Methods and compositions for cell surface co-localization of chimeric proteins

Abstract

The present disclosure provides compositions and methods related to co-localization of chimeric proteins on a cell surface. In particular, the present disclosure provides methods and compositions for facilitating functional interactions of chimeric polypeptides using dimerization domains to assist in cell surface co-localization of chimeric polypeptides.

Description

Technical Field

[0001] The present disclosure provides compositions and methods related to the co-localization of chimeric proteins on cell surfaces. Specifically, the present disclosure provides methods and compositions for facilitating the cell surface co-localization of chimeric polypeptides (e.g., chimeric antigen receptors) by using dimerization domains to promote functional interactions (e.g., inhibition of activating signals).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 423,364, filed on November 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing Statement

[0005] 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 its entirety. Background Art

[0006] The immune system is crucial for detecting and combating human cancers. Most transformed cells are rapidly detected by immune sentinels and destroyed through activation of immune effector cells, which include T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage. Therefore, cancer can be considered an immune disorder - the failure of the immune system to generate the necessary anti-tumor response to durably suppress and eliminate the disease. To more effectively combat cancer, certain immunotherapy interventions developed over the past few decades have focused specifically on enhancing one or more aspects of immune effector cells and engineering these cells to target specific tumor antigens (e.g., using chimeric antigen receptors).

[0007] Chimeric antigen receptor (CAR) can target and activate immune effector cells in vivo. These recombinant membrane receptors generally have an antigen binding domain and one or more signal transduction domains to allow effector cells to recognize specific protein antigens on tumor cells and induce activation and signaling pathways. The latest results of clinical trials with T cells expressing chimeric receptors provide convincing support for their practicality as agents for cancer immunotherapy. However, despite these promising results, many side effects associated with CAR T cell therapeutics have been found, which have caused major safety issues. One side effect is the "target / non-target tissue (on-target / off-tissue)" adverse event from the effector cells of TCR and CAR engineering, which occurs when CAR effector cells bind to their ligands outside the target tumor tissue and induce an immune response. Therefore, there is a need for effective targeting and treatment of tumors without damaging the method for normal cells expressing the same target antigen. Summary of the Invention

[0008] Embodiments of the present disclosure include chimeric protein systems. According to these embodiments, the chimeric protein system includes 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 be combined with the second dimerization domain. In some embodiments, the chimeric protein system including the first and second polypeptides provides pairing of chimeric antigen receptors (e.g., co-localization of receptor pairs) on the cell surface (e.g., via one or more dimerization domains).

[0009] 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 includes a dimerization domain from CD94, and the second dimerization domain includes a dimerization domain from NKG2A or a dimerization domain from NKG2C. In some embodiments, the first dimerization domain includes a dimerization domain from NKG2A or a dimerization domain from NKG2C, and the second dimerization domain includes a dimerization domain from CD94. The present disclosure is not limited to any specific dimerization domain. In fact, any one or more protein domains and / or amino acid sequences for forming a complex between the first and second polypeptides disclosed herein (e.g., to form a pair of chimeric antigen receptors) can be used. In some embodiments, the protein domain and / or amino acid sequence for forming a complex is a leucine zipper domain. In some embodiments, a first protein domain and / or amino acid sequence is used to form a 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 used to form a complex 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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 one embodiment, the antigen binding domain binds to an antigen expressed on a cancer cell. In some embodiments, the cancer comprises a glioblastoma, a neuroblastoma, a breast cancer, a colorectal cancer, a prostate cancer, a bladder cancer, a liver cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a gastric cancer, an endometrial cancer, a cervical cancer, a leukemia (e.g., a lymphoid, lymphocytic, lymphoblastic myeloid, or granulocytic leukemia), a lymphoma, or a myeloma.

[0014] In some embodiments, the antigen binding domain is specific for 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, RAGE 1, MN-CA IX, human telomerase reverse transcriptase, RUl, RU2 (AS), intestinal carboxylesterase, mut 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, TNFRSF 17), Claudin18.2, B7-H3, or Rorl.

[0015] In some embodiments, 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 from a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3ζ 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, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain. membrane domain, LAIR1 transmembrane domain, GRB-2 transmembrane domain, Dok-1 transmembrane domain, Dok-2 transmembrane domain, SLAP1 transmembrane domain, SLAP2 transmembrane domain, CD200R transmembrane domain, SIRPα 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.

[0016] In some embodiments, the first polypeptide and / or the second polypeptide comprises one or more intracellular signaling domains. In some embodiments, the one or more intracellular signaling domains are selected from the group consisting of: CD3 ζ-chain intracellular signaling domain, 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, GI TR 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0017] 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 GI TR 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0018] In some embodiments, the first polypeptide and / or the second polypeptide includes a hinge domain located between the antigen binding domain and the transmembrane domain.

[0019] 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.

[0020] Embodiments of the present disclosure also include engineered polynucleotides encoding any of the first polynucleotide and / or second polynucleotide described herein.

[0021] Embodiments of the present disclosure also include expression vectors comprising any of the engineered polynucleotides described herein.

[0022]

[0014] Embodiments of the present disclosure also include engineered cells comprising any of the disclosed engineered polynucleotides, vectors, or first and / or second polypeptides described herein.

[0023] In some embodiments, the cell is selected from the group consisting of: T cells, CD4+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphocytes, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, ESC-derived cells and iPSC-derived cells.

[0024] In some embodiments, the cells are engineered to express effector molecules. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0025] Embodiments of the present disclosure also include pharmaceutical compositions comprising any of the engineered cells described herein, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0026] Embodiments of the present disclosure also include methods for treating a subject having cancer, the methods 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., lymphoid, lymphocytic, lymphoblastic myeloid or granulocytic leukemia), lymphoma or myeloma.

[0027] Embodiments of the present disclosure also include methods for enhancing immune cell-mediated killing of cancer cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of any composition described herein or any cell described herein. In some embodiments, cancer includes solid tumors. In some embodiments, cancer includes 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., lymphoid, lymphocytic, lymphoblastic myeloid or granulocytic leukemia), lymphoma or myeloma.

[0028] Embodiments of the present disclosure also include methods for reducing off-target killing of healthy cells of a subject, the methods comprising administering to the subject a therapeutically effective dose of any composition described herein or any cell described herein, wherein the subject has been diagnosed with cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer includes 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., lymphoid, lymphocytic, lymphoblastic myeloid or granulocytic leukemia), lymphoma or myeloma.

[0029] Other aspects and embodiments of the present disclosure will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1A-1E : Representative data illustrating successful transduction of an exemplary aCAR / iCAR combination with a dimerization domain in NK cells ( Figure 1A ). Representative data of target cell killing assay when target cells expressed VSIG2 are provided; data are expressed as normalized E:T ratio ( Figures 1B-1C ) and the percentage of individual target cells ( Figures 1D-1E ).

[0031] Figures 2A-2C : Representative data from an experiment performed to establish baseline levels of fluorescence per well based on the percentage of individual target cells, using non-dimerized aCAR / iCAR ( Figure 2A ), CAR containing NKG2C dimerization domain ( Figure 2B ) and CAR containing CD94 dimerization domain ( Figure 2C ) for normalization.

[0032] Figures 3A-3C : Representative data of a killing assay in which three rounds of killing were performed under all specified conditions. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; and each color reflects the use of the same NK effector cells (with or without target cells expressing VSIG2). The data for the first round are shown in Figure 3A The second round of data is shown in Figure 3B The third round of data is shown in Figure 3C middle.

[0033] Figures 4A-4C : Representative data from a killing assay in which three rounds of killing were performed for a subset of conditions tested involving aCD94.

[0034] Figures 5A-5C: Representative data from a killing assay in which three rounds of killing were performed for a subset of conditions tested involving aNKG2C.

[0035] Figures 6A-6C : Representative control data from killing assay. Figure 6A Data for non-dimerized CAR and no-virus controls are presented; Figure 6B Data for single transduction controls are provided; and Figure 6C Data are presented for non-dimerized CAR conditions (second and third rounds shown).

[0036] Figures 7A-7C : Representative data from a killing assay comparing aCAR with a CD94 dimerization domain + iCAR with a NKG2C dimerization domain pairing with a non-dimerizing CAR and a no-virus control. Figure 7A The data comes from two rounds of killing, and Figure 7B The data comes from three rounds of kills. Figure 7C Represented in the form of a bar graph Figure 7B data. DETAILED DESCRIPTION

[0037] The present disclosure provides compositions and methods related to the co-localization of chimeric proteins on cell surfaces. Specifically, the present disclosure provides methods and compositions for facilitating the cell surface co-localization of chimeric polypeptides (e.g., chimeric antigen receptors) by using dimerization domains to promote functional interactions (e.g., inhibition of activating signals).

[0038] The section headings used in this section and the overall disclosure herein are for organizational purposes only and are not intended to be limiting.

[0039] 1. Definition

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to the methods and materials described herein may be used in the practice or testing of the present disclosure. As used herein, the phrase "in one embodiment" may refer to the same embodiment, but does not necessarily refer to the same embodiment. In addition, as used herein, the phrase "in another embodiment" may not refer to different embodiments, although it may refer to different embodiments. Therefore, as described below, the various embodiments of the present disclosure can be easily 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 herein by reference in their entirety. The materials, methods and embodiments disclosed herein are illustrative only and are not restrictive.

[0041] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to represent open transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates additional embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements provided herein, whether or not explicitly stated.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations of + / - 20% or + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, and still more preferably + / - 0.1% from the stated value, as such variations are suitable for practicing the disclosed methods.

[0044] Range: Throughout the disclosure, various aspects of the present disclosure may be presented in the form of ranges. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as strict limitations on the scope of the present disclosure. Therefore, the description of a range should be considered to explicitly disclose all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to explicitly disclose 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 ranges 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 applies regardless of the width of the range.

[0045] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural or recombinant source, and can be an immunoreactive portion of an intact immunoglobulin. An antibody can be a multimer of single immunoglobulin molecules. The antibodies described in the present disclosure can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, single-chain variable fragments (scFv), 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, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: 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).

[0046] The term "antibody fragment" refers to a portion of an intact antibody and preferably refers to the antigen-determining variable region of an 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.

[0047] As used herein, an "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their native conformation. As used herein, an "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their native conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0048] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are continuously connected via a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv can include VL-linker-VH or can include VH-linker-VL.

[0049] The terms "synthetic antibody" and "recombinant antibody" are used interchangeably herein to refer to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a phage or yeast expression system. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein, or an amino acid sequence specific for the antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0050] As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes called B cell receptors (BCRs) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the main antibody present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response of most subjects. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is also important in defending against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing mediators to be released from mast cells and basophils after exposure to allergens.

