune-responsive cells or their offspring of the Disclosure may be derived from peripheral blood cells (e.g., in vivo, ex vivo, or in vitro) and may be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the therapeutic compositions of the Disclosure (e.g., pharmaceutical compositions comprising the genetically modified cells of the Disclosure), they are generally formulated in unit dose injectable forms (solutions, suspensions, emulsions).

[0267] formulation Some aspects of this disclosure relate to formulations of compositions comprising the EMCN-specific protein of this disclosure (e.g., chimeric receptor) or genetically modified cells expressing such proteins (e.g., immune-responsive cells of this disclosure). In some embodiments, the compositions of this disclosure comprising genetically modified cells may be provided as sterile liquid preparations, including but not limited to isotonic aqueous solutions, suspensions, emulsions, dispersions, and viscous compositions that can be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions can be administered more conveniently, particularly by injection. In some embodiments, viscous compositions may be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions may include carriers, which may be solvents or dispersion media, comprising, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0268] In some embodiments, sterile injectable solutions can be prepared by incorporating the genetically modified cells of this disclosure into a sufficient amount of a suitable solvent along with various desired amounts of any other components. Such compositions can be mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, and dextrose. In some embodiments, the compositions can also be lyophilized. Depending on the administration route and the desired preparation, the compositions may include auxiliary substances such as wetting agents, dispersants, pH buffers, and antimicrobial agents.

[0269] In some embodiments, the compositions of the present disclosure may further include a variety of additives that can enhance the stability and sterility of the compositions. Examples of such additives include, but are not limited to, antimicrobial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination can be prevented by including, but not limited to, a variety of antimicrobial and antifungal agents, including parabens, chlorobutanol, phenol, and sorbic acid. Long-term absorption of the injectable formulations of the present disclosure can be achieved by using suitable agents that delay absorption, such as aluminum monostearate and gelatin.

[0270] 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.

[0271] In some embodiments, the components of the formulations of the Disclosure are selected to be chemically inert and not affect the viability or efficacy of the genetically modified cells of the Disclosure.

[0272] One consideration relating to the therapeutic use of genetically modified cells in this disclosure is the amount of cells required to achieve optimal efficacy. In some embodiments, the amount of cells administered varies depending on the target being treated. In certain embodiments, the amount of genetically modified cells administered to the target requiring it is 1 × 10⁻⁶ 4 Individual cells ~1 × 10 10 The dose may range from a single cell. In some embodiments, the exact amount of cells considered an effective dose may be based on individual factors for each subject, including size, age, sex, weight, and condition of the particular subject. The dosage can be readily determined by those skilled in the art based on this disclosure and technical knowledge.

[0273] Heterogeneous parts and modifications In a further series of embodiments, the EMCN-specific proteins of this specification (e.g., EMCN-specific chimeric proteins comprising an antigen-binding domain having one or more of the amino acid sequences listed in Table A) include additional moieties and / or modifications.

[0274] Drug conjugate In various embodiments, a protein containing the EMCN-specific antigen-binding domain described herein is conjugated to a therapeutic agent (i.e., a drug) to form an antibody-drug conjugate. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, contrast 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 conjugated to the antigen-binding domain via a linker peptide, as will be discussed in more detail herein.

[0275] Methods for preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate a drug to an antigen-binding domain (e.g., having one or more of the amino acid sequences listed in Table A) disclosed herein include, for example, U.S. Patent No. 8,624,003 (Pot Method), U.S. Patent No. 8,163,888 (One-Step Method), U.S. Patent No. 5,208,020 (Two-Step Method), and U.S. Patent No. 8,337,85 No. 6, 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, International Publication No. 2017 / 136623, International Publication No. 2017 / 015502, International Publication No. 2017 / 015496, International Publication No. 2017 / 015495, International Publication No. 2004 / 010957, International Publication No. 2005 / 077090, International Publication No. 2005 / 0820 Issue 23, International Publication No. 2006 / 065533, International Publication No. 2007 / 030642, International Publication No. 2007 / 103288, International Publication No. 2013 / 173337, International Publication No. 2015 / 057699, International Publication No. 2015 / 095755, International Publication No. 2015 / 123679, International Publication No. 2015 / 157286, International Publication No. 2017 / 165851, International Publication No. 2009 / 073445, International Publication No. International Publication No. 2010 / 068759, International Publication No. 2010 / 138719, International Publication No. 2012 / 171020, International Publication No. 2014 / 008375, International Publication No. 2014 / 093394, International Publication No. 2014 / 093640, International Publication No. 2014 / 160360, International Publication No. 2015 / 054659, International Publication No. 2015 / 195925, International Publication No. 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 This is described in 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, with respect to all of its teachings, is incorporated herein by reference in its entirety.

[0276] Further connecting parts In various embodiments, the EMCN-specific chimeric protein comprises an antigen-binding domain having one or more of the amino acid sequences listed in Table A and one or more further binding sites. In certain embodiments, the binding site is an antibody fragment or antibody format, non-limitingly including full-length antibodies, Fab fragments, Fv, scFv, tandem scFv, diabodies, sc-diabodies, DART, tandAb, minibodies, camel VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), and all of their teachings are incorporated herein by reference.

[0277] In certain embodiments, one or more additional binding moieties are bound to the C-terminus of one or more peptides with an EMCN-specific antigen-binding domain, such as VH and / or VL, Fab heavy chain and / or light chain fragments, or scFv. In certain embodiments, one or more additional binding moieties are bound to the N-terminus of one or more peptides with an EMCN-specific antigen-binding domain, such as VH and / or VL, Fab heavy chain and / or light chain fragments, or scFv.

[0278] In certain embodiments, one or more further binding sites are specific to an antigen or epitope different from EMCN. In certain embodiments, one or more further binding sites are specific to EMCN.

[0279] In certain embodiments, one or more additional binding moieties are bound to the antigen-binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) using, non-limiting, in vitro methods including reactive chemistry (e.g., click chemistry) and affinity tagging systems. In certain embodiments, one or more additional binding moieties are bound to the antigen-binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) via Fc-mediated binding (e.g., protein A / G). In certain embodiments, one or more additional binding moieties are bound to the antigen-binding domain described herein (e.g., having one or more of the amino acid sequences listed in Table A) using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the antigen-binding domain described herein and the additional binding moieties of the same expression vector (e.g., plasmid).

[0280] Functional / reactive group In various embodiments, the antigen-binding domains described herein (for example, having one or more of the amino acid sequences listed in Table A) have functional groups or chemical reaction groups that can be used in downstream processes and downstream purification processes, such as by binding to further parts (e.g., drug conjugates and further binding parts).

[0281] In certain embodiments, the modification is a chemically reactive group, non-limitingly, including reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemical" groups (e.g., reactive alkyne groups), and aldehydes that produce formylglycine (FGly). In certain embodiments, the modification is a functional group, non-limitingly, including affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and streptosystems). 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, non-limitingly, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In certain embodiments, protease cleavage is carried out by an intracellular protease. In certain embodiments, protease cleavage is carried out 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.), and all of their instructions are incorporated herein by reference.

