Methods and compositions for cell surface colocalization of chimeric proteins

Abstract

The present disclosure provides compositions and methods related to co-localization of chimeric proteins at the cell surface. In particular, the present disclosure provides methods and compositions for promoting cell surface co-localization of chimeric polypeptides through the use of dimerization domains to promote functional interactions.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 423,364, filed November 7, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure provides compositions and methods related to colocalization of chimeric proteins on the cell surface. In particular, the present disclosure provides methods and compositions for promoting cell surface colocalization of chimeric polypeptides (e.g., chimeric antigen receptors) through the use of dimerization domains to promote functional interactions (e.g., inhibition of activation signals).

[0003] Sequence Listing The contents of the electronic sequence listing entitled SENTI_41244_601_ST26.xml (size: 253,510 bytes and creation date: November 6, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] The immune system is essential for detecting and combating human cancer. Most transformed cells are rapidly detected by immune sentinels and destroyed through the activation of immune effector cells, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage. Cancer can therefore be considered an immunological disorder, a failure of the immune system to mount the antitumor responses necessary to durably suppress and eliminate disease. To combat cancer more effectively, certain immunotherapeutic interventions developed over the past several decades have focused specifically on enhancing one or more aspects of immune effector cells and modifying these cells to target specific tumor antigens (e.g., using chimeric antigen receptors).

[0005] Chimeric antigen receptors (CARs) enable targeted in vivo activation of immune effector cells. These recombinant membrane receptors generally contain an antigen-binding domain and one or more signaling domains, enabling effector cells to recognize specific protein antigens on tumor cells and induce activation and signaling pathways. Recent clinical results using chimeric receptor-expressing T cells have provided strong support for their potential use as therapeutic agents in cancer immunotherapy. However, despite these promising results, several side effects associated with CAR T cell therapy have been identified, raising serious safety concerns. One side effect is "on-target / off-tissue" adverse events mediated by TCR- and CAR-modified effector cells, which occur when CAR effector cells bind to ligands present outside the target tumor tissue and induce an immune response. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, methods are needed to effectively target and treat tumors without damaging normal cells that express the same target antigen. [Means for solving the problem]

[0007] (Abstract) Embodiments of the present disclosure include chimeric protein systems. According to these embodiments, the chimeric protein systems include a first polypeptide comprising a first antigen-binding domain, a first transmembrane domain, and a first dimerization domain, and a second polypeptide comprising a second antigen-binding domain, a second transmembrane domain, and a second dimerization domain. In some embodiments, the first dimerization domain can bind to the second dimerization domain. In some embodiments, the chimeric protein systems comprising the first and second polypeptides provide for chimeric antigen receptor pairing (e.g., colocalization of the receptor pair) on the cell surface (e.g., via one or more dimerization domains).

[0008] In some embodiments, the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A, and NKG2C. In some embodiments, the first dimerization domain comprises the dimerization domain of CD94, and the second dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C. In some embodiments, the first dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C, and the second dimerization domain comprises the dimerization domain of CD94. The present disclosure is not limited to any particular dimerization domain. Indeed, any one or more protein domains and / or amino acid sequences useful for forming a complex between a first and second polypeptide disclosed herein (e.g., to form a chimeric antigen receptor pair) may be used. In some embodiments, the protein domain and / or amino acid sequence used to form the complex is a leucine zipper domain. In some embodiments, a first protein domain and / or amino acid sequence is used to complex with a second protein domain and / or amino acid sequence to form a heterodimer (e.g., via a disulfide bond). Non-limiting examples of protein domains and / or amino acid sequences useful for complex formation include, but are not limited to, CD94, NKG2A, NKG2C, R34(GS)3, E34-I(GS)3, E34-V(GS)3, E34-N(GS)3, CD94-rev, NKG2A-rev, NKG2C-rev, (GS3)3R34, (GS3)3E34-I, (GS3)3E34-V, and (GS3)3E34-N.

[0009] In some embodiments, the first dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. In some embodiments, the second dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

[0010] In some embodiments, the first polypeptide is a chimeric antigen receptor. In some embodiments, the first polypeptide is an activating chimeric antigen receptor. In some embodiments, the first polypeptide is an inhibitory chimeric antigen receptor.

[0011] In some embodiments, the second polypeptide is a chimeric antigen receptor. In some embodiments, the second polypeptide is an activating chimeric antigen receptor. In some embodiments, the second polypeptide is an inhibitory chimeric antigen receptor.

[0012] In some embodiments, the antigen-binding domain comprises a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), a single-domain antibody, a diabody, a VHH fragment, or a synthetic epitope. In some embodiments, the antigen-binding domain binds to an antigen expressed on a cancer cell. In some embodiments, the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia (e.g., lymphocytic, lymphocytic, lymphoblastic myeloid, or granulocytic leukemia), lymphoma, or myeloma.

[0013] In some embodiments, the antigen binding domain is selected from the group consisting of carcinoembryonic antigen (CEA), mesothelin, Axl, GPC3, FLT3, CD33, TROP2, MUCl, MUC16, IL13Ra, ErbB2 (HER2 / neu), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE1, MN-CA IX, human telomerase reverse transcriptase, RUL, RU2 (AS), intestinal carboxylesterase, mut Specific for hsp70-2, M-CSF, prostate, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF17), Claudin18.2, B7-H3, or Rorl.

[0014] In some embodiments, the transmembrane domain is a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain of a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3 zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIRl transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain. The polypeptide is selected from the group consisting of an IR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, a SIRPa transmembrane domain, a HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain.

[0015] In some embodiments, the first polypeptide and / or the second polypeptide comprise one or more intracellular signaling domains. In some embodiments, the one or more intracellular signaling domains are selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-lBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0016] In some embodiments, the one or more intracellular signaling domains comprise a costimulatory domain selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0017] In some embodiments, the first polypeptide and / or the second polypeptide comprises a hinge domain disposed between the antigen binding domain and the transmembrane domain.

[0018] In some embodiments, the first polypeptide and / or the second polypeptide comprise one or more linkers, hi some embodiments, the one or more linkers comprise a GSG linker, a Whitlow linker, an eGK linker, or any derivative thereof.

[0019] Embodiments of the present disclosure also include modified polynucleotides that encode any of the first and / or second polynucleotides described herein.

[0020] Embodiments of the present disclosure also include expression vectors that include any of the modified polynucleotides described herein.

[0021] Embodiments of the present disclosure also include modified cells comprising any of the disclosed modified polynucleotides, vectors, or first and / or second polypeptides described herein.

[0022] In some embodiments, the cell is selected from the group consisting of a T cell, a CDS+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.

[0023] In some embodiments, the cells are modified to express an effector molecule. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0024] Embodiments of the present disclosure also include pharmaceutical compositions comprising any of the modified cells described herein and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0025]

[0013] Embodiments of the present disclosure also include methods of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of any of the compositions described herein or any of the cells described herein. In some embodiments, the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia (e.g., lymphocytic, lymphocytic, lymphoblastic myeloid, or granulocytic leukemia), lymphoma, or myeloma.

[0026]

[0010] Embodiments of the present disclosure also include methods of enhancing immune cell-mediated killing of cancer cells in a subject in need thereof, comprising administering a therapeutically effective dose of any of the compositions described herein or any of the cells described herein to the subject. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia (e.g., lymphocytic, lymphocytic, lymphoblastic myeloid, or granulocytic leukemia), lymphoma, or myeloma.

[0027]

[0010] Embodiments of the present disclosure also include methods of reducing off-target killing of healthy cells in a subject, comprising administering to the subject a therapeutically effective dose of any of the compositions described herein or any of the cells described herein, wherein the subject has been diagnosed with cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia (e.g., lymphocytic, lymphocytic, lymphoblastic myeloid, or granulocytic leukemia), lymphoma, or myeloma.

[0028] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description. [Brief explanation of the drawings]

[0029] [Figure 1A] Representative data demonstrating successful transduction of exemplary aCAR / iCAR combinations with dimerization domains in NK cells (Figure 1A). Representative data from a target cell killing assay where target cells express VSIG2 are presented, with data expressed as normalized E:T ratios (Figures 1B-1C) and percent target cells only (Figures 1D-1E). [Figure 1B]Representative data demonstrating successful transduction of exemplary aCAR / iCAR combinations with dimerization domains in NK cells (Figure 1A). Representative data from a target cell killing assay where target cells express VSIG2 are presented, with data expressed as normalized E:T ratios (Figures 1B-1C) and percent target cells only (Figures 1D-1E). [Figure 1C] Representative data demonstrating successful transduction of exemplary aCAR / iCAR combinations with dimerization domains in NK cells (Figure 1A). Representative data from a target cell killing assay where target cells express VSIG2 are presented, with data expressed as normalized E:T ratios (Figures 1B-1C) and percent target cells only (Figures 1D-1E). [Figure 1D] Representative data demonstrating successful transduction of exemplary aCAR / iCAR combinations with dimerization domains in NK cells (Figure 1A). Representative data from a target cell killing assay where target cells express VSIG2 are presented, with data expressed as normalized E:T ratios (Figures 1B-1C) and percent target cells only (Figures 1D-1E). [Figure 1E] Representative data demonstrating successful transduction of exemplary aCAR / iCAR combinations with dimerization domains in NK cells (Figure 1A). Representative data from a target cell killing assay where target cells express VSIG2 are presented, with data expressed as normalized E:T ratios (Figures 1B-1C) and percent target cells only (Figures 1D-1E). [Figure 2A] Representative data from experiments performed to establish baseline levels of fluorescence per well based on percent target cells only normalized to non-dimerized aCAR / iCAR (Figure 2A), NKG2C dimerization domain containing CAR (Figure 2B), and CD94 dimerization domain containing CAR (Figure 2C). [Figure 2B]Representative data from experiments performed to establish baseline levels of fluorescence per well based on percent target cells only normalized to non-dimerized aCAR / iCAR (Figure 2A), NKG2C dimerization domain containing CAR (Figure 2B), and CD94 dimerization domain containing CAR (Figure 2C). [Figure 2C] Representative data from experiments performed to establish baseline levels of fluorescence per well based on percent target cells only normalized to non-dimerized aCAR / iCAR (Figure 2A), NKG2C dimerization domain containing CAR (Figure 2B), and CD94 dimerization domain containing CAR (Figure 2C). [Figure 3A] Representative data from a killing assay performed with triplicate killing under all indicated conditions. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; each color reflects the use of the same NK effector cells (with or without VSIG2-expressing target cells). Data from the first round are shown in Figure 3A, data from the second round in Figure 3B, and data from the third round in Figure 3C. [Figure 3B] Representative data from a killing assay performed with triplicate killing under all indicated conditions. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; each color reflects the use of the same NK effector cells (with or without VSIG2-expressing target cells). Data from the first round are shown in Figure 3A, data from the second round in Figure 3B, and data from the third round in Figure 3C. [Figure 3C] Representative data from a killing assay performed with triplicate killing under all indicated conditions. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; each color reflects the use of the same NK effector cells (with or without VSIG2-expressing target cells). Data from the first round are shown in Figure 3A, data from the second round in Figure 3B, and data from the third round in Figure 3C. [Figure 4A]Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aCD94. [Figure 4B] Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aCD94. [Figure 4C] Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aCD94. [Figure 5A] Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aNKG2C. [Figure 5B] Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aNKG2C. [Figure 5C] Representative data from a killing assay performed with triplicate killing under a subset of test conditions involving aNKG2C. [Figure 6A] Representative control data from the killing assay: Figure 6A presents data for the non-dimerized CAR and no virus control, Figure 6B presents data for the single transduction control, and Figure 6C presents data under non-dimerized CAR conditions (second and third transduction rounds shown). [Figure 6B] Representative control data from the killing assay: Figure 6A presents data for the non-dimerized CAR and no virus control, Figure 6B presents data for the single transduction control, and Figure 6C presents data under non-dimerized CAR conditions (second and third transduction rounds shown). [Figure 6C] Representative control data from the killing assay: Figure 6A presents data for the non-dimerized CAR and no virus control, Figure 6B presents data for the single transduction control, and Figure 6C presents data under non-dimerized CAR conditions (second and third transduction rounds shown). [Figure 7A]7A and 7B are representative data from a killing assay performed to compare an iCAR with an NKG2C dimerization domain paired with an aCAR with a CD94 dimerization domain plus a non-dimerized CAR with a virus-free control. The data in Figure 7A are from two killings, and the data in Figure 7B are from three killings. Figure 7C presents the data in Figure 7B as a bar graph. [Figure 7B] 7A and 7B are representative data from a killing assay performed to compare an iCAR with an NKG2C dimerization domain paired with an aCAR with a CD94 dimerization domain plus a non-dimerized CAR with a virus-free control. The data in Figure 7A are from two killings, and the data in Figure 7B are from three killings. Figure 7C presents the data in Figure 7B as a bar graph. [Figure 7C] 7A and 7B are representative data from a killing assay performed to compare an iCAR with an NKG2C dimerization domain paired with an aCAR with a CD94 dimerization domain plus a non-dimerized CAR with a virus-free control. The data in Figure 7A are from two killings, and the data in Figure 7B are from three killings. Figure 7C presents the data in Figure 7B as a bar graph. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure provides compositions and methods related to colocalization of chimeric proteins on the cell surface. In particular, the present disclosure provides methods and compositions for promoting cell surface colocalization of chimeric polypeptides (e.g., chimeric antigen receptors) through the use of dimerization domains to promote functional interactions (e.g., inhibition of activation signals).