[0051] The term "antigen" or "Ag" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies, or the activation of specific immunocompetent cells, or both. The skilled person will understand that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. The skilled person will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response thus encodes the term "antigen" as used herein. In addition, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that trigger a desired immune response. Moreover, the skilled person will understand that an antigen need not be encoded by a "gene" at all. It is apparent that an antigen can be synthesized, or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid with other biological components.

[0052] The term "chimeric receptor," "chimeric antigen receptor," or alternatively "CAR" refers to a recombinant polypeptide that includes at least an extracellular antigen-binding portion, a transmembrane domain, and an intracellular signaling domain (also referred to as a "cytoplasmic signaling domain") that contains a functional signaling domain.

[0053] As used herein, the term "antigen binding domain" refers to a protein that binds to a target antigen. An antigen binding domain may include, but is not limited to, a protein (e.g., an immunoglobulin chain or a fragment thereof) comprising at least one immunoglobulin variable domain sequence. The term "antigen binding domain" includes antibodies and antibody fragments. In some embodiments, the antigen binding domain is a multispecific antibody molecule (e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity to a first epitope, and a second immunoglobulin variable domain sequence in the plurality has binding specificity to a second epitope). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In some embodiments, an antigen binding domain may refer to an effector protein, wherein the effector protein can bind to its cognate protein partner (such as a receptor).

[0054] As used herein, the term "extracellular domain" or "ECD" refers to a polypeptide or combination of polypeptides that form an external portion of a larger polypeptide (e.g., a chimeric antigen receptor), which extends beyond the outer membrane of the cell or organelle expressing it. In some embodiments, the extracellular domain includes more than one domain. In some embodiments, the extracellular domain includes different domains that can interact with different ligands. In other aspects, the extracellular domain includes different domains that can be cut by proteases. In still other aspects, the extracellular domain is connected to other domains (e.g., a transmembrane domain) to form a larger polypeptide (e.g., a chimeric antigen receptor). In some embodiments, the extracellular domain includes a hinge region that connects the extracellular domain to the transmembrane domain.

[0055] As used herein, the term "membrane spaning domain", "TM" or "TMD" refers to a polypeptide or combination of polypeptides that pass through the membrane of a cell or organelle in which it is expressed. In some embodiments, the membrane spaning domain is connected to the extracellular domain and / or the intracellular domain to form a larger polypeptide (e.g., a chimeric antigen receptor) that can transmit external signals to the intracellular response. In some embodiments, the membrane spaning domain is mainly composed of non-polar amino acid residues and can pass through the bilayer once (e.g., one way) or many times (multi-chain). In some embodiments, the membrane spaning domain single-pass transmembrane protein includes three domains, an extracellular domain, a membrane spaning domain and an intracellular domain. In some embodiments, the membrane spaning domain forms an alpha helix and is inserted into the membrane bilayer.

[0056] As used herein, the term "intracellular signaling domain", "cytoplasmic domain" or "ICD" refers to a polypeptide or combination of polypeptides located in the lumen of a cell or organelle in which it is expressed. In some embodiments, the intracellular signaling domain is connected to the extracellular domain via a transmembrane domain to form a larger polypeptide (e.g., a chimeric antigen receptor) that can transmit external signals to an intracellular response. In some embodiments, the intracellular signaling domain includes a single signaling domain that can provide an intracellular activation signal when a ligand binds to the extracellular domain to which it is attached. In other aspects, the intracellular signaling domain includes more than one signaling domain that can provide one or more intracellular activation signals when a ligand binds to the extracellular domain to which it is attached. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain and a costimulatory domain.

[0057] As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual. "Allogeneic" refers to a transplant derived from a different animal of the same species. "Xenogeneic" refers to a transplant derived from an animal of a different species.

[0058] As used herein, the term "therapeutic" means treating and / or preventing. A therapeutic effect is achieved by suppressing, alleviating, or eradicating a disease state.

[0059] 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 specific biological outcome. For example, a "therapeutically effective amount" can be the amount of a compound or composition that will elicit a biological or medical response in a tissue, system, or subject that is being 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 development of one or more signs or symptoms of the condition or disease being treated, or to alleviate to some extent one or more signs or symptoms of the condition 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.

[0060] As used herein, the term "treatment" disease means to reduce the frequency or severity of at least one sign or symptom of the disease or illness experienced by the subject.For example, treatment can refer to reducing or improving the progress, severity and / or duration of proliferative disorders, or improving one or more symptoms (preferably one or more discernible symptoms) of the proliferative disorders caused by the administration of one or more therapies. In some embodiments, the terms "treatment (treat)", "treatment (treatment)" and "treating" refer to improving at least one measurable physical parameter of the proliferative disorders that the patient may not be aware of, such as the growth of a tumor. In other respects, the terms "treatment (treat)", "treatment (treatment)" and "treating" refer to physically suppressing the development of proliferative disorders by, for example, stabilizing discernible symptoms, or refer to physiologically suppressing the development of proliferative disorders by, for example, stabilizing physical parameters, or both suppressing. In other respects, the terms "treatment (treat)", "treatment (treatment)" and "treating" refer to the minimizing or stabilizing of tumor size or cancer cell counts.

[0061] As used herein, "endogenous" refers to any material that comes from or is produced within an organism, cell, tissue, or system. As used herein, the term "exogenous" refers to any material that is introduced or produced from outside an organism, cell, tissue, or system.

[0062] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When the same base or amino acid monomer subunit occupies a position in both of the two compared sequences, for example, if adenine occupies a position in each of the two DNA molecules, the molecules are homologous at that position. The percentage 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 compared positions × 100. For example, if 6 out of 10 positions of two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, comparisons are made when the two sequences are aligned to give maximum homology.

[0063] "Isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its native state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment such as, for example, a host cell.

[0064] As used herein, "substantially purified" cells are cells that are substantially 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 substantially purified cell population refers to a homogenous cell population. In other cases, the term simply refers to cells that have been separated from 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.

[0065] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. A phrase nucleotide sequence encoding a protein or RNA may also include introns, and to some extent, a nucleotide sequence encoding a protein may contain one or more introns in certain forms.

[0066] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results 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 has 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. Typically, operably linked DNA sequences are contiguous and, in the case of two protein coding regions, are in the same reading frame.

[0067] "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0068] 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.

[0069] About antibody or extracellular target binding portion, as used herein, the term "specific binding" means an antibody or extracellular target binding portion that recognizes a specific antigen, but does not substantially recognize or bind to other molecules in the sample. For example, an antibody specifically bound to an antigen from a species can also be bound to an antigen from one or more species. However, this cross-species reactivity itself does not change the specific classification of the antibody. In another example, an antibody specifically bound to an antigen can also be bound to different allele forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody. In some cases, the term "specific binding (specific binding)" or "specifically binding (specifically binding)" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than a general protein. If the 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 bound to the antibody.

[0070] The term "immune effector cell" as used herein refers to a cell that participates in an immune response, e.g., a cell that participates in promoting an immune effector cell response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0071] As used herein, the term "immune effector function or immune effector response" refers to, for example, a function or response of an immune effector cell that is capable of enhancing or promoting 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 killing or inhibits target cell growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0072] The term "effector function" refers to a specific 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.

[0073] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or spread to other parts of the body through the blood and lymphatic system. Examples of various cancers described herein include, but are 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, for example, both terms cover solids and liquids, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-malignant, as well as malignant cancers and tumors.

[0074] The terms "cancer antigen," "cancer-associated antigen," or "tumor antigen" refer interchangeably to molecules (typically proteins, carbohydrates, or lipids) expressed on the surface of cancer cells, either intact or as fragments (e.g., MHC / peptides), and can be used to preferentially target agents to cancer cells. In some embodiments, tumor antigens are markers expressed by normal and cancer cells, such as lineage markers, such as CD19 or CD123 on B cells. In some embodiments, tumor antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells, for example, by 1-fold, 2-fold, 3-fold, or more overexpressed compared to normal cells. In some embodiments, tumor antigens are cell surface molecules that are inappropriately synthesized in cancer cells, for example, molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, tumor antigens are expressed exclusively on the cell surface of cancer cells, either intact or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells.

[0075] As used herein, the term "anti-tumor effect" refers to a biological effect that can 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" can also be demonstrated by the ability of the disclosed peptides, polynucleotides, antibodies (or antigen-binding portions thereof), and engineered immune cells to prevent tumorigenesis in the first place.

[0076] The term "anti-cancer effect" refers to a biological effect that can be manifested in various ways, including, but not limited to, for example, 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-tumor effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of cancer in the first place.

[0077] As used herein, the term "subject suspected of having cancer" refers to a subject who presents one or more symptoms indicating cancer (e.g., an obvious lump or mass) or is undergoing cancer screening (e.g., during a routine physical examination). A subject suspected of having cancer may also have one or more risk factors that develop into cancer. Subjects suspected of having cancer are not typically tested for cancer. However, "subject suspected of having cancer" encompasses individuals who have received a preliminary diagnosis (e.g., a CT scan shows a mass) but have not yet conducted confirmatory testing (e.g., biopsy and / or histology) or cancer type and / or stage unknown. The term also includes people who previously suffered from cancer (e.g., individuals in remission). "Subject suspected of having cancer" is sometimes diagnosed with cancer, and sometimes is found to not have cancer.

[0078] As used herein, the term "a subject diagnosed with cancer" refers to a subject that has been tested 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.

[0079] As used herein, the term "subject at risk for cancer" refers to a subject who has one or more risk factors for developing a particular cancer. Risk factors include, but are not limited to, sex, age, genetic predisposition, environmental exposures, and previous cancer events, pre-existing non-cancer diseases, and lifestyle.

[0080] As used herein, the term "characterizing a cancer in a subject" refers to identifying one or more characteristics of a cancer sample from a subject, including but not limited to the presence of benign, precancerous, or cancerous tissue and the stage of the cancer.

[0081] As used herein, the term "tumor microenvironment" refers to the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix (ECM) (see, e.g., Pattabiraman, DR & Weinberg, RANature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, FR et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007).

[0082] As used herein, "refractory" refers to a disease, such as cancer, that does not respond to treatment. In some aspects, a refractory cancer can be resistant to treatment before or at the start of treatment. In other aspects, a refractory cancer can become resistant during treatment. Refractory cancers are also called resistant cancers.

[0083] As used herein, "relapsed" or "recurrence" refers to the recurrence or reappearance of signs and symptoms of a disease (e.g., cancer) or a disease (such as cancer) after a period of improvement or response (e.g., after treatment with a previous therapy (e.g., cancer therapy)). The initial phase of response may involve cancer cell levels falling below a certain threshold, for example, less than 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. Reappearance may involve cancer cell levels rising above a certain threshold, for example, greater than 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. For example, as in the case of B-ALL, reappearance may involve, for example, after a complete response, the reappearance of primitive cells (blast) in blood, bone marrow (>5%) or any extramedullary sites. In this case, a complete response may involve <5% BM primitive cells. 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 aspect, the initial phase of response lasts 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, 8, 10, or 12 years.