[0282] Treatment method Certain aspects of the present disclosure relate to methods for using the proteins (e.g., chimeric receptors) and genetically modified cells (e.g., immune-responsive cells) of the present disclosure expressing such proteins (e.g., chimeric receptors) to treat subjects that require such treatment. In some embodiments, the methods of the present disclosure are useful for treating cancers of subjects such as myelopathy. In some embodiments, the myelopathy is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myeloid leukemia, or polycythemia vera. In some embodiments, the myelopathy is AML. Other aspects of the present disclosure relate to the use of the chimeric receptors and genetically modified cells (e.g., immune-responsive cells) of the present disclosure expressing such chimeric receptors in methods for treating pathogen infections or other infectious diseases in subjects such as immunocompromised human subjects. In some embodiments, the methods of the Disclosure may include administering the gene-modified cells of the Disclosure in an amount effective to achieve a desired effect, including but not limited to mitigating an existing condition, preventing a condition, treating an existing condition, managing an existing condition, or preventing recurrence or relapse of a condition. In some embodiments, the effective amount may be provided in a single or series of administrations of the gene-modified cells of the Disclosure (e.g., immune-responsive cells). In some embodiments, the effective amount may be provided by a bolus or continuous perfusion.

[0283] As disclosed herein, an “effective dose” or “therapeutic effective dose” is an amount sufficient to produce a beneficial or desired clinical outcome at the time of treatment. An effective dose may be administered to a subject in one or more doses. With respect to treatment, an effective dose is an amount sufficient to mitigate, improve, stabilize, reverse, or delay the progression of a disease, or to reduce the pathological outcomes of the disease. The effective dose is generally determined on a case-by-case basis by a physician and is within the scope of the skill of those skilled in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective dose. These factors include the subject’s age, sex, and weight, the condition being treated, the severity of the condition, and the morphology and effective concentration of the immune-responsive cells being administered.

[0284] In adoptive immunotherapy using antigen-specific cells (e.g., immune response cells such as T cells), approximately 1 × 10⁻⁶ 6 ~1 × 10 10 Individual cells (for example, about 1 × 10⁻⁶) 9 Cell doses ranging from a few cells are typically injected. During cell administration to a target and subsequent differentiation, immune-responsive cells are induced to be specifically directed toward a particular antigen. In some embodiments, induction of immune-responsive cells may include, but is not limited to, inactivation of antigen-specific cells by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance in autoimmune disorders, etc. Genetically modified cells may be administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodular, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

[0285] In some embodiments, the method of use includes a method of inhibiting an immune response. Inhibition of an immune response may refer to, for example, the prevention, attenuation, or inhibition of a cell-mediated immune response induced by chimeric receptors expressed on the surface of immunomodulatory cells. In embodiments, the method includes the prevention, attenuation, or inhibition of the activation of activated chimeric receptors expressed on the surface of immunomodulatory cells.

[0286] In some embodiments, inhibitory chimeric receptors of this disclosure are used to prevent, attenuate, inhibit, or suppress an immune response initiated by a tumor-targeting chimeric receptor (e.g., activated CAR). For example, immunomodulatory cells express 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 the exemplary immunomodulatory cells come into contact with the target cells, the inhibitory chimeric receptor and the tumor-targeting chimeric receptor may or may not bind to their respective congener antigens. In this example scenario, where the target cells are non-tumor cells 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 by the tumor-targeting chimeric receptor, and the immunomodulatory cells are not activated. Similarly, in an exemplary example where the target cells are non-tumor cells expressing only antigen target 1, only the inhibitory chimeric receptor can be activated. In contrast, in exemplary cases where the target cells are tumor cells expressing only antigen target 2, inhibitory chimeric receptors cannot be activated, but tumor-targeting chimeric receptors can be activated, leading to signaling that results in the activation of immunomodulatory cells.

[0287] Inhibition of the immune response initiated by tumor-targeting chimeric receptors can be an inhibition or reduction of tumor-targeting chimeric receptor activation, an inhibition or reduction of tumor-targeting chimeric receptor signaling, or an inhibition or reduction of immunomodulatory cell activation. Inhibitory chimeric receptors can inhibit tumor-targeting chimeric receptor activation, tumor-targeting chimeric receptor signaling, or tumor-targeting chimeric receptor-mediated immunomodulatory cell activation by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more compared to tumor-targeting chimeric receptor activation, immunomodulatory cell signaling, or activation in immunomodulatory cells lacking the inhibitory chimeric receptor. In some embodiments, inhibition refers to a decrease or reduction in the activity of the tumor-targeting chimeric receptor before and after its activation.

[0288] An immune response can be the production and secretion of cytokines or chemokines from activated immunomodulatory cells. An immune response can also be a cell-mediated immune response against target cells.

[0289] In some embodiments, chimeric inhibitory receptors can suppress cytokine production from activated immunomodulatory cells. In some embodiments, chimeric inhibitory receptors can suppress cell-mediated immune responses against target cells, in which the immune response is induced by the activation of immunomodulatory cells.

[0290] Treatment In some embodiments, the methods of the present disclosure increase the immune response in subjects requiring it. In some embodiments, the methods of the present disclosure include methods for treating and / or preventing myelopathy in subjects. In some embodiments, the subjects are human. In some embodiments, human subjects suitable for treatment may include two therapeutic groups that can be distinguished by clinical criteria. Subjects having “progressive disease” or “high tumor burden” are subjects having clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., based on the percentage of leukemia cells by palpation, CAT scan, ultrasound, mammography, or X-ray; positive biochemical or histopathological markers are insufficient in themselves to identify this population). In some embodiments, the pharmaceutical compositions of the present disclosure are administered to these subjects to induce an antitumor response for the purpose of alleviating their condition. In some embodiments, a reduction in tumor load 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 reduction in the risk of pathological consequences of the tumor or a reduction in the rate of progression. In some embodiments, a second group of preferred human subjects is the “adjuvant group” subjects. These subjects are individuals with a history of bone marrow disorders but who have responded to other treatments. Previous treatments may include, but are not limited to, surgical resection, radiotherapy, and / or conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk of disease progression, either near the original tumor site or due to metastasis. In some embodiments, this group can be further subdivided into high-risk and low-risk individuals. Subdivision can be made based on features observed before or after initial treatment. These features are known in clinical practice and are preferably defined for each different bone marrow disorder. Typical features of the high-risk subgroup include tumor infiltration into adjacent tissue or lymph node involvement.

[0291] In any and all embodiments of the increase in immune response described herein, any increase, decrease, or change in any aspect of the characteristic or function is compared to cells that have not been in contact with the immune-responsive cells described herein.

[0292] An increase in the immune response can be either an enhancement or an induction of the immune response. For example, increasing the immune response encompasses both initiating or starting an immune response, or increasing or amplifying an ongoing or existing immune response. In some embodiments, a 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, a treatment enhances the 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, a treatment increases the immune response. In some embodiments, the increased immune response is an adaptive immune response. In some embodiments, the increased immune response is an innate immune response.

[0293] In some embodiments, a further group of subjects may be those who have a genetic predisposition to myelopathy but have not yet demonstrated clinical signs of myelopathy. For example, a woman who has been detected as positive for an AML-related gene mutation but is still of childbearing age may benefit from receiving one or more cells (e.g., immune-responsive cells) of the Disclosure in prophylactic treatment to prevent the development of AML until she is suitable to undergo prophylactic surgery. In some embodiments, subjects may have a disease progression morphology, in which case the treatment objectives may include mitigating or reversing disease progression and / or improving side effects. In some embodiments, subjects may have a history of a condition for which they are already being treated, in which case the treatment objectives may typically include reducing or delaying the risk of relapse.

[0294] Combination therapy In some embodiments, genetically modified cells (e.g., immune response cells) expressing one or more proteins containing the antigen-binding domain (e.g., scFv) of the Disclosure, such as the chimeric receptor of the Disclosure, may be used in combination with other known agents and therapies. In some embodiments, the combination therapy of the Disclosure includes genetically modified cells of the Disclosure, which may be administered in combination with one or more additional therapeutic agents. In some embodiments, the recombinant 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 substance may be administered first, and one or more additional agents may be administered second, or the order of administration may be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[0295] In some embodiments, the gene-modified cells of this disclosure may be used in therapeutic 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), cytoxin, fludarabume, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation methods, and peptide vaccines.