[0031] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0032] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The phrase "in one embodiment," when used herein, does not necessarily refer to the same embodiment, but may. Furthermore, the phrase "in another embodiment," when used herein, does not necessarily refer to a different embodiment, but may. Thus, as described below, various embodiments of the present disclosure can be readily combined without departing from the scope or spirit of the embodiments provided herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0033] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and their derivatives are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0034] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] "About," as used herein, when referring to a measurable value, e.g., amount, duration, etc., is intended to encompass a variation of + / - 20% or + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, and still more preferably + / - 0.1% from the particular value, where such variation is appropriate for practicing the methods of the present disclosure.

[0036] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description of the range format is merely for simplicity and convenience and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This is true regardless of the breadth of the range.

[0037] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural sources or from recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody can be a multimer of individual immunoglobulin molecules. The antibodies described in this disclosure may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fvs, single-chain variable fragments (scFvs), Fab and F(ab)2 fragments, VHH fragments, diabodies, synthetic epitopes, single-domain antibodies, human antibodies, and humanized antibodies (see, e.g., Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0038] The term "antibody fragment" refers to a portion of an intact antibody, preferably the antigenic determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0039] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0040] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0041] The terms "synthetic antibody" and "recombinant antibody" are used interchangeably herein and refer to antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed in bacteriophage or yeast expression systems. The terms should also be taken to mean antibodies produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence techniques available and well known in the art.

[0042] The term "immunoglobulin" or "Ig" as used herein refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called B cell receptors (BCRs) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal fluids, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced by the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by inducing mediator release from mast cells and basophils upon exposure to allergens.

[0043] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including almost any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. The present disclosure will readily appreciate that it includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Furthermore, those skilled in the art will readily appreciate that an antigen need not be encoded by a "gene" at all. Antigens can be synthetically produced or derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological fluids.

[0044] The terms "chimeric receptor," "chimeric antigen receptor," or alternatively, "CAR" refer to a recombinant polypeptide comprising at least an extracellular antigen-binding portion, a transmembrane domain, and an intracellular signaling domain (also called a "cytoplasmic signaling domain") that includes a functional signaling domain.

[0045] As used herein, the term "antigen-binding domain" refers to a protein that binds to a target antigen. Antigen-binding domains include, but are not limited to, proteins comprising at least one immunoglobulin variable domain sequence (e.g., an immunoglobulin chain or fragment thereof). The term "antigen-binding domain" encompasses antibodies and antibody fragments. In some embodiments, an antigen-binding domain is a multispecific antibody molecule (e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope). In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antibodies. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, an antigen binding domain can refer to an effector protein, which can bind to a cognate protein partner, such as a receptor.

[0046] As used herein, the term "extracellular domain" or "ECD" refers to a polypeptide or combination of polypeptides that form the exterior of a larger polypeptide (e.g., a chimeric antigen receptor) that extends outward from the outer membrane of a cell or organelle in which the larger polypeptide is expressed. In some embodiments, an extracellular domain comprises two or more domains. In some embodiments, an extracellular domain comprises unique domains that can interact with different ligands. In other aspects, an extracellular domain comprises unique domains that can be cleaved by a protease. In still other aspects, an extracellular domain is linked to other domains (e.g., transmembrane domains) to form a larger polypeptide (e.g., a chimeric antigen receptor). In some embodiments, an extracellular domain comprises a hinge region that connects the extracellular domain to the transmembrane domain.

[0047] As used herein, the terms "transmembrane domain," "TM," or "TMD" refer to a polypeptide or combination of polypeptides that traverse the membrane of a cell or organelle in which the polypeptide is expressed. In some embodiments, a transmembrane domain is linked to an extracellular domain and / or an intracellular domain to form a larger polypeptide (e.g., a chimeric antigen receptor) that can transduce an external signal into an intracellular response. In some embodiments, a transmembrane domain is composed primarily of non-polar amino acid residues and may traverse the bilayer once (e.g., single pass) or several times (multiple chains). In some embodiments, a single-pass transmembrane protein comprises three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the transmembrane domain forms an alpha helix and is inserted into the membrane bilayer.

[0048] As used herein, the terms "intracellular signaling domain," "cytoplasmic domain," or "ICD" refer to a polypeptide or combination of polypeptides that are located within the lumen of a cell or organelle in which the polypeptide is expressed. In some embodiments, an intracellular signaling domain is linked to an extracellular domain via a transmembrane domain to form a larger polypeptide (e.g., a chimeric antigen receptor) that can transduce an external signal into an intracellular response. In some embodiments, an intracellular signaling domain comprises a single signaling domain that can provide an intracellular activation signal to the ligand-linked extracellular domain upon binding with a ligand. In other aspects, an intracellular signaling domain comprises two or more signaling domains that can provide one or more intracellular activation signals to the ligand-linked extracellular domain upon binding with a ligand. In some embodiments, an intracellular signaling domain comprises a primary intracellular signaling domain as well as a costimulatory domain.

[0049] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into that individual. "Allogeneic" refers to a graft derived from a different animal of the same species. "Xenogeneic" refers to a graft derived from an animal of a different species.

[0050] The term "treatment" as used herein means treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, amelioration, or eradication of the disease state.

[0051] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result. For example, a "therapeutically effective amount" can be an amount of a compound or composition that elicits a biological or medical response in a tissue, system, or subject that is sought by a researcher, veterinarian, physician, or other clinician. A therapeutically effective amount includes an amount of a compound or composition that, when administered, is sufficient to prevent the occurrence of, or alleviate to some extent, one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0052] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. For example, treating can refer to reducing or ameliorating the progression, severity, and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies. In some embodiments, the terms "treat," "treatment," and "treating" refer to the improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other aspects, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disorder, either physically, e.g., by stabilization of discernible symptoms, physiologically, e.g., by stabilization of physical parameters, or both. In other aspects, the terms "treat," "treatment," and "treating" refer to the reduction or stabilization of tumor size or cancerous cell number.

[0053] As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system. As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue, or system.

[0054] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed by aligning the two sequences to obtain maximum homology.

[0055] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the materials with which it coexists in the natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0056] As used herein, "substantially purified" cells are cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0057] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that a nucleotide sequence that encodes a protein may contain introns in some versions.

[0058] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0059] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0060] The terms "patient," "subject," "individual," and the like are used interchangeably herein and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0061] The term "specifically binds," as used herein with respect to an antibody or extracellular target binding moiety, means an antibody or extracellular target binding moiety that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen of one species may also bind to antigens of one or more species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" are used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, it can mean that an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that becomes bound to the antibody.

[0062] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., that are involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes.

[0063] "Immune effector function or immune effector response," as that term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack on a target cell. For example, an immune effector function or response refers to a property of a T or NK cell that promotes the killing or inhibition of the growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0064] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0065] The term "cancer" refers to a disease characterized by the rapid and uncontrollable growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0066] The terms "cancer antigen," "cancer-associated antigen," or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either entirely or as fragments (e.g., MHC / peptides), and that is useful for preferentially targeting pharmacological agents to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 or CD123 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., by 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more, compared to normal cells. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, a tumor antigen is expressed exclusively on the cell surface of cancer cells, either entirely or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells.

[0067] The term "anti-tumor effect," as used herein, refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the ability of the peptides, polynucleotides, antibodies (or antigen-binding portions thereof), and modified immune cells of the present disclosure in preventing the development of tumors in the first place.

[0068] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies in preventing the development of cancer in the first place.

[0069] As used herein, the term "subject suspected of having cancer" refers to a subject who presents with one or more symptoms indicative of cancer (e.g., a discernible lump or mass) or who is being screened for cancer (e.g., during a routine checkup). A subject suspected of having cancer may also have one or more risk factors for developing cancer. A subject suspected of having cancer generally has not been tested for cancer. However, "subject suspected of having cancer" encompasses individuals who have had a preliminary diagnosis (e.g., a CT scan showing a mass) but have not had a confirmatory test (e.g., biopsy and / or histology) or whose type and / or stage of cancer is unknown. The term further includes those who previously had cancer (e.g., individuals in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and sometimes found to be free of cancer.

[0070] As used herein, the term "subject diagnosed with cancer" refers to a subject who has been tested for and found to have cancerous cells. Cancer can be diagnosed using any suitable method, including, but not limited to, biopsy, X-ray, blood test, etc.

[0071] As used herein, the term "subject at risk of cancer" refers to a subject who has one or more risk factors for developing a particular cancer, including, but not limited to, gender, age, genetic predisposition, environmental exposure, previous cancer occurrence, existing non-cancer diseases, and lifestyle habits.

[0072] As used herein, the term "characterizing cancer in a subject" refers to the determination of one or more characteristics of a cancer sample from a subject, including, but not limited to, the presence of benign, pre-cancerous, or cancerous tissue and the stage of the cancer.

[0073] As used herein, the term "tumor microenvironment" refers to the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.A. Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. et al., J. Cell Sci. 125, 5591-5596, 2012; and Li, H. et al., J. Cell Biochem. 101(4), 805-15, 2007).

[0074] "Refractory," as used herein, refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer may also be referred to as a resistant cancer.

[0075] "Relapsed" or "recurring," as used herein, refers to the return or recurrence of a disease (e.g., cancer) or signs and symptoms of a disease, such as cancer, after a period of improvement or responsiveness, e.g., after prior treatment with a therapy, e.g., a cancer therapy. Initial response may involve levels of cancer cells below a certain threshold, e.g., less than 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may involve levels of cancer cells above a certain threshold, e.g., greater than 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, recurrence may involve the reappearance of blasts, e.g., in the blood, bone marrow (>5%), or any extramedullary site, after a complete response. A complete response, in this context, may involve <5% BM blasts. More generally, in one aspect, a response (e.g., complete response or partial response) may involve the absence of detectable MRD (minimal residual disease). In one embodiment, the initial period of response lasts for at least 1, 2, 3, 4, 5 or 6 days, at least 1, 2, 3 or 4 weeks, at least 1, 2, 3, 4, 6, 8, 10 or 12 months, or at least 1, 2, 3, 4 or 5 years.

[0076] The term "derived from," as used herein, refers to the relationship between a first and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include limitations on the process or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the required function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the particular process by which the intracellular signaling domain is produced, e.g., it does not mean that one must start with the CD3ζ sequence and delete unnecessary sequences or make mutations to obtain the intracellular signaling domain.

[0077] "Activated," as used herein, refers to the state of an immune cell (e.g., an NK cell or a T cell) that has been stimulated sufficiently to induce a detectable change, such as, but not limited to, detectable cell proliferation or an immune response. Activation may also be associated with induced cytokine production and / or detectable effector function. The term "activated NK cell" refers, inter alia, to an NK cell that has undergone cellular differentiation and / or enhanced cytokine production and / or secretion.