[0084] As used herein, the term "derived from" refers to the relationship between a first and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include any limitation on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any limitation on a particular process for generating the intracellular signaling domain. For example, it does not imply that, in order to provide an intracellular signaling domain, one must start with the CD3 zeta sequence and delete unwanted sequences, or impart mutations, to arrive at the intracellular signaling domain.

[0085] As used herein, "activation" refers to a state in which immune cells (e.g., NK cells or T cells) have been sufficiently stimulated to induce detectable changes, such as, but not limited to, detectable cell proliferation or immune response. Activation can also be associated with induced cytokine production and / or detectable effector functions. The term "activated NK cells" especially refers to NK cells that undergo cell division and / or enhanced cytokine production and / or secretion.

[0086] As used herein, the term "immune response" refers to a response by a subject's immune system. For example, an immune response includes, but is not limited to, detectable changes (e.g., increases) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th17, or Th2 type cytokine) 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., growth of a cell population) of cells of the immune system (e.g., T cells, B cells (e.g., B cells at any developmental stage (e.g., plasma cells)), and increased processing and presentation of antigens by antigen-presenting cells. The 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 meant to encompass all aspects of the subject's immune system's ability to respond to an antigen and / or immunogen (e.g., both the initial response to the immunogen and the acquired (e.g., memory) response as a result of an adaptive immune response).

[0087] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method as recited unless expressly stated otherwise.

[0088] 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. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not limiting.

[0089] 2. Chimeric protein systems including dimerization domains

[0090] Embodiments of the present disclosure include chimeric protein systems. According to these embodiments, the chimeric protein system includes 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 is capable of binding to the second dimerization domain.

[0091] According to various embodiments as described herein, there is complementary or opposite function (for example, activation and inhibition function) immune receptor (for example, chimeric antigen receptor or CAR) can be engineered to include dimerization domain, so as to promote their co-location in immune effector cells (for example, at immune synapse). In some embodiments, dimerization domain forms the heterodimer complex of immune receptor (for example, receptor with complementary function, receptor with non-complementary function or receptor with opposite function). As further described herein, without being bound by theory, some CAR (for example, activation CAR and inhibitory CAR) show maximum efficacy when expressing in close proximity to each other to perform desired function (for example, suppressing activation signal). Data and results provided herein show that including dimerization domain as a part of CAR helps their co-location and enhances their synergistic effect and / or competitive effect. In some embodiments, dimerization domain can be located in the extracellular or intracellular domain of CAR.

[0092] As will be appreciated by those of ordinary skill in the art based on the present disclosure, the dimerization domains contained within the first and second polypeptides of the chimeric protein system of the present disclosure can be any dimerization domain that promotes the colocalization of the first and second polypeptides. In fact, the present disclosure provides that the first and second polypeptides disclosed herein include 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 is bound to a second protein domain via a disulfide bond or other type of bond). In some embodiments, the dimerization domains present in the first and second polypeptides promote pairing / complexation of chimeric antigen receptors. In the non-limiting example of a first polypeptide and a second polypeptide that dimerize (form a complex and / or colocalize) to form a pair of chimeric antigen receptors, a dimerization domain from CD94 and a dimerization domain selected from NKG2A and NKG2C are used.

[0093] 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 a dimerization domain from CD94, and the second dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C. In some embodiments, the first dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C, and the second dimerization domain comprises a dimerization domain from CD94.

[0094] In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any one 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.

[0095] In some embodiments, the second dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any one 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.

[0096] In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:70. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:72. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:74. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:76. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:78.In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:80. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:82. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:84. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:86. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:88.In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:90.

[0097] In some embodiments, the second dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:70. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:72. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:74. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:76. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:78.In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:80. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:82. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:84. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:86. In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:88.In some embodiments, the first dimerization domain comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:90.

[0098] In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system comprises an antigen binding domain, wherein the antigen binding domain is selected from the group consisting of: F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFv), single-domain antibodies, diabodies, VHH fragments, and synthetic epitopes. In some embodiments, scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, VH and VK are separated by a peptide linker. Typically, scFv has a light chain variable domain (VL) that is connected to the N-terminus of the heavy chain variable domain (VH) from its C-terminus via a polypeptide chain. Alternatively, scFv comprises a polypeptide chain in which the C-terminus of VH is connected to the N-terminus of VL via a polypeptide chain. In some embodiments, scFv comprises the structure VH-L-VL or VL-L-VH, in which VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. An sdAb is a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. A F(ab) fragment comprises the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains VL and VH located on the light and heavy chains, respectively. A F(ab') fragment differs from a Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A F(ab')2 fragment comprises two Fab' fragments connected by a disulfide bond near the hinge region. As used herein, a synthetic epitope refers to a non-naturally occurring epitope component located within (e.g., inserted into) the extracellular portion of a chimeric protein system (e.g., in an antigen-binding domain, spacer, linker, or elsewhere). The present disclosure is not limited to any particular synthetic epitope, nor is it limited to how a synthetic epitope is utilized. For example, a synthetic epitope can be used to facilitate the binding of an epitope-specific antibody to a chimeric protein system (e.g., for separation and / or purification during manufacturing, or to delete / remove a protein system from a source).

[0099] Chimeric antigen receptor

[0100] As described in detail herein, certain aspects of the present disclosure relate to chimeric antigen receptors (CARs), which are co-localized on the cell surface by forced binding via one or more dimerization domains present in the CAR polypeptide sequence. CARs disclosed herein containing one or more dimerization domains are not limited by specificity and / or function. In some embodiments, CARs containing one or more dimerization domains of the present disclosure include an extracellular antigen binding domain fused to a transmembrane domain, which is fused to one or more intracellular signaling domains. In some embodiments, CAR includes a spacer region or a hinge domain.

[0101] Extracellular antigen-binding domain

[0102] In some embodiments, the first and / or second polypeptide of the chimeric protein system of the present disclosure includes an antigen binding domain targeting any tumor-associated antigen of interest. In some embodiments, the first and / or second polypeptide of the present disclosure is an activated CAR (e.g., an activated CAR including a dimerization domain). In some embodiments, the antigen binding domains of the present disclosure bind to antigens expressed on the surface of cancer cells. In some embodiments, cancer cells are from solid tumors. In some embodiments, 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.

[0103] In some embodiments, the antigen binding domain is specific for at least one 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, RAGE 1, MN-CAIX, human telomerase reverse transcriptase, RUl, RU2 (AS), intestinal carboxylesterase, mut 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, TNFRSF 17), Claudin18.2, B7-H3 or Rorl.

[0104] 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., it is specific for two target antigens). In some embodiments, the two target antigens are the same. In some embodiments, the two target antigens are different.

[0105] According to the above-mentioned embodiment, the first and / or second polypeptide (for example, CAR) of the chimeric protein system of the present disclosure includes the extracellular domain containing antigen binding domain and dimerization domain.In some embodiments, dimerization domain promotes the co-localization (that is, combination) of the first and second polypeptides on the cell surface of immune effector cells, so that the antigen binding domains of the first and second polypeptides can be combined with their corresponding antigens.In some embodiments, for example, the first polypeptide can be a kind of aCAR including dimerization domain and antigen binding domains with specificity to the antigen on the cancer cell surface, and the second polypeptide can be a kind of iCAR including dimerization domain and antigen binding domains with specificity to the antigen on the surface of non-cancerous cells (for example, healthy cells).In the presence of non-cancerous cells expressing two kinds of antigens, the first and second polypeptides can cause iCAR to suppress the activation of aCAR via the combination and co-localization of their dimerization domains on the cell surface of immune effector cells (for example, via preventing the intracellular signal activation of aCAR).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 the off-target effects of non-cancerous cells.

[0106] transmembrane domain

[0107] In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system include a transmembrane domain. The present disclosure is not limited by the transmembrane domain used. In some embodiments, the CAR polypeptide sequence includes a transmembrane domain from: 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, SI GLEC-10, KLRG1, NKG2A, CD72, PECAM-1, CEACAM-1, PCDHGC3, PCDHGC5, CDH11, PTPRO, PTPRZ1, SLAMF1, SLAMF5, CD45, IMPG2, DSCAM, LIFR, E RMAP, IL1RAP, IL1RAPL2, CDH5, MPZL1, MPZ, MPIG6B, VSIG4, SLAP1, SLAP2, dok-1, Dok-2, GRB-2, CD200R, SIRPa, HAVR, GITR, PDL-1 and / or CD94.

[0108] Table 1: Exemplary transmembrane domain sequences.

[0109]

[0110] In some embodiments, 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 from a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3ζ 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. The pluripotent stem cell regulatory domain comprises the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain, the pluripotent stem cell regulatory domain

[0111] In some embodiments, the transmembrane domain of the first and / or second polypeptide of the inventive chimeric protein system is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide may be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide may be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide 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 can 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 can 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 can 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.

[0112] In some embodiments, the transmembrane domain comprises a moiety comprising a moiety corresponding to NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_00 5205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a fragment thereof. In some embodiments, the polypeptide can include one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some aspects, the polypeptide can have a polypeptide identified as NCBI reference number NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length.

[0113] Additional examples of suitable polypeptides from which a transmembrane domain can be derived include, but are not limited to, one or more transmembrane regions of the α, β, or ζ chain of a T cell receptor, CD27, CD3ε, 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, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, I TGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile) , CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D and NG2C.

[0114] spacer

[0115] In some embodiments, the CAR of the present disclosure includes a spacer or hinge domain connecting the extracellular antigen-binding domain to the transmembrane domain. The spacer or hinge domain can be any oligopeptide or polypeptide, and its function is to connect the transmembrane domain to the extracellular domain and / or intracellular signaling domain in the polypeptide chain. The spacer or hinge domain provides flexibility for the chimeric receptor or its domain, or prevents the steric hindrance of the chimeric receptor or its domain. For example, the spacer can have enough flexibility to allow the antigen-binding domain to be oriented in different directions, thereby promoting antigen recognition. In some embodiments, the spacer can be a hinge from a human protein. In some embodiments, the spacer domain or hinge domain can include up to 300 amino acids (for example, 10 to 100 amino acids or 5 to 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 IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge. In some embodiments, the spacer comprises an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-β extracellular linker. In some embodiments, the spacer is located between the antigen binding domain and the transmembrane domain.

[0116] Table 2: Exemplary spacer or finger domain sequences

[0117]

[0118]

[0119] Table 3. Exemplary linker sequences.