[0296] In some embodiments, the gene-modified cells of this disclosure may be used in combination with lymphocyte depletion agents. Suitable lymphocyte depletion agents reduce or decrease lymphocytes, such as B-cell lymphocytes and / or T-cell lymphocytes, before immunotherapy. Examples of suitable lymphocyte depletion agents include, but are not limited to, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[0297] In some embodiments, the gene-modified cells of this disclosure may be used in combination with chemotherapeutic agents. 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., folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., acralubicin A, gliotoxin, bortezomib), and immunodermal derivatives, e.g., thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0298] Examples of common chemotherapeutic agents suitable for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (bleomycin®), busulfan (Milleran®), busulfan infusion (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), and chloram. Bucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Citoxane® or Neosal®), cytarabine, cytosine arabinoside (Citosal-U®), cytarabine liposome injection (Depotyte®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Servidine®), daunorubicin citrate liposome Injection (Daunozom®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepsid®), fludarabine phosphate (Fludarabine®), 5-fluorouracil (Adolsyl®, Efdex®), flutamide (Eurexin®), tezacytibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idani) Syn (registered trademark), Ifosfamide (IFEX (registered trademark)), Irinotecan (Camptosal (registered trademark)), L-asparaginase (ELSPAR (registered trademark)), Leucovorin calcium, Melphalan (Alkeran (registered trademark)), 6-mercaptopurine (Printol (registered trademark)), Methotrexate (Forex (registered trademark)), Mitoxantrone (Novantrone (registered trademark)), Mylotarg, Paclitaxel (Taxol (registered trademark)), Phoenix (Yttrium90 / MX-DTPA), Pentostatin,This includes, but is not limited to, Polyfeprosan 20 (Giliadel®) containing carmustine implants, tamoxifen citrate (Nolvadex®), teniposide (Bumon®), 6-thioguanine, thiotepa, tirapazamine (Thirazon®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velvon®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0299] Examples of suitable alkylating agents include nitrogen mustard, ethyleneimine 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) Examples include, but are not limited to, immune®, ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambutyl (Leukeran®), pipobromane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozosin (Zanosar®), and dacarbazine (DTIC-Dome®).Examples of additional exemplary alkylating agents include oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolicin, and phenylalanine master, Alkeran®), altoretamine (also known as hexamethylmelamine (HMM), Hexalen®), and carmustine (B iCNU(registered trademark), bendamustine (Treanda(registered trademark)), busulfan (Busulfex(registered trademark) and Myleran(registered trademark)), carboplatin (Paraplatin(registered trademark)), lomustine (also known as CCNU, CeeNU(registered trademark)), cisplatin (also known as CDDP, Platinol(registered trademark) and Platinol(registered trademark)-AQ), chlorambucil (Leukeran(registered trademark)), cyclophosphamide (Cytoxan(registered trademark) and Neo sar(registered trademark)), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome(registered trademark)), Aitretamine (also known as hexamethylmelamine (HMM), Hexalen(registered trademark)), Ifosfamide (Ifex(registered trademark)), Prednumustine, Procarbazine (Matulane(registered trademark)), Mechloretamine (also known as nitrogen mustard, mustine, and mechloretamine hydrochloride) This includes, but is not limited to, Mustargen®, streptozocin (Zanosar®), thiotepa (thiophosphoamide, also known as TESPA and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine HC1 (Treanda®).

[0300] Examples of suitable mTOR inhibitors include, but are not limited to, temsirolimus, ridafololimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RAD001), rapamycin (AY22989, Silolumius®), and XL765.

[0301] Examples of suitable immunomodulatory agents include, but are not limited to, aftuzumab, pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Salomid®), actimide (CC4047), and IRX-2.

[0302] Examples of suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin® and Rubex®), bleomycin (lenoxane®), daunorubicin (daunorubicin hydrochloride, DaunoXome, and rubidomycin hydrochloride, Cerubidine®), daunorubicin liposome (daunorubicin triglyceride liposome, DaunoXome®), mitoxantrone (DHAD, Novantrone®), epirubicin (Ellence®), idarubicin (idamycin®, idamycin PES®), mitomycin C (mutamycin®), geldanamycin, harbimycin, rabidomycin, and desacetramycin.

[0303] Suitable examples of vinca alkaloids include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldicine®), and vinblastine (also known as vinblastine sulfate, vincalucoblastine and VLB, Alkaban-AQ® and Verban®).

[0304] 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.

[0305] In some embodiments, the gene-modified cells of this 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, bertuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), okalatuzumab, and Prol31921.

[0306] In some embodiments, the genetically modified cells of this disclosure are administered in combination with an oncolytic virus. In some embodiments, the oncolytic virus can selectively replicate in cancer cells, inducing cancer cell death or delaying their proliferation. 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 adenoviruses, oncolytic herpes simplex viruses, oncolytic retroviruses, oncolytic parvoviruses, oncolytic vaccinia viruses, oncolytic cymbisviruses, oncolytic influenza viruses, or oncolytic RNA viruses (e.g., oncolytic reoviruses, 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.

[0307] In some embodiments, the gene-modified cells of this disclosure are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, such as an SHP-1 inhibitor or an SHP-2 inhibitor. In one embodiment, the gene-modified cells of this disclosure can be used in combination with a kinase inhibitor. Examples of suitable kinase inhibitors include, but are not limited to, CDK4 inhibitors, CDK4 / 6 inhibitors, BTK inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, mTOR inhibitors, MNK inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors.

[0308] In some embodiments, the gene-modified cells of this disclosure are administered to a subject in combination with a myeloid-derived suppressor cell (MDSC) modulator. MDSCs accumulate in the periphery and tumor sites of many solid tumors. These cells suppress the T cell response, thereby hindering the effectiveness of chimeric receptor-expressing cell therapy. While not bound by theory, administration of MDSC modulators is thought to enhance the effectiveness of the gene-modified cells of this disclosure. Examples of preferred MDSC modulators include, but are not limited to, MCS110 and BLZ945.

[0309] In some embodiments, the gene-modified cells of this 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, immunosuppressive plasma cells may express one or more of IgA, interleukin (IL)-10, and PD-L1.

[0310] In some embodiments, the gene-modified cells of the Disclosure are administered to a subject in combination with interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both IL-15 polypeptide and IL-15Ra polypeptide. In some embodiments, the gene-modified cells of the Disclosure are further modified to express interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both IL-15 polypeptide and IL-15Ra polypeptide.

[0311] In some embodiments, subjects with bone marrow disorders (e.g., AML) are administered the gene-modified cells of the Disclosure in combination with a drug, e.g., a cytotoxic agent or chemotherapeutic agent, a biological therapy (e.g., an antibody, e.g., a monoclonal antibody, or cell therapy), or an inhibitor (e.g., a kinase inhibitor). In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin, bosaroxine (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is a liposomal formulation containing cytarabine and daunorubicin in a 5:1 molar ratio. In some embodiments, subjects are 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, subjects are administered the gene-modified cells of the Disclosure in combination with a biological therapy, e.g., an antibody or cell therapy, e.g., 225Ac-lintuzumab (Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma / Bristol Myers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer). In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with an FLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo), clenolanib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), or ASP2215 (Astelias).In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with an isocitrate dehydrogenase (IDH) inhibitor, such as AG-221 (Celgene / Agios) or AG-120 (Agios / Celgene). In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with a cell cycle regulator, such as a polo-like kinase 1 (Plkl) inhibitor, such as boracertib (Boehringer Ingelheim), or a cyclin-dependent kinase 9 (Cdk9) inhibitor, such as arbocidib (Tolero Pharmaceuticals / Sanofi Aventis). In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with a B-cell receptor signaling network inhibitor, e.g., a B-cell lymphoma 2 (Bcl-2) inhibitor, e.g., venetoclax (Abbvie / Roche), or a Bruton's tyrosine kinase (Btk) inhibitor, e.g., ibrutinib (Pharmacyclics / Johnson & Johnson Janssen Pharmaceutical). In some embodiments, subjects are administered the gene-modified cells of the Disclosure in combination with an M1 aminopeptidase inhibitor, a histone deacetylase (HDAC) inhibitor, e.g., prasinostat (MEI Pharma), a multi-kinase inhibitor, e.g., rigosertib (Onconova Therapeutics / Baxter / SymBio), or a peptide CXCR4 reverse agonist, e.g., BL-8040 (BioLineRx).