[0078] As used herein, the term "immune response" refers to a response by a subject's immune system. For example, immune responses include, but are not limited to, a detectable change (e.g., an increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th17, or Th2-type cytokines), or chemokine) expression and / or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and / or B cell activation (e.g., antibody production and / or secretion). Additional examples of immune responses include binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule and induction of a cytotoxic T lymphocyte ("CTL") response, induction of a B cell response (e.g., antibody production) and / or a T helper lymphocyte response and / or a delayed-type hypersensitivity (DTH) response to the antigen from which the immunogenic polypeptide is derived, expansion (e.g., proliferation of cell populations) of cells of the immune system (e.g., T cells, B cells (e.g., at any developmental stage (e.g., plasma cells)), and increased antigen processing and presentation by antigen-presenting cells. An immune response can be to an immunogen that the subject's immune system recognizes as foreign (e.g., a non-self antigen from a microorganism (e.g., a pathogen) or a self-antigen that is recognized as foreign). The term "immune response" is intended to encompass all aspects of the ability of a subject's immune system to respond to an antigen and / or immunogen (e.g., both an initial response to the immunogen and an acquired (e.g., memory) response that is a result of an adaptive immune response).

[0079] It should also be understood that, unless specifically and clearly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0080] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0081] 2. Chimeric protein systems containing dimerization domains Embodiments of the present disclosure include chimeric protein systems. According to these embodiments, the chimeric protein system comprises a first polypeptide comprising a first antigen-binding domain, a first transmembrane domain, and a first dimerization domain, and a second polypeptide comprising a second antigen-binding domain, a second transmembrane domain, and a second dimerization domain. In some embodiments, the first dimerization domain can bind to the second dimerization domain.

[0082] According to various embodiments described herein, immune receptors (e.g., chimeric antigen receptors or CARs) with complementary or opposing functions (e.g., activating function vs. inhibitory function) can be engineered to include a dimerization domain to promote colocalization in immune effector cells (e.g., at the immune synapse). In some embodiments, the dimerization domain forms a heterodimeric complex of immune receptors (e.g., receptors with complementary functions, receptors with non-complementary functions, or receptors with opposing functions). As further described herein, certain CARs (e.g., activating CARs and inhibitory CARs) exhibit maximal efficacy for carrying out a desired function (e.g., inhibiting an activating signal) when expressed in close proximity to each other, without being bound by theory. The data and results presented herein demonstrate that including a dimerization domain as part of a CAR promotes colocalization and enhances synergistic and / or competitive effects. In some embodiments, the dimerization domain can be located in the extracellular or intracellular domain of the CAR.

[0083] The dimerization domain contained within the first and second polypeptides of the chimeric protein system of the present disclosure can be any dimerization domain that promotes colocalization of the first and second polypeptides, as will be recognized by those of skill in the art based on this disclosure. Indeed, the present disclosure provides that the first and second polypeptides disclosed herein can comprise any one or more protein domains and / or amino acid sequences that form a complex (e.g., a leucine zipper domain and / or a first protein domain that binds to a second protein domain via a disulfide or other type of bond). In some embodiments, the dimerization domains present in the first and second polypeptides promote chimeric antigen receptor pairing / complex formation. Non-limiting examples of first and second polypeptides that dimerize (complex and / or colocalize) to form a chimeric antigen receptor pair include the dimerization domain of CD94 and a dimerization domain selected from NKG2A and NKG2C.

[0084] In some embodiments, the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A, and NKG2C. In some embodiments, the first dimerization domain comprises the dimerization domain of CD94, and the second dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C. In some embodiments, the first dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C, and the second dimerization domain comprises the dimerization domain of CD94.

[0085] In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. In some embodiments, the first dimerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

[0086] In some embodiments, the second dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. In some embodiments, the second dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

[0087] In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 70. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 72. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 74. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 76.In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 78. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:80. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:82. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:84.In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:86. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:88. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:90.

[0088] In some embodiments, the second dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 70. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 72. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 74. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 76.In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 78. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:80. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:82. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:84.In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:86. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:88. In some embodiments, the first dimerization domain comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:90.

[0089] In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system comprises an antigen-binding domain, and the antigen-binding domain is selected from the group consisting of an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), a single-domain antibody, a diabody, a VHH fragment, and a synthetic epitope. In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. Generally, an scFv has a light chain variable domain (VL) connected by a polypeptide chain from the C-terminus to the N-terminus of the heavy chain variable domain. Alternatively, an scFv comprises a polypeptide chain, and the C-terminus of the VH is connected to the N-terminus of the VL by a polypeptide chain. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is a peptide linker, and VL is the light chain variable domain. An sdAb is a molecule in which one antibody variable domain specifically binds to an antigen without the presence of other variable domains. The F(ab) fragment contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), along with the variable domains VL and VH of the light and heavy chains, respectively. The F(ab') fragment differs from the Fab fragment by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines of the antibody hinge region. The F(ab')2 fragment contains two Fab' fragments joined near the hinge region by disulfide bonds. As used herein, a synthetic epitope refers to a non-naturally occurring epitope moiety present (e.g., inserted) within the extracellular portion of a chimeric protein system (e.g., within an antigen-binding domain, spacer, linker, or other portion). The present disclosure is not limited to any particular synthetic epitope or how the synthetic epitope is utilized. For example, synthetic epitopes can be used to facilitate binding of epitope-specific antibodies to a chimeric protein system (e.g., for isolation and / or purification during manufacturing or depletion / removal of the protein system from a source).

[0090] Chimeric Antigen Receptor As detailed herein, certain aspects of the present disclosure relate to chimeric antigen receptors (CARs) that are co-localized on the cell surface by forced association via one or more dimerization domains present within the CAR polypeptide sequence. CARs containing one or more dimerization domains disclosed herein are not limited in specificity and / or function. In some embodiments, CARs containing one or more dimerization domains of the present disclosure comprise an extracellular antigen-binding domain fused to a transmembrane domain fused to one or more intracellular signaling domains. In some embodiments, the CAR comprises a spacer region or hinge domain.

[0091] Extracellular antigen-binding domain In some embodiments, the first and / or second polypeptide of the chimeric protein system of the present disclosure comprises an antigen-binding domain that targets any tumor-associated antigen of interest. In some embodiments, the first and / or second polypeptide of the present disclosure is an activating CAR (e.g., an activating CAR comprising a dimerization domain). In some embodiments, the antigen-binding domain of the present disclosure binds to an antigen expressed on the surface of a cancer cell. In some embodiments, the cancer cell is derived from a solid tumor. In some embodiments, the cancer includes, but is not limited to, glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, and cervical cancer.

[0092] In some embodiments, the antigen binding domain is selected from the group consisting of carcinoembryonic antigen (CEA), mesothelin, Axl, GPC3, FLT3, CD33, TROP2, MUCl, MUC16, IL13Ra, ErbB2 (HER2 / neu), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE1, MN-CA IX, human telomerase reverse transcriptase, RUL, RU2 (AS), intestinal carboxylesterase, mut The antibodies are specific for at least one of hsp70-2, M-CSF, prostate, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF17), Claudinl8.2, B7-H3, or Rorl.

[0093] In some embodiments, the first polypeptide of the chimeric protein system is a chimeric antigen receptor (CAR). In some embodiments, the first polypeptide is an activating chimeric antigen receptor (aCAR). In some embodiments, the first polypeptide is an inhibitory chimeric antigen receptor (iCAR). In some embodiments, the second polypeptide of the chimeric protein system is a chimeric antigen receptor (CAR). In some embodiments, the second polypeptide is an activating chimeric antigen receptor (aCAR). In some embodiments, the second polypeptide is an inhibitory chimeric antigen receptor (iCAR). In some embodiments, the activating chimeric antigen receptor (aCAR) is bivalent (e.g., specific for two target antigens). In some embodiments, the two target antigens are the same. In some embodiments, the two target antigens are different.

[0094] According to the above embodiments, the first and / or second polypeptide (e.g., a CAR) of the chimeric protein system of the present disclosure comprises an extracellular domain comprising an antigen-binding domain and a dimerization domain. In some embodiments, the dimerization domain promotes co-localization (i.e., binding) of the first and second polypeptides to the cell surface of an immune effector cell such that the antigen-binding domains of the first and second polypeptides can bind to corresponding antigens. In some embodiments, for example, the first polypeptide can be an aCAR comprising a dimerization domain and an antigen-binding domain specific for an antigen on the surface of a cancer cell, and the second polypeptide can be an iCAR comprising a dimerization domain and an antigen-binding domain specific for an antigen on the surface of a non-cancerous cell (e.g., a healthy cell). Binding and co-localization of the first and second polypeptides to the cell surface of an immune effector cell via the dimerization domains can result in an iCAR that inhibits activation of the aCAR (e.g., via prevention of intracellular signal activation of the aCAR) in the presence of non-cancerous cells expressing both antigens. In this way, the chimeric protein system of the present disclosure provides a more specific and effective means for targeting cancer cells and preventing or reducing off-target effects on non-cancerous cells.

[0095] Transmembrane domain In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system comprises a transmembrane domain. The present disclosure is not limited with respect to the transmembrane domain utilized. In some embodiments, the CAR polypeptide sequence is selected from the group consisting of LIR1, LIR2, LIR3, LIR5, LIR8, IRTA1, IRTA2, IRTA4, LAIR1, BTLA, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, NKIR, TLT1, PD-1, CTLA4, TIM-3, LAG3, TIGIT, FCRH3, FCGR2B, SIGLEC-2, SIGLEC-6, SIGLEC-7, SIGLEC-12, SIGLEC-10, K and / or CD94.

[0096] [Table 1]

[0097] In some embodiments, the transmembrane domain is a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain of a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3 zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIRl transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain. The polypeptide is selected from the group consisting of an IR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, a SIRPa transmembrane domain, a HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain.

[0098] In some embodiments, the transmembrane domain of the first and / or second polypeptide of the chimeric protein system of the present invention 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 a CTLA-4 polypeptide.Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide can 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 can be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0099] In some embodiments, the transmembrane domain is selected from the group consisting of NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_00500 9, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a fragment thereof. In some embodiments, the polypeptide 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 is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length. NCBI Reference Nos. NP_001139345, AAA92533.1, NP_006130.1, ... NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0100] Further examples of suitable polypeptides from which a transmembrane domain may be derived include, but are not limited to, T cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, Examples include the transmembrane regions of the alpha, beta, or zeta chains of ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NG2C.

[0101] Spacer region In some embodiments, a CAR of the present disclosure comprises a spacer region or hinge domain that links the extracellular antigen-binding domain to the transmembrane domain. The spacer or hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or intracellular signaling domain of a polypeptide chain. The spacer or hinge domain provides flexibility to the chimeric receptor or its domains or prevents steric hindrance of the chimeric receptor or its domains. For example, the spacer region can be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region can be a hinge derived from a human protein. In some embodiments, the spacer or hinge domain can comprise up to 300 amino acids (e.g., 10-100 amino acids or 5-20 amino acids). In some embodiments, one or more spacer domains can be included in other regions of the chimeric receptor. For example, the hinge can be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region comprises an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer region is located between the antigen-binding domain and the transmembrane domain.

[0102] [Table 2]

[0103] [Table 3]

[0104] In some embodiments, one or more linker regions are present between various domains of the CAR. For example, in some embodiments, the linker region may be located between the dimerization domain and the protease cleavage domain in the extracellular domain of the first polypeptide and / or the second polypeptide. In some embodiments, the linker region may be located between the dimerization domain and the VH or VL region of the antigen-binding domain in the extracellular domain of the first polypeptide and / or the second polypeptide. In some embodiments, the linker region may be located between the VH and VL regions of the antigen-binding domain in the extracellular domain of the CAR. In some embodiments, the linker region may be located between the effector molecule and any of the above domains in the extracellular domain of the CAR. In some embodiments, the linker sequence may be repeated one or more times within the linker region. In some embodiments, the first polypeptide and / or the second polypeptide comprises one or more linkers. In some embodiments, the one or more linkers comprise a GSG linker, a Whitlow linker, an eGK linker, or any derivative thereof.

[0105] Intracellular domain In some embodiments, a CAR of the present disclosure comprises one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of a CAR can comprise an intracellular signaling domain.

[0106] Examples of intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act cooperatively to modulate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capability.

[0107] Without wishing to be bound by theory, it is believed that signals generated via the TCR alone are insufficient to fully activate T cells, and thus secondary and / or costimulatory signals are typically also utilized for full activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences: cytoplasmic signaling sequences that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and cytoplasmic signaling sequences that act in an antigen-dependent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). In addition, T cell signaling and function (e.g., activation signaling cascades) can be negatively regulated by inhibitory receptors present on T cells via intracellular inhibitory co-signaling domains.