[0120]

[0121]

[0122] In some embodiments, one or more joint regions are present between different domains of CAR. For example, in some embodiments, the joint region can 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 joint region can 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 joint region can be located between the VH or VL region of the antigen-binding domain in the extracellular domain of CAR. In some embodiments, the joint region can be located between any of the above-mentioned domains in the extracellular domain of the effector molecule and CAR. In some embodiments, the joint sequence can be repeated once or more times in the joint region. In some embodiments, the first polypeptide and / or the second polypeptide include one or more joints. In some embodiments, one or more joints include GSG joints, Whitlow joints, eGK joints, or any derivatives thereof.

[0123] Intracellular domain

[0124] In some embodiments, the CAR of the present disclosure includes one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain.

[0125] Examples of intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which act synergistically to modulate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capability.

[0126] Without wishing to be bound by theory, it is believed that the signal generated by TCR alone is not enough to fully activate T cells, and therefore secondary and / or co-stimulatory signals are also generally used for full activation. Therefore, T cell activation can be mediated by two different types of cytoplasmic signaling sequences, one type of which is to initiate antigen-dependent primary activation by TCR (primary intracellular signaling domain), and to act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, for example, co-stimulatory domains). In addition, T cell signaling and function (for example, activation signaling cascades) can be negatively regulated by inhibitory receptors present in T cells through intracellular inhibitory co-signaling domains.

[0127] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure includes an inhibitory intracellular signaling domain. In some embodiments, the inhibitory intracellular signaling domain includes one or more intracellular inhibitory co-signaling domains. In some embodiments, one or more intracellular inhibitory co-signaling domains are connected to other domains (e.g., transmembrane domains) by a peptide linker (e.g., see Table 6) or a spacer or hinge sequence (e.g., see Table 7). In some embodiments, when there are two or more intracellular inhibitory co-signaling domains, two or more intracellular inhibitory co-signaling domains can be connected by a peptide linker or a spacer or hinge sequence (e.g., see Table 7). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, one or more intracellular inhibitory co-signaling domains of the chimeric protein include one or more ITIM-containing proteins or one or more fragments thereof. ITIM is a conserved amino acid sequence found in the cytoplasmic tail of many inhibitory immune receptors. 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 scaffold proteins or one or more fragments thereof. In some embodiments, the one or more non-ITIM scaffold proteins or fragments thereof are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, GITR, and PD-L1. The inhibitory intracellular signaling domain may further comprise an enzymatic inhibitory domain. In some embodiments, the enzymatic 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 signaling are described in more detail in Pavel Otáhal et al. (Biochim Biophys Acta. 2011 Feb; 1813(2): 367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 Jun; 14(6): 669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep; 137(1): 1-19), each of which is incorporated herein by reference for all purposes.

[0128] In some embodiments, the intracellular signaling domain of CAR of the present disclosure includes a primary signaling domain for regulating the primary activation of the TCR complex in a stimulating manner or an inhibitory manner. The primary intracellular signaling domain that works in a stimulating manner can include a signaling motif, which is referred to as an activation motif (ITAM) based on immunoreceptor tyrosine. Examples of suitable primary intracellular signaling domains containing ITAM that can be used for CAR of the present disclosure include but are not limited to CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, DAP10, DAP12 and CD66d.

[0129] In some embodiments, the CARs of the present disclosure include an intracellular signaling domain, such as a primary signaling domain of a CD3-zeta polypeptide. The CD3-zeta polypeptides of the present disclosure may have an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI reference number NP_932170 or NP_001106864.2, or a fragment thereof. In some embodiments, the CD3-zeta polypeptide may include one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a contiguous portion of NCBI reference number NP_932170 or NP_001106864.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acid sequences in length.

[0130] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, which has altered (e.g., increased or decreased) activity compared to a native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0131] 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 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GI TR 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0132] The chimeric polypeptide of the present disclosure can be a first generation, second generation or third generation CAR. The first generation CAR includes a single intracellular signaling domain, usually derived from a T cell receptor chain. The first generation CAR usually has an intracellular signaling domain from the CD3ζ (CD3ζ) chain, which is the main transmitter of signals from endogenous TCR. The first generation CAR can provide de novo antigen recognition and induce CD4 through the CD3ζ chain signaling domain in its single fusion molecule. + and CD8 + The activation of both T cells does not depend on HLA-mediated antigen presentation. The second generation CAR adds the second intracellular signaling domain from one of various costimulatory molecules (for example, CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of CAR to provide additional signals to T cells. The second generation CAR provides costimulation (for example, CD28 or 4-1BB) and activation (CD3 ζ). The third generation CAR has multiple intracellular costimulatory signaling domains (for example, CD28 and 4-1BB) and intracellular activation signaling domains (CD3 ζ).

[0133] In some embodiments, the intracellular signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-ζ). In some embodiments, the intracellular signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule defined below. In some embodiments, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the chimeric polypeptide of the present disclosure includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the chimeric polypeptide of the present disclosure includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the chimeric polypeptides of the present disclosure include chimeric fusion proteins comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising two functional signaling domains derived from one or more stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the chimeric polypeptides of the present disclosure include chimeric fusion proteins comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the 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.

[0134] 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 aspects, the CAR comprises a CD3 zeta chain intracellular signaling domain and one or more additional intracellular signaling domains (e.g., 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 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 ... a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, a 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0135] In some embodiments, the first polypeptide and / or the second polypeptide of the chimeric protein system of the present disclosure comprises an intracellular cytoplasmic domain from: 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, SILG ... GLEC-12, SIGLEC-10, KLRG1, NKG2A, CD72, PECAM-1, CEACAM-1, PCDHGC3, PCDHGC5, CDH11, PTPRO, PTPRZ1, SLAMF1, SLAMF5, CD45, IMPG2, DSCAM, L IFR, ERMAP, IL1RAP, IL1RAPL2, CDH5, MPZL1, MPZ, MPIG6B, VSIG4, SLAP1, SLAP2, dok-1, Dok-2, GRB-2, CD200R, SIRPa, HAVR, GITR, PDL-1 and / or CD94.

[0136] Preamble / Signal Sequence

[0137] In some embodiments, the CAR of the present disclosure includes a leader sequence (also referred to as a signal sequence) at the amino terminus (N-terminus) of the CAR. In some embodiments, the CAR also includes a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein, during cell processing and positioning of the CAR on the cell membrane, the leader sequence is optionally cut from the antigen recognition domain (e.g., scFv).

[0138] Table 6 below provides exemplary structures of chimeric polypeptides of the chimeric protein system of the present disclosure. In addition, Table 7 below provides exemplary amino acid sequences of various domains of the first polypeptide and / or second polypeptide of the chimeric protein system of the present disclosure.

[0139] Polynucleotide constructs encoding chimeric receptors

[0140] According to the above embodiments, the first and / or second polypeptide (for example, CAR) of the chimeric protein system of the present disclosure can be encoded by a nucleic acid molecule. The present disclosure provides nucleic acid molecules encoding any polypeptide including a dimerization domain as described herein. In some embodiments, the present disclosure provides engineered polynucleotides including an expression cassette, the expression cassette including a promoter operably connected to an exogenous polynucleotide sequence encoding CAR, the CAR including a dimerization domain. As used herein, the term "promoter" generally refers to the control region of a nucleic acid sequence, in which the initiation and rate of transcription of the remainder of the nucleic acid sequence are controlled. The promoter can also include a subregion in which regulatory proteins and molecules can be combined, such as RNA polymerases and other transcription factors. The promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. The promoter drives the expression or transcription of the nucleic acid sequence it regulates. In this article, when the promoter is in the correct functional position and orientation relative to the nucleic acid sequence that controls ("drives") the transcription initiation and / or expression of the sequence, the promoter is considered to be "operably connected".

[0141] A promoter can be one naturally associated with a gene or sequence, such as one that can be obtained by isolating a 5' non-coding sequence upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as "endogenous". In some embodiments, the coding nucleic acid sequence can be under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a coding sequence in its natural environment. Such promoters can include promoters of other genes; promoters isolated from any other cell; and non-"naturally occurring" synthetic promoters or enhancers, such as, for example, those containing different elements of different transcriptional regulatory regions and / or promoters or enhancers with mutations that alter expression by genetic engineering methods known in the art. In addition to synthesizing nucleic acid sequences for promoters and enhancers, sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques (including polymerase chain reaction (PCR)) (see, for example, U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906).

[0142] The promoter of the engineered nucleic acid of the present disclosure can be an "inducible promoter," which refers to a promoter characterized by regulating (e.g., starting or activating) transcriptional activity when there is a signal, is affected by a signal, or is contacted by a signal. The signal can be an endogenous or typically exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine) that contacts the inducible promoter and thereby plays a role in regulating 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. In contrast, inactivation of transcription can involve acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor, which then acts on the promoter.

[0143] If, in the presence of a local tumor state (e.g., inflammation or hypoxia), transcription from the promoter is activated, inactivated, increased, or decreased, then the promoter is "responsive" to or "regulated by" the state or signal. In some embodiments, the promoter includes a response element. A "response element" is a short DNA sequence within the promoter region that binds to a specific molecule (e.g., a transcription factor) that regulates (modulates) gene expression in the promoter. Response elements that can be used according to the present disclosure include, but are not limited to, phloretin regulatable control element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activated 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-κB 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, incorporated herein by reference). Other response elements are also contemplated herein. Response elements can also comprise tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase 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 times the repeat occurs.

[0144] Non-limiting examples of promoters include cytomegalovirus (CMV) promoter, elongation factor 1-alpha (EF1a) promoter, elongation factor (EFS) promoter, MND promoter (a synthetic promoter comprising the U3 region of the modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), phosphoglycerate kinase (PGK) promoter, spleen focus forming virus (SFFV) promoter, simian virus 40 (SV40) promoter, and ubiquitin C (UbC) promoter. In some embodiments, the promoter is a constitutive promoter.

[0145] In some embodiments, the promoter sequence is derived from a promoter selected from the group consisting of: 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, inducer molecule response 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 the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0146] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") are listed in Table 4, which shows the design of promoters and transcription factors, and the effects of inducer molecules on transcription factors (TFs) and transgene transcription (T) (B, binding; D, dissociation; nd, not determined) (A, activation; DA, deactivation; DR, derepression) (see 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.