[0312] In some embodiments, subjects may be administered agents that enhance the activity or compatibility of the genetically modified cells of this disclosure. For example, the agent may inhibit molecules that modulate or control T cell function, such as inhibitory molecules. In some embodiments, the molecules that modulate or control T cell function are inhibitory molecules. In some embodiments, inhibitory molecules such as programmed death 1 (PD-1) can reduce the ability of genetically modified cells to initiate an immune effector response. Examples of suitable inhibitory molecules include, but are not limited to, PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta. For example, inhibition of molecules that modulate or control T cell function by inhibition at the DNA, RNA, or protein level can optimize the performance of the genetically modified cells of this disclosure. In some embodiments, a drug, such as an inhibitory nucleic acid, such as dsRNA, such as siRNA or shRNA, clustered and regularly arranged short palindromic sequence repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), may be used to inhibit the expression of an inhibitory molecule in genetically modified cells. In one embodiment, the inhibitor is shRNA. In some embodiments, the genetically modified cells of the present disclosure may be further modified to express an inhibitory nucleic acid, such as dsRNA, such as siRNA or shRNA, clustered and regularly arranged short palindromic sequence repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), which may be used to inhibit the expression of an inhibitory molecule in genetically modified cells.

[0313] In one embodiment, a drug that modulates or controls, for example, T cell function, is inhibited in the genetically modified cells of the Disclosure. In such embodiments, a dsRNA molecule that inhibits the expression of a molecule that modulates or controls, for example, T cell function, is ligated to a component of the chimeric receptor of the Disclosure, for example, a nucleic acid encoding all of its components. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or controls T cell function is operably ligated to a promoter, for example, an HI or U6-derived promoter, so that the dsRNA molecule that inhibits the expression of a molecule that modulates or controls T cell function is expressed, for example, in genetically modified cells. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or controls, for example, T cell function, is present on the same vector, for example, a lentiviral vector, that contains the components of the chimeric receptor, for example, a nucleic acid molecule encoding all of its components. In such embodiments, a nucleic acid molecule encoding a dsRNA molecule that modulates or controls T cell function, for example, inhibits the expression of an inhibitory molecule, is located on a 5'- or 3'- vector of the components of a chimeric receptor, for example, a nucleic acid encoding all components, such as a lentiviral vector. The nucleic acid molecule encoding a dsRNA molecule that modulates or controls T cell function, for example, inhibits the expression of an inhibitory molecule, may be transcribed in the same or different direction as the components of the chimeric receptor, for example, the nucleic acid encoding all components. In one embodiment, the nucleic acid molecule encoding a dsRNA molecule that modulates or controls T cell function, for example, inhibits the expression of an inhibitory molecule, is located on a vector other than the vector containing the components of the chimeric receptor, for example, a nucleic acid molecule encoding all components. In one embodiment, the nucleic acid molecule encoding a dsRNA molecule that modulates or controls T cell function, for example, inhibits the expression of an inhibitory molecule, is transiently expressed in a gene-modified cell. In one embodiment, the nucleic acid molecule encoding a dsRNA molecule that modulates or controls T cell function, for example, inhibits the expression of an inhibitory molecule, is stably incorporated into the genome of the gene-modified cell of this disclosure.

[0314] In one embodiment, a drug that modulates or controls, for example, T cell function, may be an antibody or antibody fragment that binds to an inhibitory molecule. For example, the drug may be an antibody or antibody fragment that binds to PD-1, PD-L1, PD-L2, or CTLA4. In one embodiment, the drug is an antibody or antibody fragment that binds to TIM3. In one embodiment, the drug is an antibody or antibody fragment that binds to LAG3.

[0315] 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 CEACAM inhibitor is an anti-CEACAM antibody molecule. In one embodiment, the agent that enhances the activity of the genetically modified cells of this 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 this disclosure are further modified to express an antibody or antibody fragment that binds to the inhibitory molecule of this disclosure.

[0316] In one embodiment, the agent that enhances the activity of the genetically modified cells of this disclosure is a cytokine. Cytokines have important functions related to the expansion, differentiation, survival, and homeostasis of immune-responsive cells. Cytokines that can be administered to subjects receiving the genetically modified cells of this disclosure include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21, or combinations thereof. Cytokines can be administered once daily, or more than once daily, for example, twice daily, three times daily, or four times daily. Cytokines can be administered for more than one day, for example, cytokines can be administered for two days, three days, four days, five days, six days, one week, two weeks, three weeks, or four weeks. For example, cytokines can be administered once daily for seven days. In some embodiments, the gene-modified cells of this 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.

[0317] In some embodiments, cytokines may be administered simultaneously with or concurrently with genetically modified cells, for example, on the same day. Cytokines may be prepared in the same pharmaceutical composition as the genetically modified cells, or in a separate pharmaceutical composition. Cytokines may be administered immediately after administration of genetically modified cells, for example, on day 1, 2, 3, 4, 5, 6, or 7 days after administration of the genetically modified cells. In some embodiments in which cytokines are administered in a drug regimen that results in more than one day of administration, the first day of the cytokine drug regimen may be the same day as the administration with the genetically modified cells, or the first day of the cytokine drug regimen may be day 1, 2, 3, 4, 5, 6, or 7 days after administration of the genetically modified cells. In one embodiment, genetically modified cells are administered on the first day, and cytokines are administered once daily for the next 7 days on the second day. In some embodiments, cytokines are administered for a period following the administration of 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 more than 1 year after administration of genetically modified cells. In one embodiment, cytokines are administered after evaluation of the subject's response to the genetically modified cells.

[0318] 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 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 cells of the present disclosure (e.g., immune-responsive cells), in an effective amount. In some embodiments, the kit comprises a sterile container. In some embodiments, such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, 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 drugs.

[0319] In some embodiments, the therapeutic or prophylactic composition is provided with instructions for administering the therapeutic or prophylactic composition to subjects who have or are 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 the disorder. In some embodiments, the instructions may include, but are not limited to, a description of the therapeutic or prophylactic composition, a dosing schedule, a dosing schedule for the treatment or prevention of the disease or its symptoms, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical trials, and / or references. In some embodiments, the instructions may be printed directly on the container (if any), or as a label affixed to the container, or as a separate sheet, brochure, card, or folder provided inside or with the container.