[0108] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises an inhibitory intracellular signaling domain. In some embodiments, the inhibitory intracellular signaling domain comprises one or more intracellular inhibitory co-signaling domains. In some embodiments, the one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., transmembrane domains) via peptide linkers (see, e.g., Table 6) or via spacer or hinge sequences (see, e.g., Table 7). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains may be linked via peptide linkers or spacer or hinge sequences (see, e.g., Table 7). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains of the chimeric protein comprise one or more ITIM-containing proteins or fragments thereof. ITIMs are conserved amino acid sequences found in the cytoplasmic tails of many inhibitory immunoreceptors. In some embodiments, the one or more ITIM-containing proteins or fragments thereof are selected from PD-1, CTLA4, TIGIT, BTLA, and LAIR1. In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffolding proteins or fragments thereof. In some embodiments, the one or more non-ITIM scaffolding proteins or fragments thereof are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, GITR, and PD-L1. The inhibitory intracellular signaling domain can further comprise an enzyme inhibitory domain. In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain. In some embodiments, the enzyme catalytic domain is derived from an enzyme including, but not limited to, CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, or RasGAP.Examples of enzymatic regulation of signal transduction are described in more detail in Pavel Otahal et al. (Biochim Biophys Acta. 2011 February;1813(2):367-76), Kosugi A. et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 June;14(6):669-80), and Stanford et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 September;137(1):1-19), each of which is incorporated herein by reference for all purposes.

[0109] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a primary signaling domain that regulates primary activation of the TCR complex, either in a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in a CAR of the present disclosure include, but are not limited to, those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcΣRI, DAP10, DAP12, and CD66d.

[0110] In some embodiments, a CAR of the present disclosure comprises an intracellular signaling domain, such as the primary signaling domain of a CD3 zeta polypeptide. The CD3 zeta polypeptide of the present disclosure may have an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of NCBI reference numbers NP_932170 or NP_001106864.2, or a fragment thereof. In some embodiments, the CD3 zeta polypeptide 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 can have an amino acid sequence that is a contiguous portion of NCBI Reference No. NP_932170 or NP_001106864.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids in length.

[0111] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary intracellular signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0112] In some embodiments, the one or more intracellular signaling domains are selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-lBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0113] The chimeric polypeptide of the present disclosure can be a first-, second-, or third-generation CAR. First-generation CARs contain a single intracellular signaling domain generally derived from a T cell receptor chain. First-generation CARs generally have an intracellular signaling domain derived from the CD3 zeta (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. First-generation CARs provide de novo antigen recognition independent of HLA-mediated antigen presentation and transduce CD4 ζ via the CD3ζ chain signaling domain in a single fusion molecule. + T cells and CD8 +Second-generation CARs can induce both activation of T cells and activation of T cells. Second-generation CARs add a second intracellular signaling domain from one of various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide an additional signal to the T cell. Second-generation CARs provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Third-generation CARs have multiple intracellular costimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0114] In some embodiments, the intracellular signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3 zeta). In some embodiments, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, a chimeric polypeptide of the present disclosure comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, a chimeric polypeptide of the present disclosure comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a chimeric polypeptide of the present disclosure comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a chimeric polypeptide of the present disclosure comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0115] In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system of the present disclosure comprises a costimulatory domain, including, but not limited to, a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.In some embodiments, the CAR comprises a CD3 zeta chain intracellular signaling domain and one or more additional intracellular signaling domains (e.g., costimulatory domains) selected from a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0116] In some embodiments, the first and / or second polypeptide of the chimeric protein system of the present disclosure is selected from the group consisting of LIR1, LIR2, LIR3, LIR5, LIR8, IRTA1, IRTA2, IRTA4, LAIR1, BTLA, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, NKIR, TLT1, PD-1, CTLA4, TIM-3, LAG3, TIGIT, FCRH3, FCGR2B, SIGLEC-2, SIGLEC-6, SIGLEC-7, SIGLEC-12, SIG and / or CD94.

[0117] Leader / Signal Sequence In some embodiments, a CAR of the present disclosure comprises a leader sequence (also called a signal sequence) at the amino-terminus (N-terminus) of the CAR. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-recognition domain (e.g., scFv) upon cellular processing and localization of the CAR to the cell membrane.

[0118] Exemplary structures of the chimeric polypeptides of the chimeric protein system of the present disclosure are provided below in Table 6. Additionally, exemplary amino acid sequences of various domains of the first polypeptide and / or second polypeptide of the chimeric protein system of the present disclosure are provided below in Table 7.

[0119] Chimeric receptor encoding polynucleotide constructs According to the above embodiments, the first and / or second polypeptide (e.g., a CAR) of the chimeric protein system of the present disclosure can be encoded by a nucleic acid molecule. The present disclosure provides a nucleic acid molecule encoding any of the polypeptides comprising a dimerization domain described herein. In some embodiments, the present disclosure provides a modified polynucleotide comprising an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence encoding a CAR comprising a dimerization domain. As used herein, the term "promoter" generally refers to the regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remaining nucleic acid sequence. A promoter also contains small regions where regulatory proteins and molecules may bind, for example, RNA polymerase and other transcription factors. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence it regulates and controls ("drives") the initiation and / or expression of the nucleic acid sequence.

[0120] A promoter may be one naturally associated with a gene or sequence or may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter may be referred to as "endogenous." In some embodiments, a coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coding sequence in its natural environment. Such promoters may include promoters of other genes, promoters isolated from any other cell, and synthetic promoters or enhancers that are not "naturally occurring," such as those containing different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic modification known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0121] The promoter of a modified nucleic acid of the present disclosure may be an "inducible promoter," which refers to a promoter characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence of, under the influence of, or contact with a signal. The signal can be an endogenous or usually exogenous condition (e.g., light), compound (e.g., a chimeric or non-chimeric compound), or protein (e.g., a cytokine) that contacts the inducible promoter so as to be active in modulating the transcriptional activity of the inducible promoter. Activation of transcription can involve acting directly on the promoter to drive transcription or indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription can involve acting directly on the promoter to prevent transcription or indirectly on the promoter by activating a repressor that then acts on the promoter.

[0122] A promoter is "responsive to" or "modulated by" a local tumor condition or signal (e.g., inflammation or hypoxia) if, in the presence of the signal, transcription by the promoter is activated, inactivated, increased, or decreased. In some embodiments, a promoter contains a response element. A "response element" is a short sequence of DNA within the promoter region that binds to a specific molecule (e.g., a transcription factor) that modulates (regulates) gene expression by the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin-regulatable control element (PEACE), zinc finger DNA-binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al., J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated herein by reference), interferon-stimulated response element (ISRE) (Han, KJ et al., J Biol Chem. 2004 Apr. 9;279(15):15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al., Cell Reports. 2012;2(4):824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al., J of Biol. Chem. 1996;271:9503-9509). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element), generally increasing the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2x, 3x, 4x, 5x, etc. to indicate the number of repeats present.

[0123] Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1 alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter. In some embodiments, the promoter is a constitutive promoter.

[0124] In some embodiments, the promoter sequence is derived from a promoter selected from minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, an inducible molecule responsive promoter, and tandem repeats thereof. In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the constitutive promoter is selected from CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0125] Non-limiting examples of responsive promoters (also called "inducible promoters") are listed in Table 4, which shows the promoter and transcription factor design and the effect of the inducing molecule on the transcription factor (TF) and transgene transcription (T) (B = binding; D = dissociation; nd = undetermined) (A = activation; DA = inactivation; DR = repression) (Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m and references cited therein). Non-limiting examples of inducible promoter components include those shown in Table 5.

[0126] [Table 4] TIFF2025542083000006.tif98169

[0127] [Table 5] TIFF2025542083000008.tif89165

[0128] Multicistronic and multiple promoter systems In some embodiments, modified nucleic acids of the present disclosure can be multicistronic or polycistronic (e.g., polynucleotides encoding two or more CARs containing a dimerization domain produced from a single mRNA transcript). Modified nucleic acids can be multicistronic or polycistronic through the use of various linkers, such as a 5' to 3' first gene: linker: second gene (e.g., a polynucleotide sequence encoding a CAR containing a dimerization domain can be linked to a nucleotide sequence encoding a second exogenous polynucleotide). The linker polynucleotide sequence can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosome skipping element allows for the production of distinct polypeptides encoded by the first and second genes upon translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, and after expression, the cleavable linker polypeptide is cleaved to produce separate polypeptides encoded by the first and second genes. The cleavable linker can include polypeptide sequences such as flexible linkers (e.g., Gly-Ser-Gly sequences) to further facilitate cleavage. The linker can encode an internal ribosome entry site (IRES) to produce separate polypeptides encoded by the first and second genes upon translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0129] The linker may be a combination of linkers, such as a furin 2A linker, which produces separate polypeptides via 2A ribosomal skipping, followed by further cleavage of the furin site to allow complete removal of the 2A residue. In some embodiments, the linker combination may comprise a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-T2A fusion polypeptide. Generally, multicistronic or polycistronic systems can use any number or combination of linkers to express any number of genes or portions thereof (e.g., a modified nucleic acid can encode a first, second, and third chimeric polypeptide, each separated by a linker such that separate chimeric polypeptides are produced). As used herein, "linker" can refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, or to the multicistronic linker described above.

[0130] In some embodiments, the modified nucleic acids of the present disclosure comprise a post-transcriptional regulatory element (PRE). PREs can enhance gene expression by enabling tertiary RNA structure stability and 3' end formation. Non-limiting examples of PREs include Hepatitis B virus PRE (HPRE) and Woodchuck Hepatitis virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE comprises the alpha, beta, and gamma components of a WPRE element. In some embodiments, the WPRE comprises the alpha component of a WPRE element.

[0131] 3. Engineered Immune Effector Cells Also provided herein are cells and methods for producing cells that contain one or more modified nucleic acids of the present disclosure. These cells are referred to herein as "modified cells." These cells, which typically contain one or more modified nucleic acids, do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express one or more modified nucleic acids. In some embodiments, the modified one or more nucleic acids are expressed from one or more vectors or from a selected locus in the genome of the cell. In some embodiments, the cells are modified to contain a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) comprising any of the dimerization domains described herein.

[0132] The modified cells of the present disclosure can contain modified nucleic acids integrated into the genome of the cell. The modified cells can contain modified nucleic acids that can be expressed without being integrated into the genome of the cell, for example, modified nucleic acids via transient expression systems such as plasmids or mRNA. In some embodiments, the modified nucleic acids are selected from DNA, cDNA, RNA, mRNA, and naked plasmids. Also provided herein are expression vectors containing the modified nucleic acids.

[0133] The modified or isolated cells of the present disclosure can be human cells. The modified or isolated cells can be human primary cells. The modified primary cells can be tumor-infiltrating primary cells. The modified primary cells can be primary T cells. The modified primary cells can be hematopoietic stem cells (HSCs). The modified primary cells can be natural killer (NK) cells. The modified primary cells can be any somatic cell. The modified primary cells can be MSCs. In some embodiments, the modified cells are derived from a subject. In some embodiments, the modified cells are allogeneic with respect to the subject.

[0134] The modified cells of the present disclosure can be isolated from a subject, such as a subject known or suspected to have cancer. Cell isolation methods are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. The modified cells can be allogeneic with respect to the subject to whom the therapy is being administered. Allogeneic modified cells can be HLA-matched with respect to the subject to whom the therapy is being administered. The modified cells can be cultured cells, such as ex vivo cultured cells. The modified cells can be ex vivo cultured cells, such as primary cells isolated from a subject. The cultured cells can be cultured with one or more cytokines.

[0135] In some embodiments, the modified or isolated cells of the present disclosure are selected from T cells, CD8+ T cells, CD4+ T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory 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 modified cells are natural killer (NK) cells. In some embodiments, the modified cells are CD3 - and / or CD56 + In some embodiments, the modified cells are autologous. In some embodiments, the modified cells are allogeneic.

[0136] In some embodiments, the modified cells of the present disclosure are tumor cells selected from adenocarcinoma cells, bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colorectal tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, mesothelioma cells, ovarian tumor cells, pancreatic tumor cells, gastric tumor cells, testicular yolk sac tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0137] Also provided herein are methods comprising culturing the modified cells of the present disclosure. Methods for culturing the modified cells described herein are known. Those skilled in the art will recognize that the culture conditions depend on the specific modified cells of interest. Those skilled in the art will recognize that the culture conditions depend on the specific downstream use of the modified cells, for example, will recognize specific culture conditions for subsequent administration of the modified cells to a subject.