[0147] Table 4. Exemplary inducible promoters

[0148]

[0149]

[0150]

[0151] Table 5. Exemplary components of inducible promoters

[0152]

[0153]

[0154]

[0155] Polycistronic and multi-promoter systems

[0156] In some embodiments, the engineered nucleic acid of the present invention can be a multicistronic (multicistronic or polycistronic) (e.g., a polynucleotide encoding more than one CAR, the CAR including a dimerization domain produced by a single mRNA transcript). The engineered nucleic acid can be a multicistronic, which is achieved by using various linkers (e.g., a polynucleotide sequence encoding a CAR including a dimerization domain can be connected to a nucleotide sequence encoding a second exogenous polynucleotide), such as in a first gene: linker: second gene 5' to 3' orientation. The linker polynucleotide sequence can encode a 2A ribosomal skipping element, such as T2A. Other 2A ribosomal skipping elements include but are not limited to E2A, P2A, and F2A. The 2A ribosomal skipping element allows the production of independent polypeptides encoded by the first and second genes to be provided during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, wherein the cleavable linker polypeptide is cut after expression, thereby producing an independent polypeptide encoded by the first and second genes. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), which further facilitates cleavage. The linker can encode an internal ribosome entry site (IRES), thereby generating independent polypeptides encoded by the first and second genes during translation. The linker can encode a splicing receptor, such as a viral splicing receptor.

[0157] In some embodiments, the combination of joints can include furin sequences, flexible joints, and 2A joints. Therefore, in some embodiments, the joint is furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the joint is furin-Gly-Ser-Gly-T2A fusion polypeptide. Generally, the polycistronic system can use any number of joints or the combination of joints to express any number of genes or parts thereof (for example, the nucleic acid of through engineering approaches can encode the first, second, and third chimeric polypeptide, each separated by a joint, thereby producing an independent chimeric polypeptide). As used herein, " joint " can refer to a polypeptide or the above-mentioned polycistronic joint connecting the first polypeptide sequence and the second polypeptide sequence.

[0158] In some embodiments, the engineered nucleic acids of the present disclosure include a post-transcriptional regulatory element (PRE). A PRE can enhance gene expression by stabilizing the tertiary RNA structure and forming a 3' end. Non-limiting examples of PRE 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 includes the α, β, and γ components of the WPRE element. In some embodiments, the WPRE includes the α component of the WPRE element.

[0159] 3. Engineered immune effector cells

[0160] Also provided herein are cells and methods for producing cells, which include one or more engineered nucleic acids of the present disclosure. These cells are referred to herein as "engineered cells". These cells that typically contain one or more engineered nucleic acids do not exist in nature. In some embodiments, the cell is a cell that recombinantly expresses the separation of one or more engineered nucleic acids. In some embodiments, one or more engineered nucleic acids are expressed from a selected locus of one or more vectors or cell genomes. In some embodiments, the cell is engineered to include a nucleic acid containing a promoter, which is operably connected to a nucleic acid sequence encoding a chimeric polypeptide (e.g., CAR) comprising any dimerization domain as described herein.

[0161] The engineered cells of the present disclosure can include the engineered nucleic acids that are integrated into the cell genome. The engineered cells can include the engineered nucleic acids that can be expressed when being integrated into the cell genome, for example, by transient expression systems (such as plasmids or mRNA) engineering. In some embodiments, the engineered nucleic acids are selected from: DNA, cDNA, RNA, mRNA and naked plasmids. An expression vector comprising the engineered nucleic acids is also provided herein.

[0162] The engineered cells or isolated cells of the present disclosure can be human cells. The engineered cells or isolated cells can be human primary cells. The engineered primary cells can be tumor-infiltrating primary cells. The engineered primary cells can be primary T cells. The engineered primary cells can be hematopoietic stem cells (HSCs). The engineered primary cells can be natural killer (NK) cells. The engineered primary cells can be any somatic cells. The engineered primary cells can be MSCs. In some embodiments, the engineered cells are derived from a subject. In some embodiments, the engineered cells are allogeneic relative to the subject.

[0163] The engineered cells of the present disclosure can be separated from a subject, such as a subject known or suspected of having cancer. Cell separation methods are well 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 separation techniques, and negative separation, magnetic separation, and combinations thereof. For a subject administered for treatment, the engineered cells can be allogeneic. The modified cells of the allogeneic can be HLA matched with the subject administered for treatment. The engineered cells can be cultured cells, such as cells cultured in vitro. The engineered cells can be cells cultured in vitro, such as primary cells isolated from a subject. The cultured cells can be cultivated together with one or more cytokines.

[0164] In some embodiments, the engineered or isolated cells of the present disclosure are selected from the group consisting of: T cells, CD8+ T cells, CD4+ T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphocytes, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelets, 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 engineered cells are natural killer (NK) cells. In some embodiments, the engineered cells are CD3 - and / or CD56 + In some embodiments, the engineered cells are autologous. In some embodiments, the engineered cells are allogeneic.

[0165] In some embodiments, the engineered cells of the present disclosure are tumor cells selected from the group consisting of 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.

[0166] Also provided herein are methods comprising culturing the engineered cells of the present disclosure. Methods for culturing the engineered cells described herein are known. Those skilled in the art will recognize that the culture conditions will depend on the specific engineered cells of interest. Those skilled in the art will recognize that the culture conditions will depend on the specific downstream use of the engineered cells, for example, the specific culture conditions for subsequently administering the engineered cells to a subject.

[0167] Embodiments of the present disclosure also include compositions and methods for engineering cells to produce chimeric polypeptides (e.g., CAR) including dimerization domains. Typically, by importing (i.e., delivering) one or more polynucleotides of the present disclosure into the cytosol and / or nucleus of cells, cells are engineered to produce chimeric polypeptides (e.g., CAR) including dimerization domains, and the polynucleotides include promoters and coding for exogenous polynucleotide sequences of chimeric polypeptides including dimerization domains. For example, the polynucleotide expression cassette encoding the chimeric polypeptides including dimerization domains can be any engineered nucleic acid as described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, ultrasonic treatment, and the cell membrane deformation achieved by physical means. It will be understood by those skilled in the art that delivery methods may be selected depending on the specific cell type to be engineered.

[0168] 4. Delivery System

[0169] Virus-mediated delivery. Delivery platforms based on viral vectors can be used for cell engineering. Generally, delivery platforms based on viral vectors are engineered by introducing (i.e., delivering) into host cells. For example, delivery platforms based on viral vectors can be engineered by introducing any engineered nucleic acid described herein. Delivery platforms based on viral vectors can be nucleic acids, and therefore, engineered nucleic acids can also encompass engineered virus-derived nucleic acids. Such engineered virus-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0170] The delivery platform based on viral vector can encode more than one engineered nucleic acid, gene or transgene in the same nucleic acid.For example, engineered virus-derived nucleic acid (for example, recombinant virus or engineered virus) can encode one or more transgenes, including but not limited to any chimeric polypeptide including one or more dimerization domains as described herein.In addition to one or more chimeric polypeptides, the delivery platform based on viral vector can also encode one or more genes, such as viral genes (for example, capsid protein, envelope protein, viral polymerase, viral transcriptase, etc.) required for viral infectivity and / or viral production, referred to as cis-acting elements or genes. Generally, any of the systems based on viral vectors can be used for the in vitro production of chimeric polypeptides, or for in vivo and ex vivo gene therapy programs, such as in vivo delivery of engineered nucleic acids encoding chimeric polypeptides. The selection of suitable viral vector-based systems will depend on various factors, such as carrier (cargo) / payload size, immunogenicity of viral systems, target cells of interest, gene expression intensity and time, and other factors understood by those skilled in the art.

[0171] The delivery platform based on a viral vector can be an RNA-based virus or a DNA-based virus. Exemplary delivery platforms based on a viral vector include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesicular stomatitis virus (Indiana vesiculovirus), Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Maraba virus (Maraba virus), rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, measles virus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus (Seneca Valley virus), Sindbis virus (sindbis virus) and any variants or derivatives thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentivirus, including but not limited to 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): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0172] The delivery platform based on a viral vector can 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 virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesicular stomatitis virus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic measles virus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variants or derivatives thereof. Any oncolytic virus described herein can be a recombinant oncolytic virus comprising one or more transgenes (e.g., engineered nucleic acids) encoding a chimeric polypeptide comprising 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).

[0173] Viral-based delivery platforms can be retroviral-based. Typically, retroviral vectors consist of cis-acting long terminal repeats (LTRs) with a packaging capacity of up to 6-10 kb of exogenous sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate one or more engineered nucleic acids into target cells to provide permanent transgene expression. Retroviral delivery systems include, but are not limited to, those based on 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 retroviral system.

[0174] The delivery platform based on viral vector can be based on lentivirus. Generally, lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells, and generally produce high viral titers. The delivery platform based on lentivirus can be based on HIV, such as ViraPower system (ThermoFisher) or pLenti system (Cell Biolabs). The delivery platform based on lentivirus can be based on SIV or FIV. Other exemplary delivery platforms based on lentivirus 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; 6,955,919, each of which is incorporated herein by reference for all purposes.

[0175] Viral vector-based delivery platforms can be based on adenoviruses. Generally, adenovirus-based vectors are able to achieve very high transduction efficiencies in many cell types, do not require cell division, achieve high titers and high levels of expression, and can be produced in large quantities using relatively simple systems. Adenoviruses are often used for transient expression of transgenes within infected cells because they do not typically integrate into the host genome. 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; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 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. Patent 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 WO 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0176] The viral vector-based delivery platform can be adeno-associated virus (AAV)-based.Adeno-associated virus ("AAV") vectors can be used to transduce cells with engineered nucleic acids (eg, any engineered nucleic acid described herein). The AAV system can be used for the in vitro production of chimeric polypeptides comprising a dimerization domain, or for in vivo and ex vivo gene therapy procedures, such as for the in vivo delivery of engineered nucleic acids encoding one or more chimeric polypeptides comprising a dimerization domain (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; U.S. Patent Application Nos. US2003-0138772, US2007 / 0036760, and US2009 / 0197338; Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al., J. Virol, 78(12):6381-6388 (June 2004). al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and international patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each of which is incorporated herein by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No. 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes. Typically, AAV-based vectors include a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof.

[0177] The delivery platform based on viral vector can be virus-like particle (VLP) platform. Generally, VLP is constructed by producing viral structural protein and purifying the obtained viral particles. Then, after purification, vehicle / payload (for example, any engineered nucleic acid as described herein) is encapsulated in the purified particle in vitro. Therefore, the generation of VLP maintains the separation of nucleic acid encoding viral structural protein and nucleic acid encoding vehicle / payload. The viral structural protein for VLP production can be produced in a variety of expression systems, including mammals, yeast, insects, bacteria or in vivo translation expression systems. Using methods known to those skilled in the art, purified viral particles can be denatured and recombined to produce VLP in the presence of the desired vehicle. The generation of VLP 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.

[0178] The delivery platform based on viral vector can be engineered to target (i.e., infect) a series of cells, a narrower cell subpopulation of the targeting range, or target specific cells. Generally, the envelope protein selected for the delivery platform based on viral vector will determine the tropism of the virus. The virus used in the delivery platform based on viral vector can be pseudotyped to target specific cells of interest. The delivery platform based on viral vector can be pan-tropic and infect a series of cells. For example, the delivery platform based on a pan-tropic viral vector can include the VSV-G envelope. The delivery platform based on viral vector can be bitropic and infect mammalian cells. Therefore, those skilled in the art can select suitable tropism, pseudotype and / or envelope protein to target the desired cell type.