[0320] Exemplary Embodiments 1. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH is a chimeric protein containing heavy chain complementarity-determining region 3 (CDR-H3) which has the amino acid sequence of RIKD (SEQ ID NO: 4). 2. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH includes heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3), The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. A chimeric protein in which the amino acid sequences of the reference antibody CDR-H1, CDR-H2, and CDR-H3 are optionally defined based on the Kabat or Chothia numbering scheme. 3. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) including, Chimeric protein. 4. A chimeric protein according to any one of Embodiments 1 to 3, wherein the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. 5. VL is, Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) A chimeric protein according to any one of Embodiments 1 to 4, including the above. 6. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3), The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. Chimeric protein. 7. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) including, Chimeric protein. 8. The chimeric protein according to Embodiment 6 or Embodiment 7, wherein VH comprises a heavy chain complementarity determination region 1 (CDR-H1), a heavy chain complementarity determination region 2 (CDR-H2), and a heavy chain complementarity determination region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. 9. VH is, (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) A chimeric protein according to any one of embodiments 6 to 8, including the chimeric protein described in any one of embodiments 6 to 8. 10. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) including, Chimeric protein. 11. A chimeric protein according to any one of Embodiments 1 to 10, wherein the VH region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of Sequence ID No. 1. 12. A chimeric protein according to any one of Embodiments 1 to 11, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1. 13. A chimeric protein according to any one of Embodiments 1 to 12, wherein the VL region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of Sequence ID No. 9. 14. A chimeric protein according to any one of Embodiments 1 to 12, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 15. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO: 1. Chimeric protein. 16. The chimeric protein according to Embodiment 15, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1. 17. The chimera according to Embodiment 15 or Embodiment 16, wherein the VL region includes 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 with the amino acid sequence of Sequence ID No. 9. 18. The chimeric protein according to Embodiment 17, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 19. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain contains an antibody or antigen-binding fragment. The antibody or antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO: 9 Chimeric protein. 20. The chimeric protein according to Embodiment 19, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 21. A chimeric protein according to Embodiment 19 or Embodiment 20, wherein the VH region contains the amino acid sequence of Sequence ID No. 1. 22. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain competes with the reference antibody or its antigen-binding fragment for binding to EMCN. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Chimeric protein. 23. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain essentially binds to the same EMCN epitope as the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Chimeric protein. 24. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous molecule or moiety, The antigen-binding domain binds to the same human EMCN epitope as the EMCN epitope bound by the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Chimeric protein. 25. A chimeric protein according to any one of Embodiments 22 to 24, wherein the VH region of the reference antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 1. 26. A chimeric protein according to any one of embodiments 22 to 25, wherein the VL region of the reference antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 9. 27. A chimeric protein according to any one of Embodiments 1 to 26, wherein the antigen-binding domain comprises an F(ab) fragment, an F(ab') fragment, or a single-stranded variable fragment (scFV). 28. The chimeric protein according to Embodiment 27, wherein the antibody or antigen-binding fragment comprises a single-strand variable fragment (scFv). A chimeric protein according to any one of Embodiments 1 to 28, wherein the VH and VL of 29.scFv are separated by a peptide linker. 30. A chimeric protein according to any one of Embodiments 1 to 29, 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. 31. The chimeric protein according to Embodiment 29 or Embodiment 30, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 39. The chimeric protein according to Embodiment 28 or Embodiment 29, wherein 32.scFv contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 17 to 22. 33. The chimeric protein according to any one of Embodiments 1 to 32, wherein the chimeric protein is an antibody-drug conjugate, and the heterologous molecule or portion comprises a therapeutic agent. 34. A chimeric antigen receptor (CAR) chimeric protein according to any one of Embodiments 1 to 32, wherein the heterogeneous molecule or 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 a combination thereof. 35. The chimeric protein according to Embodiment 34, wherein CAR includes a transmembrane domain. 36. A chimeric protein according to Embodiment 34 or Embodiment 35, wherein CAR comprises one or more intracellular signaling domains. 37. A chimeric protein according to any one of embodiments 34 to 36, wherein the CAR is an activated CAR comprising one or more intracellular signaling domains that stimulate an immune response. 38. The chimeric protein according to any one of embodiments 34 to 36, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. 39. The chimeric protein according to Embodiment 38, wherein the intracellular inhibitory domain includes an enzyme inhibitory domain. 40. The chimeric protein according to Embodiment 38, wherein the intracellular inhibitory domain includes an intracellular inhibitory co-signaling domain. 41. A chimeric protein according to any one of embodiments 35 to 40, wherein the CAR includes a spacer region between the antigen-binding domain and the transmembrane domain. 42. The chimeric protein according to Embodiment 41, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to 48. 43. A single-stranded variable fragment (scFv) specific to endomucin (EMCN), It includes a heavy chain variable (VH) region and a light chain variable (VL) region, VH contains heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4), Single-stranded variable fragment (scFv). 44. Endomucin (EMCN) specific single-stranded variable fragment (scFv), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH includes heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3), The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. Single-stranded variable fragment (scFv). 45. Endomucin (EMCN) specific single-stranded variable fragment (scFv), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) including, Single-stranded variable fragment (scFv). 46. ​​scFv according to any one of embodiments 43 to 45, wherein VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. 47.VL is, Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) scFv as described in any one of embodiments 43 to 46, including the scFv described in any one of embodiments 43 to 46. 48. A single-stranded variable fragment (scFv) specific to endomucin (EMCN), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3), The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region amino acid sequence of SEQ ID NO: 9. Single-stranded variable fragment (scFv). 49. A single-stranded variable fragment (scFv) specific to endomucin (EMCN), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) including, Single-stranded variable fragment (scFv). 50. scFv according to Embodiment 48 or Embodiment 49, wherein VH comprises heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3), and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region amino acid sequence of SEQ ID NO: 1. 51. VH is, (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) scFv as described in any one of embodiments 48 to 50, including the scFv described in any one of embodiments 48 to 50. 52. A single-stranded variable fragment (scFv) specific to endomucin (EMCN), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH (a) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4); or (b) Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) including, Single-stranded variable fragment (scFv). 53. scFv according to any one of embodiments 43 to 52, wherein the VH region includes 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 with the amino acid sequence of SEQ ID NO: 1. 54. scFv according to any one of embodiments 43 to 53, wherein the VH region includes the amino acid sequence of SEQ ID NO: 1. 55. scFv according to any one of embodiments 43 to 54, wherein the VL region includes 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 with the amino acid sequence of SEQ ID NO: 9. 56. scFv according to any one of embodiments 43 to 55, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 57. A single-stranded variable fragment (scFv) specific to endomucin (EMCN), The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO: 1. Single-stranded variable fragment (scFv). 58. scFv according to Embodiment 57, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1. 59. scFv according to Embodiment 57 or Embodiment 58, wherein the VL region includes 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 with the amino acid sequence of Sequence ID No. 9. 60. scFv according to Embodiment 59, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 61. A single-stranded variable fragment (scFv) containing an antigen-binding domain specific to endomucin (EMCN), The antigen-binding domain contains an antibody or antigen-binding fragment. The antibody or antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VL contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO: 9 Single-stranded variable fragment (scFv). 62. scFv according to Embodiment 61, wherein the VL region contains the amino acid sequence of SEQ ID NO: 9. 63. scFv according to Embodiment 61 or Embodiment 62, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1. 64. The scFv according to any one of embodiments 43 to 63, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 17 to 22. 65. A single-stranded variable fragment (scFv) containing an antigen-binding domain specific to endomucin (EMCN), The antigen-binding domain competes with the reference antibody or its antigen-binding fragment for binding to EMCN. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Single-stranded variable fragment (scFv). 66. A single-stranded variable fragment (scFv) containing an antigen-binding domain specific to endomucin (EMCN), The antigen-binding domain essentially binds to the same EMCN epitope as the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Single-stranded variable fragment (scFv). 67. A single-stranded variable fragment (scFv) containing an antigen-binding domain specific to endomucin (EMCN), The antigen-binding domain binds to the same human EMCN epitope as the EMCN epitope bound by the reference antibody or its antigen-binding fragment. The reference antibody or its antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region. VH Heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence GFSLSRY (SEQ ID NO: 2), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, VL Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) containing the amino acid sequence of QVSKLDS (SEQ ID NO: 11) Includes, The amino acid sequences of the reference antibodies CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia annotation and numbering scheme. Single-stranded variable fragment (scFv). 68. scFv according to any one of embodiments 65 to 67, wherein the VH region of the reference antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 1. 69. The scFv according to any one of embodiments 65 to 68, wherein the VL region of the reference antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 9. 70. A composition comprising a chimeric protein according to any one of Embodiments 1 to 42, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 71. A composition comprising scFv according to any one of embodiments 43 to 69, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 72. An engineered nucleic acid encoding a chimeric protein according to any one of Embodiments 1 to 42. 73. An engineered nucleic acid encoding the scFv described in any one of embodiments 43 to 69. 74. An expression vector comprising the manipulated nucleic acid described in Embodiment 72. 75. An expression vector comprising the manipulated nucleic acid described in Embodiment 73. 76. A composition comprising the manipulated nucleic acid described in Embodiment 72 or the expression vector described in Embodiment 74, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 77. A composition comprising the manipulated nucleic acid described in Embodiment 73 or the expression vector described in Embodiment 75, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 78. A method for producing engineered cells, comprising transducing isolated cells or a population of cells using the engineered nucleic acid described in Embodiment 72 or the expression vector described in Embodiment 74. 79. A method for producing engineered cells, comprising transducing isolated cells or a population of cells using the engineered nucleic acid described in Embodiment 73 or the expression vector described in Embodiment 75. 80. Manipulated cells produced by the method described in Embodiment 78. 81. Manipulated cells produced by the method described in Embodiment 79. 82. Isolated cells comprising the manipulated nucleic acid described in Embodiment 72, the expression vector described in Embodiment 74, or the composition described in Embodiment 76. 83. Isolated cells comprising the manipulated nucleic acid described in Embodiment 73, the expression vector described in Embodiment 75, or the composition described in Embodiment 77. 84. A population of manipulated cells expressing the manipulated nucleic acid described in Embodiment 72 and the expression vector described in Embodiment 74. 85. A population of manipulated cells expressing the manipulated nucleic acid described in Embodiment 73 and the expression vector described in Embodiment 75. 86. Isolated cells containing the chimeric protein described in any one of Embodiments 1 to 42. 87. Isolated cells containing scFv as described in any one of embodiments 43 to 69. 88. A population of manipulated cells expressing the chimeric protein described in any one of Embodiments 1 to 42. 89. A population of manipulated cells expressing the scFv described in any one of embodiments 43 to 69. 90. A cell or population of cells according to any one of embodiments 80, 82, 84, 86, and 88, wherein a chimeric protein is recombinantly expressed. A cell or population of cells according to any one of embodiments 81, 83, 85, 87, and 89, wherein 91.scFv is recombinantly expressed. 92. A cell or population of cells according to any one of embodiments 80, 82, 84, 86, 88, and 90, wherein the chimeric protein is expressed from a selected gene locus derived from a vector or the cell's genome. 93. A cell or population of cells according to any one of embodiments 81, 83, 85, 87, 89, and 91, wherein the chimeric protein is expressed from a selected gene locus derived from a vector or the cell's genome. 94. The cell or cell population according to any one of Embodiments 80, 82, 84, 86, 88, 90, and 92, wherein the cell or cell population further comprises one or more tumor-targeting chimeric receptors expressed on the cell surface. 95. The cells or population of cells according to Embodiment 94, wherein one or more tumor-targeting chimeric receptors are chimeric antigen receptors (CARs) or engineered T cell receptors. 96. A cell or population of cells according to Embodiment 94 or 95, comprising a first tumor-targeted chimeric receptor that targets a first tumor-associated antigen, and a second tumor-targeted chimeric receptor that targets a second tumor-associated antigen. 97. The cells or population of cells according to Embodiment 96, wherein the first tumor-associated antigen comprises CD33 and the second tumor-associated antigen comprises FLT3. 98. The cells or population of cells according to Embodiment 94 or 95, wherein one or more tumor-targeting chimeric receptors include tumor-targeting chimeric receptors that target CD33 and FLT3. 99. A cell or population of cells according to any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-98, selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. 100. A cell or population of cells according to any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-98, which is an NK cell. 101. Autologous cells or populations of cells as described in any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-100. 102. Cells or populations of cells according to any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-100, which are of the same kind. 103. A pharmaceutical composition comprising an effective amount of cells or a population of cells as described in any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 104. A pharmaceutical composition comprising an effective amount of gene-modified cells expressing any one of Embodiments 1 to 42, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. 105. A pharmaceutical composition according to Embodiment 103 or Embodiment 104, which is for treating and / or preventing tumors. 106. A method for treating a subject in need thereof, comprising administering a therapeutically effective dose of the composition described in Embodiment 70 or Embodiment 76, or any of the cells described in any one of Embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, or the composition described in Embodiment 103 or Embodiment 104. 107. A method for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a subject having a tumor a therapeutically effective dose of the composition described in Embodiment 70 or Embodiment 76, or any of the cells described in any one of Embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, or the composition described in Embodiment 103 or Embodiment 104. 108. A method for inhibiting a cell-mediated immune response against tumor cells in a subject, comprising administering to a subject with a tumor a therapeutically effective dose of the composition described in Embodiment 70 or Embodiment 76, or any of the cells described in any one of Embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, or the composition described in Embodiment 103 or Embodiment 104. 109. The method according to Embodiment 108, comprising administering to a subject any of the cells described in any one of Embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, wherein the isolated cells or population of cells express a chimeric protein containing the inhibitory CAR described in Embodiment 38. 110. A method for treating a subject having a tumor, comprising administering a therapeutically effective dose of any of the cells described in Embodiment 76 or the composition described in Embodiment 76, or any one of the cells described in any one of Embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102, or the composition described in Embodiment 103 or Embodiment 104. 111. The method according to any one of embodiments 106-107 and 110, wherein the chimeric protein comprises an inhibitory CAR as described in any one of embodiments 38-40, and the cell or population of cells further expresses one or more tumor-targeting chimeric receptors. 112. The method according to Embodiment 111, which results in a reduction of off-target effects compared to administering an equivalent composition comprising one or more tumor-targeting chimeric receptors but lacking inhibitory CARs, comprising cells or populations of cells. 113. A kit for treating and / or preventing tumors, comprising a chimeric protein as described in any one of Embodiments 1 to 42. 114. The kit according to Embodiment 113, further comprising a described instruction manual for using a chimeric protein to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject. 115. A kit for treating and / or preventing tumors, comprising cells or populations of cells as described in any one of embodiments 80, 82, 84, 86, 88, 90, 92, and 94-102. 116. The kit according to Embodiment 115, further comprising a written instruction for using cells to treat and / or prevent tumors in a subject. 117. A kit for treating and / or preventing tumors, comprising the manipulated nucleic acid described in Embodiment 72. 118. The kit according to Embodiment 117, further comprising a described instruction manual for using nucleic acids to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject. 119. A kit for treating and / or preventing tumors, comprising the vector described in Embodiment 74. 120. The kit according to Embodiment 119, further comprising a described instruction manual for using a vector to produce one or more antigen-specific cells for treating and / or preventing tumors in a subject. 121. A kit for treating and / or preventing tumors, comprising the composition described in any one of embodiments 74, 76, and 103-105. 122. The kit according to Embodiment 121, further comprising a written instruction for using the composition for treating and / or preventing tumors in a subject. [Examples]