[0138] Embodiments of the present disclosure also include compositions and methods for modifying cells to produce chimeric polypeptides (e.g., CARs) comprising a dimerization domain. Generally, cells are modified to produce chimeric polypeptides (e.g., CARs) comprising a dimerization domain via introducing (i.e., delivering) one or more polynucleotides of the present disclosure, comprising a promoter and an exogenous polynucleotide sequence encoding the chimeric polypeptide comprising a dimerization domain, into the cytosol and / or nucleus of the cell. For example, the polynucleotide expression cassette encoding the chimeric polypeptide comprising a dimerization domain can be any of the modified nucleic acids described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the particular cell type to be modified.

[0139] 4. Delivery system Viral-mediated delivery. Viral vector-based delivery platforms can be used to modify cells. Generally, viral vector-based delivery platforms modify cells through introduction (i.e., delivery) into host cells. For example, viral vector-based delivery platforms can modify cells through the introduction of any of the modified nucleic acids described herein. The viral vector-based delivery platform can be a nucleic acid, and thus, the modified nucleic acid can also encompass modified virus-derived nucleic acids. Such modified virus-derived nucleic acids can also be referred to as recombinant viruses or modified viruses.

[0140] Viral vector-based delivery platforms can encode two or more modified nucleic acids, genes, or transgenes within the same nucleic acid. For example, a modified viral-derived nucleic acid (e.g., a recombinant virus or modified virus) can encode one or more transgenes, including, but not limited to, any of the chimeric polypeptides described herein containing one or more dimerization domains. In addition to one or more chimeric polypeptides, viral vector-based delivery platforms can encode one or more genes, referred to as cis-acting elements or genes, such as viral genes necessary for viral infection and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerase, viral transcriptase, etc.). Generally, any of the viral vector-based systems can be used for in vitro production of chimeric polypeptides or for in vivo delivery of modified nucleic acids encoding chimeric polypeptides, for example, in in vivo and ex vivo gene therapy procedures. The selection of an appropriate viral vector-based system depends on various factors, such as the size of the cargo / payload, the immunogenicity of the viral system, the intended target cells, the strength and timing of gene expression, and other factors understood by those skilled in the art.

[0141] The viral vector-based delivery platform can be an RNA-based virus or a DNA-based virus. Examples of viral vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesiculovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variant or derivative thereof.Other exemplary viral vector-based delivery platforms have been described in the art and include, but are not limited to, vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, pp. 616-629), or second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): pp. 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72(12): pp. 9873-9880).

[0142] The viral vector-based delivery platform may be a virus that targets tumor cells, referred to herein as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex viruses, oncolytic adenoviruses, oncolytic measles viruses, oncolytic influenza viruses, oncolytic Indiana vesiculoviruses, oncolytic Newcastle disease viruses, oncolytic vaccinia viruses, oncolytic polioviruses, oncolytic myxoma viruses, oncolytic reoviruses, oncolytic mumps viruses, oncolytic Maraba viruses, oncolytic rabies viruses, oncolytic rotaviruses, oncolytic hepatitis viruses, oncolytic rubella viruses, oncolytic dengue viruses, oncolytic chikungunya viruses, oncolytic respiratory syncytial viruses, oncolytic lymphocytic choriomeningitis viruses, oncolytic morbilliviruses, oncolytic lentiviruses, oncolytic replicating retroviruses, oncolytic rhabdoviruses, oncolytic Seneca Valley viruses, oncolytic Sindbis viruses, and any variants or derivatives thereof. Any of the oncolytic viruses described herein can be recombinant oncolytic viruses that include one or more transgenes (e.g., modified nucleic acids) encoding chimeric polypeptides that include a dimerization domain. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0143] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats capable of packaging up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are used to integrate one or more modified nucleic acids into target cells and provide persistent transgene expression. Retroviral-based delivery systems include, but are not limited to, murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retrovirus system.

[0144] The viral vector-based delivery platform can be lentivirus-based. Generally, lentivirus vectors are retroviral vectors that can transduce or infect non-differentiated cells and typically produce high viral titers. The lentivirus-based delivery platform can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (Cell Biolabs). The lentivirus-based delivery platform can be SIV- or FIV-based. Other exemplary lentiviral-based delivery platforms are described in more detail in U.S. Patent Nos. 7,311,907, 7,262,049, 7,250,299, 7,226,780, 7,220,578, 7,211,247, 7,160,721, 7,078,031, 7,070,993, 7,056,699, and 6,955,919, each of which is incorporated herein by reference for all purposes.

[0145] The viral vector-based delivery platform can be adenovirus-based. Generally, adenovirus-based vectors can transduce many cell types with high efficiency, do not require cell differentiation, achieve high titers and expression levels, and can be produced in large quantities in a relatively simple system. Generally, adenoviruses are not typically integrated into the host genome, so adenoviruses can be used for the transient expression of transgenes in infected cells. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984 and WO95 / 00655, each of which is incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5,585,362, 6,083,716, 7,371,570, 7,348,178, 7,323,177, 7,319,033, 7,318,919, and 7,306,793, and International Patent Application No. 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0146] Viral vector-based delivery platforms can be adeno-associated virus (AAV)-based. Adeno-associated virus ("AAV") vectors can be used to transduce cells with modified nucleic acids (e.g., any of the modified nucleic acids described herein). AAV systems can be used for in vitro production of chimeric polypeptides containing a dimerization domain, or can be used in in vivo and ex vivo gene therapy procedures, for example, for in vivo delivery of modified nucleic acids encoding one or more chimeric polypeptides containing a dimerization domain (see, e.g., West et al., Virology, 2002, pp. 111-114, each of which is incorporated herein by reference for all purposes). 160:38-47 (1987) No. 7,906,111; U.S. Patent Publication Nos. 2003-0138772, 2007 / 0036760 and 2009 / 0197338; Gao et al., J. Virol, 78(12):6381-6388 (June 2004); Gao et al., Proc. Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent Applications Nos. 2010 / 138263 and 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et al., J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes.Generally, AAV-based vectors comprise a capsid protein having an amino acid sequence corresponding to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof.

[0147] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. Generally, VLPs are constructed by renaturing viral structural proteins and purifying the resulting viral particles. After purification, a cargo / payload (e.g., any of the modified nucleic acids described herein) is then encapsulated ex vivo within the purified particles. Thus, VLP production maintains the separation of nucleic acids encoding viral structural proteins from nucleic acids encoding cargo / payload. Viral structural proteins used in VLP production can be produced by a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. Purified viral particles can be denatured and reformed in the presence of the desired cargo using methods known to those skilled in the art to produce VLPs. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777), which is incorporated herein by reference for all purposes.

[0148] Viral vector-based delivery platforms can be modified to target (i.e., infect) a range of cells, target a narrow subset of cells, or target specific cells. Generally, the envelope protein selected for a viral vector-based delivery platform determines the viral tropism. Viruses used in viral vector-based delivery platforms can be pseudotyped to target specific cells of interest. Viral vector-based delivery platforms can be pantropic and capable of infecting a range of cells. For example, a pantropic viral vector-based delivery platform can include a VSV-G envelope. Viral vector-based delivery platforms can be amphotropic and capable of infecting mammalian cells. Thus, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0149] Lipid structure delivery system. The modified cells of the present disclosure (e.g., any of the modified nucleic acids described herein) can be introduced into cells using a lipid-mediated delivery system. Generally, lipid-mediated delivery systems use a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. The lipid structure delivery system can deliver cargo / payload (e.g., any of the modified nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0150] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a general term that encompasses in vitro preparations of lipid vesicles formed by encapsulating a desired cargo, such as a modified nucleic acid, such as any of the modified nucleic acids described herein, within a lipid shell or lipid aggregate. Liposomes can be characterized as having a vesicular structure with a bilayer membrane and generally containing phospholipids and an internal medium generally comprising an aqueous composition. Liposomes can include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol.The selection of lipids is generally guided by considering the desired purpose, for example, by the criteria for in vivo delivery, such as liposome size, acid lability and liposome stability in bloodstream.A variety of methods can be used for preparing liposomes, as described in, for example, Szoka et al., Ann.Rev.Biophys.Bioeng.9;467(1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028 and 5,019,369, each of which is incorporated herein by reference for all purposes.

[0151] Multilamellar liposomes spontaneously form when lipids, including phospholipids, are suspended in an excess amount of aqueous solution, resulting in multiple lipid layers separated by aqueous media. The water and dissolved solution are trapped in a closed structure between the lipid bilayers after the lipid components undergo self-rearrangement. The desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, modified nucleic acids such as any of the modified nucleic acids described herein, viral vectors, viral-based delivery systems, etc.) can be encapsulated in the aqueous interior of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, dispersed within the lipid bilayer of the liposome, entrapped in the liposome, complexed with the liposome, or otherwise associated with the liposome so that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions can also be dissolved in or associated with the lipid bilayer.

[0152] The liposomes used in the embodiments of the present disclosure can be prepared by different methods, which are known to those skilled in the art. The preparation of liposomes is described in more detail in WO2016 / 201323, International Patent Application Nos. PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706, each of which is incorporated herein by reference for all purposes. The liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Pat. Nos. 5,962,016, 5,030,453, 6,680,068, U.S. Patent Application No. 2004 / 0208921, and International Patent Applications Nos. 03 / 015757A1, 04029213A2, and 02 / 100435A1, each of which is incorporated herein by reference in its entirety. Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372, WO 93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Pat. No. 5,279,833; Rose, U.S. Pat. No. 5,279,833; WO 91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each of which is incorporated herein by reference for all purposes.

[0153] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. Exosome sizes range from 30 to 100 nm in diameter. The surface of exosomes consists of a lipid bilayer derived from the plasma membrane of the donor cell, contains cytosol derived from the cell that produced the exosome, and displays membrane proteins derived from the parent cell on its surface. Exosomes useful for nucleic acid delivery are known to those skilled in the art, for example, exosomes described in more detail in U.S. Pat. No. 9,889,210, incorporated herein by reference for all purposes.

[0154] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane enclosing an internal space. In general, extracellular vesicles include all membrane-bound vesicles with a diameter smaller than the cell from which they originate. Extracellular vesicles generally range in diameter from 20 nm to 1000 nm and can contain a variety of macromolecular cargoes that are either within the internal space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. Cargo can include nucleic acids (e.g., any of the modified nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0155] As used herein, the term "exosome" refers to small (20-300 nm diameter, more preferably 40-200 nm diameter) cell-derived vesicles comprising a membrane enclosing an internal space, which are generated from cells by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the modified nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes can be derived from producing cells or isolated from producing cells based on size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producing cells. Exosomes and the preparation of exosomes are described in more detail in WO 2016 / 201323, which is incorporated herein by reference in its entirety.

[0156] As used herein, the term "nanovesicle" (also referred to as "microvesicle") refers to small (20-250 nm diameter, more preferably 30-150 nm diameter) vesicles of cell origin that comprise a membrane enclosing an interior space and are generated from cells by direct or indirect manipulation such that nanovesicles are not produced by unmanipulated producing cells. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulations of producing cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some cases, result in the destruction of producing cells. Preferably, the population of nanovesicles is substantially free of vesicles derived from producing cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid such as any of the modified nucleic acids described herein, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. Once engineered, nanovesicles can be isolated from the producing cells based on size, density, biochemical parameters, or a combination thereof.

[0157] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that rely on the amphiphilic properties of lipids to form membranes and vesicle-like structures (Riley 2017). These vesicles generally deliver cargo / payloads, such as any of the modified nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. The lipids used in LNP formation can be cationic, anionic, or neutral. Lipids can be synthetic or naturally occurring and, in some cases, biodegradable. Lipids can include, but are not limited to, fats, cholesterol, phospholipids, polyethylene glycol (PEG) conjugates (PEGylated lipids), lipid conjugates, waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions generally contain defined mixtures of materials, such as cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent aggregation of LNPs, prevent lipid oxidation, or provide chemical functional groups that facilitate the attachment of additional moieties. The lipid composition can have an overall effect on the size and stability of LNPs. In one example, the lipid composition contains dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to contain one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. In addition, LNPs can be further modified or functionalized to facilitate targeting to specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0158] Micelles are generally spherical synthetic lipid structures formed using single-chain lipids, with the hydrophilic heads of the single-chain lipids forming the outer layer or membrane and the hydrophobic tails of the single-chain lipids forming the micellar core. Micelle typically refers to a lipid structure containing only a lipid monolayer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 July 5; 25(7):1501-1513), which is incorporated herein by reference for all purposes.