[0179] Lipid structure delivery system. The engineered nucleic acid of the present disclosure (e.g., any engineered nucleic acid described herein) can be introduced into cells using a lipid-mediated delivery system. Typically, a lipid-mediated delivery system uses a structure consisting of an outer lipid membrane encapsulating 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 engineered nucleic acid described herein) in vitro, in vivo, or ex vivo.

[0180] Lipid-based nanoparticles may include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid formulations. As used herein, "liposome" is a general term encompassing in vitro formulations of lipid carriers, which are formed by encapsulating a desired cargo, such as an engineered nucleic acid, such as any engineered nucleic acid described herein, within a lipid shell or lipid aggregates. Liposomes are characterized by having a vesicle structure with a double membrane, which typically includes a phospholipid and an internal medium typically containing an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes may be unilamellar liposomes. Liposomes may be multilamellar liposomes. Liposomes may be multivesicular liposomes. Liposomes may be positively charged, negatively charged, or neutrally charged. In some aspects, liposomes are charge-neutral. Liposomes may be formed from standard vesicle-forming lipids, which typically include neutrally and negatively charged phospholipids and sterols, such as cholesterol. The selection of lipids is generally guided by considerations of the intended purpose, for example criteria for in vivo delivery, such as liposome size, acid instability, and stability of the liposomes in the bloodstream. A variety of methods can be used to prepare 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.

[0181] In the case of lipid suspension comprising phospholipids, when multiple lipid layers are separated by aqueous medium, multilamellar liposomes are generated spontaneously. Water and dissolved solute are trapped in the closed structure between lipid bilayers after lipid component experience self-rearrangement. Expected carrier (for example, polypeptide, nucleic acid, small molecule drugs, engineered nucleic acid, such as any engineered nucleic acid as described herein, viral vectors, delivery systems based on viruses, etc.) can be encapsulated in the aqueous interior of liposomes, attached to liposomes via the connecting molecule associated with liposomes and polypeptide / nucleic acid, be dispersed in the lipid bilayer of liposomes, be wrapped in liposomes, be compounded with liposomes, or otherwise be associated with liposomes, so that it can be delivered to target entities. Lipophilic molecules or molecules with lipophilic regions can also be dissolved in lipid bilayers or be associated with lipid bilayers.

[0182] As known to those skilled in the art, the liposomes used in accordance with embodiments of the present disclosure can be prepared by different methods. The preparation of liposomes is further described in detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 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. Patent Nos. 5,962,016; 5,030,453; 6,680,068, U.S. Application No. 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each of which is incorporated herein by reference in its entirety for all purposes. Lipid-mediated gene delivery methods are described in, for example, 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.

[0183] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. Exosomes range in size from 30 to 100 nm in diameter. Their surface is composed of a lipid bilayer derived from the donor cell membrane, and they contain cytosol from the cell that produced the exosomes and display membrane proteins from the parent cell on their surface. Exosomes for nucleic acid delivery are known to those skilled in the art and are described in greater detail, for example, in U.S. Patent No. 9,889,210, which is incorporated herein by reference for all purposes.

[0184] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Typically, extracellular vesicles include all membrane-bound vesicles whose diameter is smaller than the diameter of the cell from which they are derived. Typically, the diameter of an extracellular vesicle ranges from 20 nm to 1000 nm and may include various macromolecular cargoes displayed on the outer surface of the extracellular vesicle in the internal space, and / or across the membrane. The cargo may include nucleic acids (e.g., any engineered nucleic acid described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. As an example and not 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 an alkaline solution), vesicle-like 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 may come from living or dead organisms, transplanted tissues or organs, and / or cultured cells.

[0185] As used herein, the term "exosome" refers to a small (diameter between 20-300 nm, more preferably between 40-200 nm) vesicle derived from a cell, which includes a membrane that encloses an internal space and is generated from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes include lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA, such as any engineered nucleic acid described herein), a sugar (e.g., a monosaccharide, a polysaccharide or a glycan) or other molecules. Exosomes can be derived from producer cells and isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are an extracellular vesicle. Typically, exosome production / biogenesis does not result in the destruction of producer cells. Exosomes and the preparation of exosomes are further described in detail in WO 2016 / 201323, which is incorporated herein by reference in its entirety.

[0186] As used herein, the term "nanovesicle" (also referred to as "microvesicle") refers to a cell-derived small (diameter between 20-250 nm, more preferably between 30-150 nm) vesicle that includes a membrane that closes the internal space, and that is generated from the cell by direct or indirect manipulation so that the nanovesicle is not produced by the production cell in the absence of the manipulation. Typically, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulations of production cells include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some cases, the production of nanovesicles may result in the destruction of the production cell. Preferably, the nanovesicle population is substantially free of vesicles derived from production cells by direct budding from the plasma membrane or by fusion of late endosomes with the plasma membrane. Nanovesicles include lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any engineered nucleic acid described herein), a sugar (e.g., a monosaccharide, a polysaccharide, or a polysaccharide) or other molecules. Once the nanovesicles are obtained from the producer cells according to the described procedures, the nanovesicles can be isolated from the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0187] In general, lipid nanoparticles (LNPs) are synthetic lipid structures that rely on the amphiphilicity of lipids to form membranes and vesicle-like structures (Riley 2017). Typically, these vesicles deliver cargo / payloads, such as any engineered nucleic acid or viral system described herein, by being absorbed into the target cell membrane and releasing the cargo into the cytosol. The lipids used for LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived and, in some cases, biodegradable. The lipids can include fats, cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins. The lipid composition typically includes a defined mixture of substances, such as cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. The lipid composition can affect the size and stability of the entire LNP. In an example, lipid composition comprises dilinoleyl methyl-4-dimethylaminobutyrate (MC3) or MC3 sample molecule.MC3 and MC3 sample lipid composition can be formulated to comprise one or more other lipids, such as lipid, sterol or the neutral lipid that PEG or PEG are put together.In addition, LNP can be by other engineering approaches or functionalization to promote the targeting to specific cell type.Another consideration in the LNP design is the balance between targeting efficiency and cytotoxicity.

[0188] Typically, micelles are spherical synthetic lipid structures formed using single-chain lipids, wherein the hydrophilic head of the single-chain lipid forms an outer layer or membrane, and the hydrophobic tail of the single-chain lipid forms the center of the micelle. Micelles generally refer to lipid structures containing only a lipid monolayer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), which is incorporated herein by reference for all purposes.

[0189] Nucleic acid vectors, such as expression vectors, are directly exposed to serum and can have some adverse consequences, including nucleic acid being degraded by serum nucleases or free nucleic acid stimulating the immune system to miss the target. Similarly, viral delivery systems directly exposed to serum can cause undesirable immune responses and / or neutralization of viral delivery systems. Therefore, the encapsulation of engineered nucleic acids and / or viral delivery systems can be used to avoid degradation while also avoiding potential miss-target effects. In some instances, engineered nucleic acids and / or viral delivery systems are fully encapsulated within the delivery carrier, such as within the aqueous interior of LNP. Encapsulation of engineered nucleic acids and / or viral delivery systems in LNP can be carried out by technology well known to those skilled in the art (such as microfluidic mixing and droplet generation performed on a microfluidic droplet generation device). This device includes but is not limited to a standard T-junction device or a flow focusing device. In an example, the desired lipid formulation (such as a composition containing MC3 or MC3 sample) and engineered nucleic acid or viral delivery system and any other desired medicament are provided to a droplet generating apparatus in parallel so that the delivery vehicle and the desired medicament are fully encapsulated within the inside of the LNP based on MC3 or MC3 sample. In an example, the droplet generating apparatus can control the size range and size distribution of the LNP produced. For example, the diameter size range of the LNP can be 1 to 1000 nanometer, for example, 1, 10, 50, 100, 500 or 1000 nanometer. After the droplet generates, the delivery carrier (for example, engineered nucleic acid and / or viral delivery system) of the encapsulated carrier / payload can be further processed or engineered so that it is ready for administration.

[0190] Nanoparticle delivery. Nanomaterials can be used to deliver engineered nucleic acids (e.g., any engineered nucleic acid described herein). Importantly, the nanomaterial carrier can be made of non-immunogenic materials and generally avoids eliciting immunity to the delivery carrier itself. These materials may 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.

[0191] Genome editing systems. Genome editing systems can be used to engineer a host genome to encode engineered nucleic acids, such as the engineered nucleic acids disclosed herein. Generally, a "genome editing system" refers to any system that integrates exogenous genes into the host cell genome. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms.

[0192] The transposon system can be used to integrate engineered nucleic acids (such as engineered nucleic acids of the present disclosure) into the host genome. Transposons typically include terminal inverted repeats (TIRs) flanking the carrier / payload nucleic acid and the transposase. The transposon system can provide a carrier with TIR flanks for the transposon in a cis or trans manner. The transposon system can be a retrotransposon system or a DNA transposon system. Typically, the transposon system randomly integrates carrier / payload (e.g., engineered nucleic acid) into the host genome. Examples of transposon systems include transposon systems that utilize the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which is 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 herein by reference 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 herein by reference for all purposes.

[0193] Nuclease genome editing systems can be used to engineer the host genome to encode engineered nucleic acids, such as the engineered nucleic acids of the present disclosure. Without wishing to be bound by theory, generally, the gene editing system mediated by nucleases for introducing exogenous genes utilizes the natural DNA repair mechanism of the cell, particularly homologous recombination (HR) repair pathway. In short, after genomic DNA is damaged (usually double-strand breaks), cells can solve damaged during DNA synthesis by using another DNA source with identical or substantially identical sequences at its 5' and 3' ends as a template to repair damage. Under natural background, HDR can use another chromosome present in the cell as a template. In the gene editing system, exogenous polynucleotides are introduced into the cell and used as homologous recombination templates (HRT or HR templates). Generally, during templated HDR, any additional exogenous sequence (for example, a gene or a part of a gene) not initially found in the chromosome with the damage included between the 5' and 3' complementary ends in the HRT can be incorporated into (that is, "integrated") into a given genomic locus. 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 engineered nucleic acid described herein).

[0194] In some instances, 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 specific instances, the HR template can be circular, such as a plasmid. Circular templates can include supercoiled templates.

[0195] Relative 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). The HR arms can be identical (i.e., 100% identical) to the region of the endogenous genomic target locus. In some instances, the HR arms can be substantially identical to the region of the endogenous genomic target locus. Although substantially identical HR arms can be used, it can be advantageous that the HR arms are identical because the efficiency of the HDR approach may be affected by HR arms having less than 100% identity.