[0321] The following are examples of methods and compositions of this disclosure. It will be understood that various other embodiments may be implemented in light of the general descriptions provided herein.

[0322] The following are examples of specific embodiments for carrying out the subject matter claimed in this disclosure. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. While efforts have been made to ensure accuracy in the numerical values ​​used (e.g., quantities, temperatures, etc.), a certain degree of experimental error and deviation should of course be acceptable.

[0323] Example 1: Anti-EMCN antibody sequencing method Antibody sequencing The rat anti-human EMCN monoclonal antibody clone Ab1 was sequenced. Briefly, samples containing each immunoglobulin chain were digested with various enzymes and then analyzed by LC-MS / MS. The peptides were characterized from the LC-MS / MS data using novel peptide sequencing, and then the antibody sequence was constructed.

[0324] result The Ab1 anti-EMCN antibody was sequenced. LC-MS / MS data from multiple enzymatic digestions were mapped to the constructed antibody sequence. In both the heavy and light chains, 100% of amino acid residues were covered by at least five peptide scans using significant support fragment ions (data not shown).

[0325] The sequencing results for the light chain and heavy chain variable regions are shown in Figures 1 and 2, respectively, using Chothia annotation and numbering schemes. The framework and complementarity-determining regions (CDRs) are annotated according to Chothia annotation and numbering schemes, as well as Kabat annotation and numbering schemes. The sequences are shown in Table A. Assuming that leucine (L) and isoleucine (I) have the same residue mass, the determination of the two amino acids was made through further analysis.

[0326] (Table A) Anti-EMCN antibody (Ab1) sequence TIFF0007913758000004.tif210150TIFF0007913758000005.tif158150

[0327] Example 2: Generation of EMCN target cells method lentivirus production EMCN-expressing cell lines were generated using a lentiviral vector encoding human endomucin (Origene catalog number RC215698L4; Lenti ORF clone of human endomucin (EMCN), transcription variant 1, mGFP). Lentiviruses were produced using the following: Lenti-X293T packaging cell line (Clontech, catalog number 632180); antibiotic-free LX293T complete growth medium; DMEM, high glucose; 1 mM sodium pyruvate, 10% FBS, thermal inactivation; Opti-Mem I low serum medium (Gibco / Thermo Fisher; catalog number 31985); FuGene HD (Promega, catalog number E2311); envelope, packaging, and transfer vector plasmids; VSV-G pseudotype envelope vector (pMD2.G); and a packaging vector (psMAX2) containing Gag, Pol, Rev, and Tat, which can be used in second and third generation transfer vectors. 293T(FT) cells were detached from a 10cm dish at 90% confluence, aliquoted in a 1:3 dilution late afternoon the day before transfection, and incubated overnight at 37°C with 5% CO2 as usual (cells should be 60-85% confluence the day after transfection).

[0328] Transfection reaction solutions were prepared in 10 cm dishes according to the following protocol. 1. Prepare transfection reaction solutions in separate 1.7 mL tubes, each containing a 10 cm dish. 2. Add 900uL of Opti-MemI using RT. 3. Add 9ug of vector backbone (containing the target gene) to each reaction mixture. 4. Add 8 ug of packaging vector to each reaction mixture. 5. Add 1 ug of envelope vector (pMD2.G) after each reaction. Vortex rapidly for 6.3 seconds to mix completely. 7. Add 55 μL of FugeneHD after each reaction. 8. Mix by quickly pipetting up and down 20 to 30 times. 9. Leave in RT mode for 10 minutes (to allow DNA complex to form). 10. Slowly drop the mixture onto the dish, then gently shake it back and forth and up and down for 5-10 seconds to mix (do not rotate it). 11. Place the dish in the virus incubator.

[0329] Viral supernatant was collected on days 2 and 3 using a serological pipette. Cellular debris was removed using a Millipore steriflip 0.45um filter. Lenti-X concentrators (catalog numbers 631231 and 631232) were used according to the protocol. 1) Add 1 volume of Lenti-X concentrator to 3 volumes of clarified supernatant. Mix by gently inverting. 2) Incubate the mixture on ice or at 4°C for 30 minutes to overnight. 3) Centrifuge the sample at 1,500 × g for 45 minutes at 4°C. 4) Carefully remove and discard the supernatant, taking care not to disturb the pellet. 5) Slowly resuspend the pellet to 1 / 10 to 1 / 100 of its original volume using sterile PBS + 0.1% BSA.

[0330] Lentiviral phenotype introduction The Molm13 cell line was obtained from AddexBio (catalog number C0003003), and the SEM cell line was obtained from German Collection of Microorganisms and Cell Culture GmbH (DSMZ No. ACC 546).

[0331] Lentiviral transduction of cell lines was performed according to the following protocol: 1. For each cell line and condition (RPMI + 1% FBS), seed 500,000 to 1,000,000 cells (500 μL) into a 24-well plate. 2. Except for the control cells that have not been transduced with the virus, 400,000 viruses are transduced into each cell line. 3. Transfer the virus (stored at -80°C) to a 1.5 mL Eppendorf tube. 4. Add 2.5 μL of LentiBlast-A to the virus tube and mix well. Add 2.5 μL of LentiBlast-B and mix well. 5.32℃, centrifuged at 4000×rpm for 10 minutes, then heated. 6. Add the virus to the cells in the plate and mix 10 times. 7. Cover the outside of the plate with Parafilm. Centrifuge the plate at 8,800 × g at 32°C for 1 hour. 9. After spinning, add another 500 μL of 1% FBS RPMI medium to the well. Place in an incubator at 10.37°C overnight. Add 11.3 mL of fresh complete medium (10% FBS + RPMI), and transfer to a 6-well plate the following day.

[0332] Puromycin selection Transduced cells were selected with 0.5–3.33 μg / mL puromycin in 10% FBS + RPMI medium. Puromycin was added 3 days after transduction, and the medium was refreshed every 2–4 days. Cells were monitored during selection of EMCN expression by flow cytometry. Puromycin selection was initiated 3 days after transduction and maintained thereafter during in vitro cell culture.

[0333] Stain for FACS analysis Antibody staining of transduced cells and / or puromycin-selected cells was performed according to the following protocol: Aliquot 1,500 μL of cells under each condition (secondary staining only and EMCN staining). 2. Spin once, aspirate the supernatant, resuspend in the L / D mixture, cover, and stain on ice for 30 minutes. 1:1000 L / D aqua fixative. 3. Wash once with FACS buffer, then resuspend in primary stain (1:100) or FACS buffer on ice for 1 hour. 4. Wash once with FACS buffer, and resuspend all samples in secondary staining solution (1:5000) on ice for 30 minutes. 5. Wash once with FACS buffer, resuspend in 200 μL of FACS buffer, and then acquire the sample using a Cytoflex flow cytometer.

[0334] result EMCN-expressing cell lines were generated. Specifically, these were cell lines known to express cancer targets that may be targets of CAR-mediated killing (e.g., FLT3 (CD135) and CD33 (SIGLEC3)). After lentiviral transduction and drug selection, the manipulated cells were evaluated for EMCN expression by flow cytometry. A gating strategy to establish an EMCN expression baseline was established using only secondary controls (Figure 3A). As shown in Figure 3B, none of the control cell lines that had not been transduced by the virus showed EMCN expression. As shown in Figure 3C, the transduced cell lines showed low levels of expression 3 days after transduction.

[0335] Next, the transduced cells were drug-selected for 21 days (day 24 after transduction). As shown in Figures 4A and 4B, the manipulated Molm13, Molm14, MV4-11, and SEM cells, after being cultured with 1 μg / mL puromycin or 0.5 μg / mL puromycin, showed between 83-99% of cells expressing EMCN by flow cytometry. Transduced PL-21 cells selected with 0.5 μg / mL puromycin showed over 50% of cells expressing EMCN (PL-21 cells were not viable after selection with 1 μg / mL puromycin).