[0159] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable consequences, including degradation of nucleic acids by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum can trigger undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of modified nucleic acid and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain instances, the modified nucleic acid and / or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of a LNP. Encapsulation of modified nucleic acid and / or viral delivery systems within LNPs can be performed by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation performed in a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In one example, a desired lipid formulation, such as an MC3- or MC3-like-containing composition, is provided to a droplet generating device in parallel with a modified nucleic acid or viral delivery system and any other desired agent, such that the delivery vector and desired agent are fully encapsulated within the MC3- or MC3-like-based LNPs. In one example, the droplet generating device can control the size range and size distribution of the generated LNPs. For example, the LNPs can have diameters ranging from 1 to 1,000 nanometers, e.g., 1, 10, 50, 100, 500, or 1,000 nanometers. After droplet generation, the delivery vehicle encapsulating the cargo / payload (e.g., the modified nucleic acid and / or viral delivery system) can be further processed or modified to prepare it for administration.

[0160] Nanoparticle Delivery. Nanomaterials can be used to deliver modified nucleic acids (e.g., any of the modified nucleic acids described herein). Importantly, nanomaterial vehicles are made from non-immunogenic materials, generally avoiding the induction of immunity against the delivery vector itself. These materials can include, but are not limited to, lipids (as described above), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery—A Review, Nanomaterials 2017, 7(5), 94), which is incorporated herein by reference for all purposes.

[0161] Genome editing systems. Genome editing systems can be used to modify a host genome to encode a modified nucleic acid, such as the modified nucleic acid of the present disclosure. Generally, a "genome editing system" refers to any system that integrates an exogenous gene into a host cell genome. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms.

[0162] A transposon system can be used to integrate modified nucleic acids, such as those of the present disclosure, into a host genome. Transposons generally contain terminal inverted repeats (TIRs) flanking a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or trans relative to the TIR-flanked cargo. The transposon system can be a retrotransposon system or a DNA transposon system. Generally, the transposon system randomly integrates the cargo / payload (e.g., modified nucleic acid) into the host genome. Examples of transposon systems include systems using transposons of the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752, and 7,985,739, each of which is incorporated by reference herein for all purposes. Another example of a transposon system includes the PiggyBac transposon system, which is described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is incorporated by reference herein for all purposes.

[0163] A nuclease genome editing system can be used to modify a host genome to encode a modified nucleic acid, such as the modified nucleic acid of the present disclosure. While not wishing to be bound by theory, nuclease-mediated gene editing systems used to introduce exogenous genes generally utilize a cell's natural DNA repair mechanism, particularly the homologous recombination (HR) repair pathway. Briefly, after damage to genomic DNA (typically a double-strand break), the cell can resolve the damage and repair the damage by using another DNA source with identical or substantially identical sequences at both the 5' and 3' ends as a template during DNA synthesis. In the natural context, HDR can use other chromosomes present in the cell as templates. In a gene editing system, an exogenous polynucleotide is introduced into a cell to be used as a homologous recombination template (HRT or HR template). Generally, any additional exogenous sequence (e.g., a gene or portion of a gene) not naturally found in the chromosome containing the lesion, contained between the 5' and 3' complementary ends within the HRT, can be incorporated (i.e., integrated) into a given genomic locus during template-based HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of the endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the modified nucleic acids described herein).

[0164] In some cases, the HR template can be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain cases, the HR template can be circular, for example, a plasmid. Circular templates can include supercoiled templates.

[0165] With respect to the exogenous sequence to be introduced, the identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms). HR arms can be identical (i.e., 100% identical) to regions of the endogenous genome target locus. HR arms can, in some cases, be substantially identical to regions of the endogenous genome target locus. Although substantially identical HR arms can be used, it can be advantageous for the HR arms to be identical, since the efficiency of the HDR pathway can be affected by HR arms with less than 100% identity.

[0166] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. Each HR arm can be 50, 100, 200, 300, 400, or 500 or more bases in length. The HR arms can generally be any length, although practical considerations such as the impact of HR arm length and overall template size on overall editing efficiency can also be taken into account. The HR arms can be identical or substantially identical to a region of the endogenous genomic target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to a region of the endogenous genomic target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to a region of the endogenous genomic target locus within a certain distance of the cleavage site, for example, within 1 base pair, 10 base pairs or less, 50 base pairs or less, or 100 base pairs or less of each other.

[0167] Nuclease genome editing systems can cleave target genomic loci using a variety of nucleases, including, but not limited to, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nucleases or derivatives thereof, Transcription Activator-Like Effector Nucleases (TALENs) or derivatives thereof, Zinc Finger Nucleases (ZFNs) or derivatives thereof, and Homing Endonucleases (HEs) or derivatives thereof.

[0168] A CRISPR-mediated gene editing system can be used to modify a host genome to encode a modified nucleic acid, such as a modified nucleic acid encoding one or more chimeric polypeptides described herein that include a dimerization domain. The CRISPR system is described in more detail in M. Adli ("The CRISPR tool kit for genome editing and beyond," Nature Communications; Vol. 9 (2018), Article No.: 1911), the entire teachings of which are incorporated herein by reference. Generally, a CRISPR-mediated gene editing system includes a CRISPR-associated (Cas) nuclease and RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which is composed of a Cas9 nuclease and RNA with a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains, a guide RNA sequence (gRNA) that directs specificity to a target sequence ("defined nucleotide sequence"), e.g., a genomic sequence, through base-pair hybridization, and a tracrRNA that hybridizes to the guide RNA sequence (gRNA) and tracrRNA. The tracrRNA can interact with a nuclease (e.g., Cas9) and thereby facilitate the recruitment of the nuclease to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides are a single polynucleotide and can also be referred to as a single guide RNA (sgRNA). While the Cas9 system is exemplified here, other CRISPR systems, such as the Cpfl system, can be used. The nuclease can include derivatives such as functional Cas9 mutants, e.g., Cas9 "nickase" mutants, which generally mediate cleavage of only one strand of the defined nucleotide sequence, as opposed to the complete double-stranded cleavage typically produced by the Cas9 enzyme.

[0169] Generally, the components of a CRISPR system interact with each other to form a ribonucleoprotein (RNP) complex to mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form an RNP complex. In some CRISPR systems, each component can be produced separately in vitro and contacted (i.e., complexed) with each other in vitro to form an RNP complex. The in vitro-produced RNP can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The in vitro-produced RNP complex can be delivered to cells by various methods, including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In a specific example, the in vitro-produced RNP complex can be delivered to cells using the Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. CRISPR nucleases, such as Cas9, can be produced in vitro (i.e., synthesized and purified) using various protein production techniques known to those skilled in the art. CRISPR-based RNAs, such as sgRNAs, can be produced in vitro (i.e., synthesized and purified) using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0170] The RNP complex produced in vitro can be complexed with different ratios of nuclease and gRNA.The RNP complex produced in vitro can also be used in different amounts in CRISPR-mediated editing system.For example, the total amount of RNP that is added can be adjusted according to the number of cells that are desired to be edited, for example, when editing a large number of cells in reaction, the amount of RNP complex that is added can be reduced.

[0171] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) is encoded by a separate polynucleotide, and each polynucleotide can be introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector; see the description of exemplary multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide-encoded CRISPR component in a cell (e.g., translation of a nuclease and transcription of a CRISPR RNA), an RNP complex can form within the cell and direct site-specific cleavage.

[0172] Some RNPs can be engineered to have moieties that facilitate delivery of the RNP to the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain, such that when the Cas9 RNP complex is delivered into the cell cytosol, or after translation of Cas9 and subsequent RNP formation, the NLS can facilitate further transport of the Cas9 RNP to the nucleus.

[0173] The modified cells described herein can be modified using non-viral methods, e.g., nucleases, and / or the CRISPR-mediated gene editing systems described herein can be delivered to cells using non-viral methods. The modified cells described herein can be modified using viral methods, e.g., nucleases, and / or the CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods, such as adenovirus, retrovirus, lentivirus, or any of the other viral-based delivery methods described herein.

[0174] In some CRISPR systems, two or more CRISPR compositions can be provided to target the same gene or a common genomic locus, each at more than one target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences that are within a certain distance from each other. In some CRISPR systems, two or more CRISPR compositions can be provided to target opposite strands of the same gene or a common genomic locus, each at different strands. For example, two separate CRISPR "nickase" compositions can be provided to direct cleavage at the same gene or a common genomic locus on opposite strands.

[0175] In general, the features of the CRISPR-mediated editing system described herein can be applied to other nuclease-based genome editing systems. TALENs are engineered site-specific nucleases composed of the DNA-binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region in the monomer of the DNA-binding domain, different artificial TALENs can be created to target various nucleotide sequences. The DNA-binding domain then directs the nuclease to the target sequence, creating a double-strand break. TALEN-based systems are described in more detail in U.S. Patent Application Nos. 12 / 965,590, 8,450,471, 8,440,431, 8,440,432, 10,172,880, and 13 / 738,381, all of which are incorporated herein by reference in their entireties. ZFN-based editing systems are described in more detail in U.S. Pat. Nos. 6,453,242, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,030,215, 6,794,136, 7,067,317, 7,262,054, 7,070,934, 7,361,635, 7,253,273 and U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, all of which are incorporated by reference in their entirety for all purposes.

[0176] Other Modified Delivery Systems. A variety of additional methods for introducing a modified nucleic acid (e.g., any of the modified nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0177] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing cargo / payload into the internal compartment of a target cell or entity by applying an electric field to temporarily permeabilize the outer membrane or shell of the target cell or entity. Generally, this method involves placing a cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the modified nucleic acids described herein). The lipid membrane of the cell is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the case of cells, at least some, if not most, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, cargo concentration, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, composition of electroporation solution, etc.) vary depending on several factors, including, but not limited to, the type of cell or other recipient entity, the cargo being delivered, the desired internalization efficiency, and the desired viability. Optimization of such criteria is within the skill of the art. Various devices and protocols can be used for electroporation. Examples include, but are not limited to, the Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ System, and the Bio-Rad® Electroporation System.

[0178] Compositions and methods for in vivo delivery of naked plasmids or modified mRNA, such as mRNA, are described in more detail in Kowalski et al. (Mol Ther. 2019 Apr. 10;27(4):710-728) and Kaczmarek et al. (Genome Med. 2017;9:60), each of which is incorporated herein by reference for all purposes.

[0179] Other methods for introducing modified nucleic acids (e.g., any of the modified nucleic acids described herein) into cells or other target recipient entities include, but are not limited to, sonication, gene guns, hydrodynamic injection, and cell membrane deformation by physical means.

[0180] 5. Compositions and Methods of Use

[0010] Embodiments of the present disclosure include pharmaceutical compositions comprising any of the engineered cells described herein (e.g., NK cells) comprising a chimeric polypeptide having a dimerization domain (e.g., a CAR) and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. In accordance with these embodiments, the present disclosure includes methods of treating a subject in need of treatment (e.g., a subject diagnosed with or suspected of having cancer). In some embodiments, the method comprises administering a therapeutically effective dose of any of the engineered cells of the present disclosure. In some embodiments, the subject has a proliferative disease, an immunological disease, a metabolic disease, a genetic disease, an ophthalmological disease, a cardiovascular disease, or a neurological disease.

[0181]

[0010] Embodiments of the present disclosure also include methods of treating a subject by administering a pharmaceutical composition comprising any of the engineered cells (e.g., NK cells) described herein that comprise a chimeric polypeptide having a dimerization domain (e.g., a CAR). In some embodiments, the subject has a proliferative disease, an immunological disease, a metabolic disease, a genetic disease, an ophthalmological disease, a cardiovascular disease, or a neurological disease.

[0182] Embodiments of the present disclosure also include kits for treating and / or preventing tumors, comprising any of the modified cells described herein (e.g., NK cells) comprising a chimeric polypeptide having a dimerization domain (e.g., a CAR) and a container. In some embodiments, the kit further comprises written instructions for using the modified cells to treat and / or prevent tumors in a subject. In some embodiments, the kit comprises a pharmaceutical composition comprising any of the modified cells described herein and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. In some embodiments, the kit further comprises written instructions for using the pharmaceutical composition to treat and / or prevent tumors in a subject.