[0196] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. The length of the HR arms can each be greater than or equal to 50, 100, 200, 300, 400 or 500 bases. Although the HR arms can generally be of any length, practical factors such as the effect of the HR arm length and the overall template size on the overall editing efficiency can also be considered. The HR arms can be identical or substantially identical to the region of the endogenous genomic target locus adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genomic target locus adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genomic target locus within a certain distance from the cleavage site, such as 1 base pair, less than or equal to 10 base pairs, less than or equal to 50 base pairs, or less than or equal to 100 base pairs from each other.

[0197] Nuclease genome editing systems can use a variety of nucleases to cleave target genomic loci, 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.

[0198] The gene editing system mediated by CRISPR can be used to engineer the host genome to encode engineered nucleic acids, such as nucleic acids encoding one or more chimeric polypeptides including dimerization domains as described herein. The CRISPR system is described in more detail in M.Adli ("The CRISPR tool kit for genome editing and beyond" Nature Communications; volume 9 (2018), Article number: 1911), the entire contents of which are incorporated herein by reference. Typically, the gene editing system mediated by CRISPR includes CRISPR-related (Cas) nucleases and one or more RNAs that direct cutting to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which consists of a Cas9 nuclease and one or more RNAs with a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. CrRNA typically has two RNA domains: a guide RNA sequence (gRNA), which is specifically directed to a target sequence ("determined nucleotide sequence"), such as a genomic sequence, by base pair hybridization; and an RNA domain that hybridizes with tracrRNA. TracrRNA can interact with a nuclease (e.g., Cas9) and thereby promote the recruitment of the nuclease to the genomic locus. CrRNA and tracrRNA polynucleotides can be independent polynucleotides. CrRNA and tracrRNA polynucleotides can be single polynucleotides, also referred to as single guide RNA (sgRNA). Although the Cas9 system is shown here, other CRISPR systems, such as the Cpf1 system, can also be used. Nucleases can include derivatives thereof, such as Cas9 functional mutants, for example, Cas9 "nickase" mutants, which typically mediate single-stranded cleavage of only a determined nucleotide sequence, rather than the complete double-stranded break typically produced by the Cas9 enzyme.

[0199] Typically, the components of the CRISPR system interact to form a ribonucleoprotein (RNP) complex to mediate sequence-specific cleavage. In some CRISPR systems, each component can be independently produced and used to form an RNP complex. In some CRISPR systems, each component can be independently produced in vitro and contacted with each other in vitro (ie, "compounded") to form an RNP complex. The RNP produced in vitro can then be introduced (ie, "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The RNP complex produced in vitro can be delivered to cells in a variety of ways, including but not limited to electroporation, lipid-mediated transfection, cell membrane deformation achieved by physical means, lipid nanoparticles (LNP), virus-like particles (VLP), and ultrasonic treatment. In a specific example, a method based on Electroporation delivery system The RNP complex produced in vitro is delivered to the cell.Other electroporation systems include, but are not limited to, MaxCyte electroporation system, Miltenyi CliniMACS electroporation system, Neon electroporation system and BTX electroporation system.CRISPR nuclease (for example, Cas9) can be produced in vitro (that is, synthesized and purified) using a variety of protein production techniques known to those skilled in the art.CRISPR system RNA (for example, sgRNA) can be produced in vitro (that is, synthesized and purified) using a variety of RNA production techniques known to those skilled in the art (such as in vitro transcription or chemical synthesis).

[0200] The in vitro generated RNP complexes can be complexed with different ratios of nuclease and gRNA. The in vitro generated RNP complexes can also be used in CRISPR-mediated editing systems in different amounts. For example, depending on the number of cells to be edited, the total amount of RNP added can be adjusted, such as when editing a large number of cells in the reaction, the amount of RNP complex added can be reduced.

[0201] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be independently encoded by a polynucleotide, wherein each polynucleotide is introduced into a cell together or independently. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or polycistronic vector, see description of exemplary polycistronic systems below) and introduced into a cell. After expressing each CRISPR component encoded by a polynucleotide (e.g., translation of a nuclease and transcription of a CRISPR RNA) in a cell, an RNP complex can be formed in the cell, and site-specific cutting can then be directed.

[0202] Some RNPs can be engineered to have moieties that facilitate RNP delivery to the nucleus. For example, the Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if the Cas9 RNP complex is delivered to the cytosol of the cell, or after translation of Cas9 and subsequent RNP formation, the NLS can facilitate further transport of the Cas9 RNP into the nucleus.

[0203] The engineered cells described herein can be engineered using non-viral methods, for example, nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using non-viral methods. The engineered cells described herein can be engineered using viral methods, for example, nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods such as adenovirus, retrovirus, lentivirus, or any other viral-based delivery method described herein.

[0204] In some CRISPR systems, more than one CRISPR composition can be provided so that each composition targets the same gene or general genomic locus at more than one target nucleotide sequence. For example, two independent CRISPR compositions can be provided to guide cutting at two different target nucleotide sequences at a certain distance from each other. In some CRISPR systems, more than one CRISPR composition can be provided so that each composition targets opposite strands of the same gene or general genomic locus. For example, two independent CRISPR "nickase" compositions can be provided to guide cutting at the same gene or general genomic locus on opposite strands.

[0205] In general, the features of the CRISPR-mediated editing system described herein can be applied to other genome editing systems based on nucleases. TALEN is an engineered site-specific nuclease that consists of the DNA binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease Fok1. By changing the amino acids present in the highly variable residue region of the monomer of the DNA binding domain, different artificial TALENs can be produced to target various nucleotide sequences. The DNA binding domain then guides the nuclease to the target sequence and produces a double-strand break. TALEN-based systems are described in more detail in U.S. Serial No. 12 / 965,590; U.S. Patent No. 8,450,471; U.S. Patent No. 8,440,431; U.S. Patent No. 8,440,432; U.S. Patent No. 10,172,880; and U.S. Serial No. 13 / 738,381, all of which are incorporated herein by reference in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent 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; 2005 / 0267061, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0206] Other Engineered Delivery Systems Various additional ways to introduce an engineered nucleic acid (eg, any engineered nucleic acid described herein) into a cell or other target receptor entity (such as any lipid structure described herein).

[0207] Electroporation can be used to deliver polynucleotides to receptor entities. Electroporation is a method of internalizing a carrier / payload into the internal compartment of a target cell or entity by applying an electric field to instantaneously penetrate the outer membrane or shell of a target cell or entity. Generally speaking, the method involves placing a cell or target entity between two electrodes in a solution containing a carrier of interest (e.g., any engineered nucleic acid described herein). Then, by applying a transient set voltage that allows the carrier to enter the interior of the entity (such as the cytoplasm of a cell), the lipid membrane of the cell is destroyed, i.e., infiltrated. In the example of a cell, even if not the majority, at least some cells still survive. Cells and other entities can be electroporated in vitro, in vivo, or in vitro. Electroporation conditions (e.g., cell number, carrier concentration, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, test tube length, electroporation solution composition, etc.) vary according to several factors, including but not limited to, the type of cell or other receptor entity, the carrier to be delivered, the required internalization efficiency, and the required viability. Optimization of this standard is within the capabilities of those skilled in the art. A variety of devices and protocols can be used for electroporation. Examples include, but are not limited to, Transfection system, Flow Electroporation TM 、 Nucleofector TM System and Electroporation system.

[0208] Compositions and methods for in vivo delivery of engineered mRNA (such as naked plasmids or mRNA) are described in 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.

[0209] Other methods for introducing an engineered nucleic acid (e.g., any engineered nucleic acid described herein) into a cell or other target receptor entity include, but are not limited to, sonication, gene guns, hydrodynamic injection, and physical deformation of the cell membrane.

[0210] 5. Composition and Method of Use

[0211] Embodiments of the present disclosure include a kind of pharmaceutical composition, which includes any engineered cell as described herein (for example, NK cell), and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient or a combination thereof, and the engineered cell includes a chimeric polypeptide (for example, CAR) with a dimerization domain. According to these embodiments, the present disclosure includes a method for treating a subject in need thereof (for example, a subject diagnosed with cancer or suspected of having cancer). In some embodiments, the method includes administering a therapeutically effective dose of any engineered cell of the present disclosure. In some embodiments, the subject suffers from proliferative diseases, immune diseases, metabolic diseases, hereditary diseases, ophthalmic diseases, cardiovascular diseases or neurological diseases.

[0212] Embodiments of the present disclosure also include a method for treating a subject by administering a pharmaceutical composition comprising any engineered cell as described herein (e.g., NK cells), wherein the engineered cell includes a chimeric polypeptide (e.g., CAR) with a dimerization domain. In some embodiments, the subject suffers from proliferative diseases, immune diseases, metabolic diseases, hereditary diseases, ophthalmic diseases, cardiovascular diseases, or nervous system diseases.

[0213] Embodiments of the present disclosure also include a kit for treating and / or preventing tumors, the kit including any engineered cells (e.g., NK cells) and containers as described herein, the engineered cells including chimeric polypeptides (e.g., CAR) with dimerization domains. In some embodiments, the kit also includes written instructions for treating and / or preventing subject tumors using engineered cells. In some embodiments, the kit includes a pharmaceutical composition including any engineered cells as described herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. In some embodiments, the kit also includes written instructions for treating and / or preventing subject tumors using a pharmaceutical composition.

[0214] In some embodiments, provided herein are methods of stimulating a cell-mediated immune response against tumor cells in a subject. In some embodiments, the method comprises administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein. In some embodiments, provided herein are methods of providing anti-tumor immunity in a subject. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein. In some embodiments, provided herein are methods of treating a subject having cancer. In some embodiments, the method comprises administering to a subject having a tumor a therapeutically effective dose of any of the engineered 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 method comprises administering to a subject having a tumor a composition comprising any of the engineered cells, isolated cells, or compositions disclosed herein. In some embodiments, administering comprises systemic administration. In some embodiments, administering comprises 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.

[0215] In some embodiments, the tumor is selected from the group consisting of adenocarcinoma, bladder tumor, brain tumor, breast tumor, cervical tumor, colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, melanoma, mesothelioma, ovarian tumor, pancreatic tumor, stomach tumor, testicular yolk sac tumor, prostate tumor, skin tumor, thyroid tumor, and uterine tumor.

[0216] Some methods involve selecting a subject (or patient population) having a tumor (or cancer) and treating the subject with engineered cells or delivery vehicles that modulate tumor-mediated immunosuppressive mechanisms.

[0217] The methods provided herein also include formulations for delivering engineered cells or delivery vehicles. In some embodiments, the formulation is a substantially pure formulation comprising, for example, less than 5% (e.g., less than 4%, 3%, 2% or 1%) of cells other than engineered cells. The formulation may include 1x10 5 cells / kg to 1x10 7 cells / kg of cells.