[0336] The expression characteristics of the manipulated cells were further evaluated. A gating strategy for establishing an EMCN expression baseline was established using only secondary controls (Figure 5). As shown in Table B, all Molm13 and SEM cells manipulated to express EMCN showed higher EMCN expression than the control, while also maintaining the expression of cancer targets FLT3 and CD33.

[0337] (Table B) Mean fluorescence intensity (MFI) of cell lines manipulated to express EMCN * TIFF0007913758000006.tif45148MFI = Average fluorescence intensity = Geometric mean, geometric mean calculated by FlowJo software flow cytometry software, (-) control = unstained sample

[0338] Example 3: Evaluation of anti-EMCN activated CAR method Lentiviral cloning and production The CAR constructs were cloned into lentiviral vectors. Lentiviruses were produced using the Lenti-X 293T system as described above. The antigen specificity and domain composition of the examined CAR constructs are shown in Table C below, and the scFv amino acid and nucleotide sequences are shown in Tables D and E, respectively.

[0339] (Table C) CAR construct (activation) TIFF0007913758000007.tif75149

[0340] (Table D) CAR scFv amino acid sequence TIFF0007913758000008.tif122150

[0341] (Table E) CAR scFv nucleotide sequences TIFF0007913758000009.tif68150TIFF0007913758000010.tif184150TIFF0007913758000011.tif116150

[0342] T cell assay Primary T cells were isolated from human donor PBMCs and frozen. Before transduction, the T cells were thawed, activated with the human T cell activator CD3 / CD28 DynaBeads, and cultured overnight in CTS OpTmizer T cell proliferation medium containing IL-2. Next, the T cells were transduced with a CAR lentivirus containing the selected CAR vector by removing a portion of the medium, adding an appropriate amount of lentiviral supernatant, and then gently mixing by pipetting. The cells were incubated overnight, and the virus was diluted the following day by adding further medium. The cells were then cultured as usual. Four days after transduction, CAR expression was evaluated by antibody staining and flow cytometry.

[0343] For functional assays, T cells and target cells were mixed and co-cultured 9 days after transduction (ET ratio: 1:1, 96-well plate, 200 μl total medium volume). To distinguish target cells from T cells, target cells were stained with CellTrace Violet dye.

[0344] For cytotoxic assays, cells were collected after 18 hours of co-culture and stained with Sytox Red cell viability dye to distinguish between live and dead target cells. T cell cytotoxicity against target cells was evaluated by flow cytometry (analysis performed using FlowJo software) and presented as a standardized cytotoxicity rate compared to non-transduced T cell controls.

[0345] result CAR T cells specific to various constructs of FLT3, CD33, or EMCN were generated. Each receptor contained a cytoplasmic signaling domain so that binding to the target antigen stimulates an immune response such as cytokine production and / or target cell killing. For EMCN-specific CAR constructs, various scFv linkers (G4S) were used. (Sequence ID 33) , (G4S)3 (Sequence ID 35) Along with Whitlow, we also evaluated the orientations of different VH and VL.

[0346] After lentiviral transduction of primary human T cells, CAR expression was evaluated in the manipulated cells. A gating strategy for establishing a CAR expression baseline was established using untransductioned controls (Figure 6A). As shown in Figures 6B and 6C, CAR expression was observed in 61–91% of transduced cells.

[0347] Next, various CAR T cells were evaluated for their functional activity. Each CAR T cell was co-incubated with Molm13 target cells, SEM target cells, Molm13 target cells engineered to express EMCN, or SEM target cells engineered to express EMCN. The parental and engineered Molm13 cell lines and SEM cell lines naturally express FLT3 and CD33, as described above (Figures 5C and 5D). Co-culture of FLT3-specific or CD33-specific CAR T cells with parental Molm13 (Figure 7A; upper panel) or SEM (Figure 7A; lower panel) target cells resulted in target cell killing (second and third columns, respectively), while co-culture with EMCN-specific CAR T cells did not show detectable levels of target ce...

Claims

1. A chimeric protein comprising an antigen-binding domain specific to endomucin (EMCN) and a heterologous polypeptide; The antigen-binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region. (a) The above VH is Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 102), Heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of VIWGNTHYHSALKS (SEQ ID NO: 103), and Heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, The aforementioned VL is Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), Light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and Light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the antigen-binding domains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Kabat numbering scheme, or (b) The above VH is Heavy chain complementarity determination region 1 (CDR-H1) having the amino acid sequence of GFSLSRY (SEQ ID NO: 2), heavy chain complementarity determination region 2 (CDR-H2) having the amino acid sequence of WGNGN (SEQ ID NO: 3), and heavy chain complementarity determination region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 4) Includes, The aforementioned VL is Light chain complementarity determination region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 10), light chain complementarity determination region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 11), and light chain complementarity determination region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 12) Includes, The amino acid sequences of the antigen-binding domains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Chothia numbering scheme, or (c) The above VH is Heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3) contained within the VH region amino acid sequence of SEQ ID NO:

1. Includes, The aforementioned VL is Light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3) contained within the VL region amino acid sequence of SEQ ID NO: 9 Includes, The amino acid sequences of the antigen-binding domains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined based on the Kabat or Chothia numbering scheme; The heterologous polypeptide comprises a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, and one or more peptide linkers. The aforementioned chimeric protein.

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

1.

3. The chimeric protein according to claim 1 or claim 2, wherein the VL region comprises the amino acid sequence of SEQ ID NO:

9.

4. The chimeric protein according to any one of claims 1 to 3, wherein the antigen-binding domain comprises a single-stranded variable fragment (scFv).

5. The chimeric protein according to claim 4, wherein the VH and VL of the scFv are separated by a peptide linker.

6. The chimeric protein according to claim 5, wherein the antigen-binding domain comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable region, L is the peptide linker, and VL is the light chain variable region.

7. The chimeric protein according to any one of claims 4 to 6, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 22.

8. The chimeric protein according to any one of claims 1 to 7, wherein the chimeric protein is a chimeric antigen receptor (CAR).

9. The chimeric protein according to claim 8, wherein the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response.

10. The chimeric protein according to claim 9, wherein the intracellular inhibitory domain comprises an enzyme inhibitory domain or an intracellular inhibitory co-signaling domain.

11. The chimeric protein according to claim 9, wherein the intracellular inhibitory domain is derived from LIR2, LIR3, LIR5, KIR2DL1, LAIR1, or SIGLEC-2.

12. A manipulated nucleic acid encoding a chimeric protein according to any one of claims 1 to 11.

13. An expression vector comprising the manipulated nucleic acid described in claim 12.

14. Isolated cells comprising the manipulated nucleic acid according to claim 12 or the expression vector according to claim 13.

15. A population of manipulated cells expressing the manipulated nucleic acid according to claim 12 or the expression vector according to claim 13.

16. The cell or population of cells according to claim 14 or claim 15, further comprising one or more tumor-targeting chimeric receptors expressed on the cell surface.

17. The cell or population of cells according to claim 16, wherein each of the one or more tumor-targeting chimeric receptors is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

18. A cell or population of cells according to any one of claims 14 to 17, 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.

19. A pharmaceutical composition comprising an effective amount of a population of cells or manipulated cells according to any one of claims 14 to 18, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

20. A pharmaceutical composition comprising cells according to any one of claims 14 to 18 or the pharmaceutical composition according to claim 19, for stimulating a cell-mediated immune response against tumor cells in a subject having a tumor, in a therapeutically effective dose.

21. A pharmaceutical composition comprising cells according to any one of claims 14 to 18 or the pharmaceutical composition according to claim 19, for treating a subject having a tumor, in a therapeutically effective dose.

Images