[0183] In some embodiments, provided herein are methods of stimulating a cell-mediated immune response against tumor cells in a subject. In some embodiments, the methods include administering a therapeutically effective dose of any of the modified cells, isolated cells, or compositions disclosed herein to a subject having a tumor. In some embodiments, provided herein are methods of providing anti-tumor immunity to a subject. In some embodiments, the methods include administering a therapeutically effective dose of any of the modified cells, isolated cells, or compositions disclosed herein to a subject in need thereof. In some embodiments, provided herein are methods of treating a subject having cancer. In some embodiments, the methods include administering a therapeutically effective dose of any of the modified cells, isolated cells, or compositions disclosed herein. In some embodiments, provided herein are methods of reducing tumor volume in a subject. In some embodiments, the methods include administering a composition comprising any of the modified cells, isolated cells, or compositions disclosed herein to a subject having a tumor. In some embodiments, the administration includes systemic administration. In some embodiments, the administration includes intratumoral administration. In some embodiments, the isolated cells are derived from the subject. In some embodiments, the isolated cells are allogeneic with respect to the subject.

[0184] In some embodiments, the tumor is selected from an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colorectal tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0185] Some methods involve selecting a subject (or patient population) that has a tumor (or cancer) and treating the subject with modified cells or delivery vehicles that modulate tumor-mediated immune suppression mechanisms.

[0186] The methods provided herein also include delivering preparations of modified cells or delivery vehicles. In some embodiments, the preparations are substantially pure, e.g., containing less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than the modified cells. The preparations may be 1×10 5 cells / kg~1×10 7 cells / kg.

[0187] The methods provided herein also include administering a drug or pharmaceutical composition in combination with a therapeutically effective dose of any of the modified cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition can be administered prior to, concurrently with, simultaneously with, and / or following administration of any of the modified cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition can be administered sequentially. The drug or pharmaceutical composition can be administered concurrently or simultaneously with administration of any of the modified cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition can be administered at a separate interval (i.e., before or following administration) from administration of any of the modified cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition can be administered both concurrently / simultaneously with, and at separate intervals from, any of the modified cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition and the modified cell, isolated cell or composition may be administered via different routes, for example, the drug or pharmaceutical composition may be administered orally and the modified cell, isolated cell or composition may be administered intraperitoneally, intravenously, subcutaneously or by any other route suitable for administration, as will be understood by those of skill in the art.

[0188] The methods provided herein also include delivering compositions capable of producing the modified cells described herein in vivo, e.g., compositions capable of delivering any of the modified nucleic acids described herein to cells in vivo. Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genome editing systems, or any of the other modified delivery systems described herein that can modify cells in vivo.

[0189] The modified nucleic acids or modified cells can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more modified nucleic acids or modified cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier and other materials can depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, intranasal, intramuscular, or intraperitoneal.

[0190] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.

[0191] For intravenous, cutaneous or subcutaneous injection, or injection into an affected area, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0192] Regardless of the polypeptide, nucleic acid, small molecule, or other pharmaceutically useful compound according to the present disclosure given to an individual, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (although in some cases, prevention may be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Prescription of treatment, e.g., determination of dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to medical doctors. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed.), 1980.

[0193] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated.

[0194] Aspects of the present disclosure include kits for treating and / or preventing tumors. In some embodiments, the kits include any of the immunoresponsive cells described herein. In some embodiments, the kits further include written instructions for using the immunoresponsive cells to treat and / or prevent tumors in a subject. Aspects of the present disclosure include kits for treating and / or preventing tumors. In some embodiments, the kits include any of the pharmaceutical compositions described herein. In some embodiments, the kits further include written instructions for using the pharmaceutical composition to treat and / or prevent tumors in a subject.

[0195] Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of cancer (e.g., solid tumors). In some embodiments, the kits comprise a therapeutic or prophylactic composition comprising an effective amount of immune effector cells comprising one or more chimeric polypeptides comprising a dimerization domain of the present disclosure, an isolated nucleic acid of the present disclosure, a vector of the present disclosure, and / or a cell (e.g., an immune effector cell) of the present disclosure. In some embodiments, the kits comprise a sterile container. In some embodiments, such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. The container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a medicament.

[0196] In some embodiments, the therapeutic or prophylactic composition is provided with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., a solid tumor). In some embodiments, the instructions may include information about using the composition for the treatment and / or prevention of a disorder. In some embodiments, the instructions include, without limitation, a description of the therapeutic or prophylactic composition, dosage schedules, administration schedules for the treatment or prevention of a disorder or its symptoms, precautions, warnings, indications, counter-indications, overdose information, side effects, veterinary pharmacology, clinical tests, and / or references. In some embodiments, the instructions may be printed directly on the container (if present), or may be a label affixed to the container, or may be a separate sheet, pamphlet, card, or holder supplied in or with the container.

[0197] Enumeration of Embodiments Embodiment 1: A chimeric protein system comprising a first polypeptide comprising a first antigen-binding domain, a first transmembrane domain and a first dimerization domain, and a second polypeptide comprising a second antigen-binding domain, a second transmembrane domain and a second dimerization domain, wherein the first dimerization domain is capable of binding to the second dimerization domain.

[0198] Embodiment 2: The chimeric protein system of embodiment 1, wherein the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A, and NKG2C.

[0199] Embodiment 3: The chimeric protein system of embodiment 1 or 2, wherein the first dimerization domain comprises the dimerization domain of CD94 and the second dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C.

[0200] Embodiment 4: The chimeric protein system of embodiment 1 or 2, wherein the first dimerization domain comprises the dimerization domain of NKG2A or the dimerization domain of NKG2C, and the second dimerization domain comprises the dimerization domain of CD94.

[0201] Embodiment 5: The chimeric protein system according to any one of embodiments 1 to 4, wherein the first dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90.

[0202] Embodiment 6: The chimeric protein system according to any one of embodiments 1 to 4, wherein the second dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90.

[0203] Embodiment 7: The chimeric protein system according to any one of embodiments 1 to 6, wherein the first polypeptide is a chimeric antigen receptor.

[0204] Embodiment 8: The chimeric protein system of embodiment 7, wherein the first polypeptide is an activating chimeric antigen receptor.

[0205] Embodiment 9: The chimeric protein system of embodiment 7, wherein the first polypeptide is an inhibitory chimeric antigen receptor.

[0206] Embodiment 10: The chimeric protein system according to any one of embodiments 1 to 9, wherein the second polypeptide is a chimeric antigen receptor.

[0207] Embodiment 11: The chimeric protein system of embodiment 10, wherein the second polypeptide is an activating chimeric antigen receptor.

[0208] Embodiment 12: The chimeric protein system of embodiment 11, wherein the second polypeptide is an inhibitory chimeric antigen receptor.

[0209] Embodiment 13: The chimeric protein system according to any one of embodiments 1 to 12, wherein the antigen-binding domain comprises a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), a single-domain antibody, a diabody, a VHH fragment or a synthetic epitope.

[0210] Embodiment 14: The chimeric protein system of embodiment 13, wherein the antigen-binding domain binds to an antigen expressed on a cancer cell.

[0211] Embodiment 15: The chimeric protein system of embodiment 14, wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

[0212] Embodiment 16: The antigen binding domain is selected from the group consisting of carcinoembryonic antigen (CEA), mesothelin, Axl, GPC3, FLT3, CD33, TROP2, MUCl, MUC16, IL13Ra, ErbB2 (HER2 / neu), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE1, MN-CA IX, human telomerase reverse transcriptase, RUL, RU2 (AS), intestinal carboxylesterase, mut 16. The chimeric protein system of any one of embodiments 1 to 15, wherein the chimeric protein system is specific for hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF17), Claudin18.2, B7-H3 or Rorl.

[0213] Embodiment 17: The transmembrane domain is selected from the group consisting of a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain of a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3 zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIRl transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain, a LAIRl transmembrane domain, a GRB-2 transmembrane domain. 17. The chimeric protein system according to any one of embodiments 1 to 16, wherein the chimeric protein system is selected from the group consisting of: a CD45 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, a SIRPa transmembrane domain, a HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain and a CEACAM1 transmembrane domain.

[0214] Embodiment 18: A chimeric protein system according to any one of embodiments 1 to 17, wherein the first polypeptide and / or the second polypeptide comprises one or more intracellular signalling domains.

[0215] Embodiment 19: The chimeric protein system of embodiment 18, wherein the one or more intracellular signaling domains are selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-lBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0216] Embodiment 20: The chimeric protein system of embodiment 18 or 19, wherein the one or more intracellular signaling domains comprise a costimulatory domain selected from the group consisting of: CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CDS intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, CD16a intracellular signaling domain, DNAM-1 intracellular signaling domain, KIR2DS1 intracellular signaling domain, KIR3DS1 intracellular signaling domain, NKp44 intracellular signaling domain, NKp46 intracellular signaling domain, FceRlg intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0217] Embodiment 21: A chimeric protein system according to any one of embodiments 1 to 20, wherein the first polypeptide and / or the second polypeptide comprises a hinge domain located between the antigen-binding domain and the transmembrane domain.

[0218] Embodiment 22: A chimeric protein system according to any one of embodiments 1 to 21, wherein the first polypeptide and / or the second polypeptide comprises one or more linkers.

[0219] Embodiment 23: The chimeric protein system of embodiment 22, wherein the one or more linkers comprise a GSG linker, a Whitlow linker, an eGK linker, or any derivative thereof.

[0220] Embodiment 24: A modified polynucleotide encoding the first polypeptide and / or the second polypeptide according to any one of embodiments 1 to 23.

[0221] Embodiment 25: An expression vector comprising the modified polynucleotide of embodiment 24.

[0222] Embodiment 26: A modified cell comprising a modified polynucleotide according to embodiment 25, a vector according to embodiment 25 or a first and / or second polypeptide according to any one of embodiments 1 to 24.

[0223] Embodiment 27: The modified cell of embodiment 26, wherein the cell is selected from the group consisting of a T cell, a CDS+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.

[0224] Embodiment 28: The modified cell of embodiment 26 or 27, wherein the cell is modified to express an effector molecule.

[0225] Embodiment 29: The modified cell of embodiment 28, wherein the cell is a natural killer (NK) cell.

[0226] Embodiment 30: The modified cells of any one of embodiments 26 to 29, wherein the cells are autologous.

[0227] Embodiment 31: The modified cells of any one of embodiments 26 to 30, wherein the cells are allogeneic.

[0228] Embodiment 32: A pharmaceutical composition comprising the modified cells of any one of embodiments 26 to 31, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0229] Embodiment 33: A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective dose of the composition of embodiment 32 or the cell of any one of embodiments 26 to 31.

[0230] Embodiment 34: The method of embodiment 35, wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

[0231] Embodiment 35: A method of enhancing immune cell-mediated killing of cancer cells in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of the composition of embodiment 32 or the cell of any one of embodiments 26 to 31.

[0232] Embodiment 36: The method of embodiment 35, wherein the cancer comprises a solid tumor.

[0233] Embodiment 37: The method of embodiment 35 or embodiment 36, wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

[0234] Embodiment 38: A method for reducing off-target killing of healthy cells in a subject, comprising administering a therapeutically effective dose of the composition of embodiment 32 or the cell of any one of embodiments 26 to 31 to the subject, wherein the subject has been diagnosed with cancer.

[0235] Embodiment 39: The method of embodiment 38, wherein the cancer comprises a solid tumor.

[0236] Embodiment 40: The method of embodiment 38 or embodiment 39, wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma. [Example]

[0237] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and understandable, and can be made using suitable equivalents without departing from the scope of the present disclosure or the scope of the aspects and embodiments disclosed herein. Having described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are intended only to illustrate some aspects and embodiments of the present disclosure and should not be considered as limiting the scope of the present disclosure. The disclosures of all journal references, U.S. patents and publications referenced herein are incorporated herein by reference in their entirety.

[0238] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0239] [Example 1] According to various embodiments described herein, experiments were performed to evaluate the effectiveness of engineered chimeric antigen receptors (CARs) with complementary or opposing functions (e.g., activating versus inhibitory) using dimerization domains to promote colocalization in effector cells (e.g., immune synapses). As further described herein, it is important that certain CARs (e.g., activating and inhibitory CARs) are expressed in close proximity to each other to perform their desired function (e.g., inhibition of an activating signal). The data provided herein demonstrate that the use of a dimerization domain within the extracellular domain of a CAR promotes colocalization, which enhances synergistic and / or competitive effects.

[0240] In particular, experiments were performed to evaluate the efficacy of generating activating CARs (aCARs) and inhibitory CARs (iCARs) with CD94 / NKG2C or CD94 / NKG2A dimerization domains, as well as their ability to modulate effector cell function. Exemplary aCAR / iCAR structures are shown in Table 6 below.

[0241] [Table 6]

[0242] According to the above, donor NK cells (donor #13 and donor #15) were transduced with various aCAR / iCAR combinations (Table 6), and their efficacy was evaluated using Ls174t target cells transduced with or without VSIG2 (V-set and Immunoglobulin Domain Containing 2 is a membrane protein uniquely expressed in CEA-positive healthy cells but not tumor cells). NK cell efficacy was evaluated using Incucyte and flow cytometry-based killing assays. Briefly, target cells were modified with or without VSIG2 to express fluorescent proteins (e.g., mKate or mCherry). They were seeded at approximately 50,000 target cells per well. Target cell area was measured and quantified using Incucyte live cell microscopy. NK donor cells were engineered to express specific aCAR / iCAR combinations (expressed via separate plasmids) capable of dimerization via the CD94 / NKG2C or CD94 / NKG2A dimerization domains. NK cells were added to wells containing target cells at a specific effector-to-target cell ratio (e.g., 1:2). Images of each well, including fluorescent images, were acquired every 4 hours using Incucyte live-cell microscopy. Every 2–3 days, engineered NK cells were harvested and added to freshly plated target cells for another round of imaging and quantification. Fluorescence area was used to measure target cell proliferation, which was normalized to t=0. Killing of target cells that do not express VSIG2 (no iCAR inhibition of aCAR) by NK cells containing the aCAR / iCAR combination was assessed along with killing of target cells that express VSIG2 (aCAR inhibition by iCAR) by NK cells containing the aCAR / iCAR combination.

[0243] Figures 1A-1E present representative data assessing the efficacy of aCAR / iCAR dimerization. Figure 1A demonstrates successful co-transduction of the indicated aCAR / iCAR heterodimerization combinations in both NK donor cells (Experiment #2396). In addition, Figures 1B-1C demonstrate reduced target cell killing when target cells express VSIG2, indicating successful inhibition of aCAR by iCAR. There was a particularly significant reduction in target cell killing by the NKG2C-aCAR + CD94-iCAR combination (Figure 1C). These data are also expressed as a percentage of target cells only (transduced with fluorescent reporter only) and are shown in Figures 1D-1E. The results in Figure 1E demonstrate particularly significant inhibition of aCAR signaling by the corresponding iCAR dimerization partner using NK cells from donor #13, regardless of which dimerization domain is present in the aCAR or iCAR.

[0244] [Example 2] Experimental killing assays were performed using aCAR / iCAR constructs transduced with vanilla non-dimerizing CARs to serve as positive (with VSIG2) and negative (without VSIG2) controls. Experiments were performed using these constructs to establish baseline levels of fluorescence per well based on the percent of target cells alone normalized to non-dimerizing CARs (Figure 2A), NKG2C dimerization domain-containing CARs (Figure 2B), and CD94 dimerization domain-containing CARs (Figure 2C).

[0245] Killing assays were performed with triplicate killing for all indicated conditions, as shown in Figures 3A-3C. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; each color reflects the use of the same NK effector cells (with or without VSIG2-expressing target cells). Data from the first round are shown in Figure 3A, data from the second round are shown in Figure 3B, and data from the third round are shown in Figure 3C. Together, these data demonstrate a general trend of more fluorescence in the iCAR-expressing group in the presence of VSIG2+ target cells, indicating successful aCAR / iCAR dimerization and significant iCAR inhibition in all conditions.

[0246] In addition, killing assays were performed with triplicate killing in a subset of conditions. Figures 4A-4C present representative data from test conditions involving CD94 dimerization domain-containing CARs, and Figures 5A-5C present representative data from test conditions involving NKG2C dimerization domain-containing CARs. Figures 6A-6C present representative data from controls (Figure 6A: nondimerized CAR and no virus control; Figure 6B: single-transduction control; Figure 6C: nondimerized CAR condition (second and third rounds shown)).

[0247] A final killing assay was performed comparing NKG2C dimerization domain-containing iCAR paired with CD94 dimerization domain-containing aCAR plus a nondimerized CAR with a no-virus control, and the data are shown in Figures 7A-7C. The data in both Figure 7A (after two killings) and Figure 7B (after three killings) demonstrate increased fluorescence in the presence of VSIG2+ target cells in the group with CD94 dimerization domain-containing CAR plus a paired NKG2C dimerization domain-containing iCAR, indicating successful aCAR / iCAR dimerization and iCAR inhibition. Figure 7C presents the data from Figure 7B in a bar graph.

[0248] Sequences. Various embodiments of the disclosure described herein may include one or more of the sequences referenced below, which can be found in the corresponding sequence listing.

[0249] Table 7 TIFF2025542083000011.tif228162TIFF2025542083000012.tif228163TIFF2025542083000013.tif227161TIFF2025542083000014.tif228163TIFF2025542083000015.tif229162TIFF2025542083000016.tif227161TIFF2025542083000017.tif228161TIFF2025542083000018.tif226165TIFF2025542083000019.tif226161TIFF2025542083000020.tif228168TIFF2025542083000021.tif232167TIFF2025542083000022.tif228160TIFF2025542083000023.tif232158TIFF2025542083000024.tif231163TIFF2025542083000025.tif228164TIFF2025542083000026.tif229161TIFF2025542083000027.tif229157TIFF2025542083000028.tif229160TIFF2025542083000029.tif229159TIFF2025542083000030.tif229161TIFF2025542083000031.tif229161TIFF2025542083000032.tif228161TIFF2025542083000033.tif230159TIFF2025542083000034.tif231161TIFF2025542083000035.tif230159TIFF2025542083000036.tif229160TIFF2025542083000037.tif228160TIFF2025542083000038.tif231160TIFF2025542083000039.tif229160TIFF2025542083000040.tif230158TIFF2025542083000041.tif229162TIFF2025542083000042.tif229158TIFF2025542083000043.tif229161TIFF2025542083000044.tif228162TIFF2025542083000045.tif229162TIFF2025542083000046.tif229162TIFF2025542083000047.tif229166TIFF2025542083000048.tif230163TIFF2025542083000049.tif229165TIFF2025542083000050.tif229167TIFF2025542083000051.tif229160TIFF2025542083000052.tif228158TIFF2025542083000053.tif228157TIFF2025542083000054.tif226163TIFF2025542083000055.tif229163TIFF2025542083000056.tif231166TIFF2025542083000057.tif224160TIFF2025542083000058.tif228165TIFF2025542083000059.tif228166TIFF2025542083000060.tif228163TIFF2025542083000061.tif228159TIFF2025542083000062.tif228162TIFF2025542083000063.tif227163TIFF2025542083000064.tif227163TIFF2025542083000065.tif228164TIFF2025542083000066.tif231164TIFF2025542083000067.tif226170TIFF2025542083000068.tif71166.

Claims

1. a first polypeptide comprising a first antigen binding domain, a first transmembrane domain and a first dimerization domain, and a second polypeptide comprising a second antigen binding domain, a second transmembrane domain and a second dimerization domain, wherein the first dimerization domain is capable of binding to the second dimerization domain, and optionally the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A and NKG2C; Chimeric protein systems.

2. 3. The chimeric protein system of claim 1 or 2, wherein the first dimerization domain comprises a dimerization domain of CD94, and the second dimerization domain comprises a dimerization domain of NKG2A or a dimerization domain of NKG2C, and optionally the second dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90.

3. 3. The chimeric protein system of claim 1 or 2, wherein the first dimerization domain comprises a dimerization domain of NKG2A or a dimerization domain of NKG2C, and the second dimerization domain comprises a dimerization domain of CD94, and optionally the first dimerization domain comprises a sequence selected from the group consisting of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90.

4. 7. The chimeric protein system of any one of claims 1 to 6, wherein the first polypeptide is a chimeric antigen receptor, and optionally the first polypeptide is an activating chimeric antigen receptor or an inhibitory chimeric antigen receptor.

5. 10. The chimeric protein system of any one of claims 1 to 9, wherein the second polypeptide is a chimeric antigen receptor, optionally wherein the second polypeptide is an activating chimeric antigen receptor or an inhibitory chimeric antigen receptor.

6. The chimeric protein system according to any one of claims 1 to 12, wherein the antigen-binding domain comprises an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), a single-domain antibody, a diabody, a VHH fragment or a synthetic epitope.

7. The antigen binding domain binds to an antigen expressed on a cancer cell, and optionally the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma, and optionally the antigen binding domain binds to an antigen expressed on a cancer cell, and optionally the antigen binding domain binds to an antigen expressed on a cancer cell, and optionally the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma, and optionally the antigen binding domain binds to an antigen expressed on a cancer cell, 13Ra, ErbB2 (HER2 / neu), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF17), Claudin18.2, B7-H3 or Rorl.

8. The transmembrane domain may be a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain of a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3 zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIR1 transmembrane domain, a NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain, a LAIR1 transmembrane domain, a GRB-2 transmembrane domain, a 17. The chimeric protein system of any one of claims 1 to 16, wherein the chimeric protein system is selected from the group consisting of: main, Dok-1 transmembrane domain, Dok-2 transmembrane domain, SLAP1 transmembrane domain, SLAP2 transmembrane domain, CD200R transmembrane domain, SIRPa transmembrane domain, HAVR transmembrane domain, GITR transmembrane domain, PD-L1 transmembrane domain, KIR2DL1 transmembrane domain, KIR2DL2 transmembrane domain, KIR2DL3 transmembrane domain, KIR3DL1 transmembrane domain, KIR3DL2 transmembrane domain, CD94 transmembrane domain, KLRG-1 transmembrane domain, PAG transmembrane domain, CD45 transmembrane domain and CEACAM1 transmembrane domain.

9. The first polypeptide and / or the second polypeptide further comprise one or more intracellular signaling domains, optionally the one or more intracellular signaling domains are a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3 ... an intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain; optionally, the one or more intracellular signaling domains are selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CDS intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain,The chimeric protein system according to any one of claims 1 to 17, comprising a costimulatory domain selected from the group consisting of a DNAM-1 intracellular signalling domain, a KIR2DS1 intracellular signalling domain, a KIR3DS1 intracellular signalling domain, an NKp44 intracellular signalling domain, an NKp46 intracellular signalling domain, an FceRlg intracellular signalling domain, an NKG2D intracellular signalling domain and an EAT-2 intracellular signalling domain.

10. The chimeric protein system according to any one of claims 1 to 20, wherein the first polypeptide and / or the second polypeptide comprises a hinge domain located between the antigen-binding domain and the transmembrane domain.

11. 22. The chimeric protein system of any one of claims 1 to 21, wherein the first polypeptide and / or the second polypeptide comprises one or more linkers, optionally wherein the one or more linkers comprise a GSG linker, a Whitlow linker, an eGK linker or any derivative thereof.

12. A modified polynucleotide encoding the first polynucleotide and / or the second polynucleotide of any one of claims 1 to 23.

13. 25. An expression vector comprising the modified polynucleotide of claim 24.

14. A modified cell comprising a modified polynucleotide according to claim 25, a vector according to claim 25 or a first and / or second polypeptide according to any one of claims 1 to 24.

15. 29. The modified cell of claim 26, wherein the cell is selected from the group consisting of a T cell, a CDS+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell, optionally wherein the cell is modified to express an effector molecule, optionally the modified cell of claim 28, wherein the cell is a natural killer (NK) cell, and optionally the cell is allogeneic or autologous.

16. A pharmaceutical composition comprising the modified cells of any one of claims 26 to 31 and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

17. 32. A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26-31, optionally wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

18. 32. A method of enhancing immune cell-mediated killing of cancer cells in a subject in need thereof, comprising administering a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26-31 to the subject, optionally wherein the cancer comprises a solid tumor, and optionally wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

19. 32. A method of reducing off-target killing of healthy cells in a subject, comprising administering a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26 to 31 to the subject, wherein the subject has been diagnosed with cancer, optionally wherein the cancer comprises a solid tumor, and optionally wherein the cancer comprises glioblastoma, neuroblastoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, or myeloma.

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