[0218] Methods provided herein also include administering a drug or pharmaceutical composition in combination with any of the engineered cells, separated cells or compositions disclosed herein for treatment of an effective dose. The drug or pharmaceutical composition can be administered before, in parallel, simultaneously and / or after any of the administration of the engineered cells, separated cells or compositions disclosed herein. The drug or pharmaceutical composition can be administered continuously. The drug or pharmaceutical composition can be administered in parallel or simultaneously with any of the administration of the engineered cells, separated cells or compositions disclosed herein. The drug or pharmaceutical composition can be administered at intervals (e.g., before or after) with any of the administration of the engineered cells, separated cells or compositions disclosed herein. The drug or pharmaceutical composition can be administered in parallel / simultaneously and at intervals separately with any of the administration of the engineered cells, separated cells or compositions disclosed herein. The drug or pharmaceutical composition and engineered cells, separated cells or compositions can be administered via different routes, for example, the drug or pharmaceutical composition can be administered orally, and the engineered cells, separated cells or compositions can be administered intraperitoneally, intravenously, subcutaneously or by any other route suitable for administration, as understood by those skilled in the art.

[0219] The methods provided herein also include in vivo delivery of compositions capable of producing engineered cells as described herein, for example, any engineered nucleic acid as described herein can be delivered to cells in vivo. Such compositions include any virally mediated delivery platform, any lipid structure delivery system, any nanoparticle delivery system, any genome editing system, or any other engineered delivery system as described herein capable of engineering cells in vivo.

[0220] Engineered nucleic acids or engineered cells can be formulated into pharmaceutical compositions. In addition to one or more of the engineered nucleic acids or engineered cells, these compositions can also include pharmaceutically acceptable excipients, vehicles, buffers, stabilizers, or other materials well known to those skilled in the art. These materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The exact nature of the vehicle or other materials can depend on the route of administration, such as oral, intravenous, cutaneous or subcutaneous, intranasal, intramuscular, and intraperitoneal routes.

[0221] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. Tablets can include solid carriers, such as gelatin or adjuvants. Liquid pharmaceutical compositions generally include liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, glucose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0222] For intravenous injection, cutaneous injection or subcutaneous injection, or injection at the affected part, the active ingredient will be in the form of an acceptable aqueous solution for parenteral administration, which does not contain pyrogens and has suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic carriers such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0223] Whether a polypeptide, nucleic acid, small molecule or other pharmaceutically useful compound according to the present disclosure is administered to an individual, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (as the case may be, although prophylaxis may be considered treatment) sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration will depend on the nature and severity of the protein aggregation disease being treated. The prescription of treatment (e.g., determination of dosage, etc.) is the responsibility of general physicians and other medical practitioners, and generally takes into account the condition to be treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the physician. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0224] The composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0225] Aspects of the present disclosure include kits for treating and / or preventing tumors. In some embodiments, the kit includes any of the immune response cells described herein. In some embodiments, the kit also includes written instructions for using the immune response 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 kit includes any of the pharmaceutical compositions described herein. In some embodiments, the kit also includes written instructions for using the pharmaceutical compositions to treat and / or prevent tumors in a subject.

[0226] Some aspects of the present disclosure relate to a test kit for treating and / or preventing cancer (for example, solid tumors). In some embodiments, the test kit includes therapeutic or preventive compositions, which include an effective amount of immune effector cells, which include one or more chimeric polypeptides containing the dimerization domain of the present disclosure, isolated nucleic acids of the present disclosure, carriers of the present disclosure and / or cells of the present disclosure (for example, immune effector cells). In some embodiments, the test kit includes a sterile container. In some embodiments, this container can be a box, an ampoule, a bottle, a vial, a pipe, a bag, a pouch (pouch), a blister pack or other suitable container forms known in the art. The container can be made of plastic, glass, laminated paper, metal foil or other materials suitable for accommodating medicine.

[0227] In some embodiments, the therapeutic or preventive composition is provided together with instructions for administering the therapeutic or preventive composition to a subject suffering from cancer (e.g., a solid tumor) or a risk of cancer. In some embodiments, the instructions may include information about the purposes of the composition for treating and / or preventing the disorder. In some embodiments, the instructions include, but are not limited to, a description of the therapeutic or preventive composition, a dosage regimen, a dosage regimen for treating or preventing the disorder or its symptoms, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or references. In some embodiments, the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, brochure, card, or foldout provided in or with the container.

[0228] Examples of implementation methods

[0229] Embodiment 1: A chimeric protein system comprises: a first polypeptide, wherein the first polypeptide comprises a first antigen binding domain, a first transmembrane domain and a first dimerization domain; and a second polypeptide, wherein the second polypeptide comprises 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.

[0230] Embodiment 2: The chimeric protein system according to embodiment 1, wherein the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A and NKG2C.

[0231] Embodiment 3: The chimeric protein system according to embodiment 1 or 2, wherein the first dimerization domain comprises a dimerization domain from CD94, and the second dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C.

[0232] Embodiment 4: The chimeric protein system according to embodiment 1 or 2, wherein the first dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C, and the second dimerization domain comprises a dimerization domain from CD94.

[0233] Embodiment 5: The chimeric protein system of 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.

[0234] Embodiment 6: The chimeric protein system of 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.

[0235] Embodiment 7: The chimeric protein system of any one of embodiments 1 to 6, wherein the first polypeptide is a chimeric antigen receptor.

[0236] Embodiment 8: The chimeric protein system of embodiment 7, wherein the first polypeptide is an activating chimeric antigen receptor.

[0237] Embodiment 9: The chimeric protein system of embodiment 7, wherein the first polypeptide is an inhibitory chimeric antigen receptor.

[0238] Embodiment 10: The chimeric protein system of any one of embodiments 1 to 9, wherein the second polypeptide is a chimeric antigen receptor.

[0239] Embodiment 11: The chimeric protein system of embodiment 10, wherein the second polypeptide is an activating chimeric antigen receptor.

[0240] Embodiment 12: The chimeric protein system of embodiment 11, wherein the second polypeptide is an inhibitory chimeric antigen receptor.

[0241] Embodiment 13: A 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.

[0242] Embodiment 14: The chimeric protein system of embodiment 13, wherein the antigen binding domain binds to an antigen expressed on cancer cells.

[0243] 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.

[0244] Embodiment 16: The chimeric protein system of any one of embodiments 1 to 15, wherein the antigen binding domain is specific for 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, RAGE 1, MN-CA IX, human telomerase reverse transcriptase, RUl, 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, ephrin B2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF 17), Claudin18.2, B7-H3 or Rorl.

[0245] Embodiment 17: The chimeric protein system according to any one of embodiments 1 to 16, wherein the transmembrane domain is selected from the group consisting of: LAX transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain, LAT transmembrane domain, transmembrane domain from LAT mutant, BTLA transmembrane domain, CDS transmembrane domain, CD28 transmembrane domain, CD3ζ transmembrane domain, CD4 transmembrane domain, 4-IBB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, 2B4 transmembrane domain, PD-1 transmembrane domain, CTLA4 transmembrane domain, BTLA transmembrane domain, TIM3 transmembrane domain, LIR1 transmembrane domain, NKG2A transmembrane domain, TIGIT transmembrane domain, and LAG3 transmembrane domain, LAIR1 transmembrane domain, GRB-2 transmembrane domain, Dok-1 transmembrane domain, Dok-2 transmembrane domain, SLAP1 transmembrane domain, SLAP2 transmembrane domain, CD200R transmembrane domain, SIRPα 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.

[0246] Embodiment 18: The chimeric protein system of any one of embodiments 1 to 17, wherein the first polypeptide and / or the second polypeptide comprises one or more intracellular signaling domains.

[0247] Embodiment 19: The chimeric protein system of claim 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 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a CD40 intracellular signaling domain, a CD54 intracellular signaling domain, a CD64 intracellular signaling domain, a CD70 intracellular signaling domain, a CD80 intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, a CD80 intracellular signaling domain, a CD70 intracellular signaling domain, a CD8 ... 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0248] 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 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 CD40 intracellular signaling domain, a CD54 intracellular signaling domain, a CD64 intracellular signaling domain, a CD70 intracellular signaling domain, a CD80 intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 ... 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, FceR1g intracellular signaling domain, NKG2D intracellular signaling domain, and EAT-2 intracellular signaling domain.

[0249] Embodiment 21: The chimeric protein system of 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.

[0250] Embodiment 22: The chimeric protein system of any one of embodiments 1 to 21, wherein the first polypeptide and / or the second polypeptide comprises one or more linkers.

[0251] 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.

[0252] Embodiment 24: An engineered polynucleotide encoding the first polynucleotide and / or the second polynucleotide of any one of embodiments 1 to 23.

[0253] Embodiment 25: An expression vector comprising the engineered polynucleotide described in embodiment 24.

[0254] Embodiment 26: An engineered cell comprising the engineered polynucleotide according to embodiment 25, the vector according to embodiment 25, or the first polypeptide and / or the second polypeptide according to any one of embodiments 1 to 24.

[0255] Embodiment 27: The engineered cells according to embodiment 26, wherein the cells are selected from the group consisting of: T cells, CD4+T cells, CD4+T cells, γ-δT cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphocytes, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, ESC-derived cells and iPSC-derived cells.

[0256] Embodiment 28: The engineered cell of embodiment 26 or 27, wherein the cell is engineered to express an effector molecule.

[0257] Embodiment 29: The engineered cell of embodiment 28, wherein the cell is a natural killer (NK) cell.

[0258] Embodiment 30: The engineered cell of any one of embodiments 26 to 29, wherein the cell is autologous.

[0259] Embodiment 31: The engineered cell of any one of Embodiments 26 to 30, wherein the cell is allogeneic.

[0260] Embodiment 32: A pharmaceutical composition comprising the engineered cells of any one of embodiments 26 to 31, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0261] 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.

[0262] Embodiment 34: The method according to 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.

[0263] 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.

[0264] Embodiment 36. The method of claim 35, wherein the cancer comprises a solid tumor.

[0265] Embodiment 37: The method according to 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.

[0266] Embodiment 38: A method of reducing off-target killing of healthy cells in a subject, the method comprising administering to the subject a therapeutically effective dose of the composition of embodiment 32 or the cells of any one of embodiments 26 to 31, wherein the subject has been diagnosed with cancer.

[0267] Embodiment 39: The method of Embodiment 38, wherein the cancer comprises a solid tumor.

[0268] Embodiment 40: The method according to 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.

[0269] Example

[0270] It will be 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 obvious, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the present disclosure 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 construed as limiting the scope of the present disclosure. The disclosures of all journal references, U.S. patents, and publications cited herein are incorporated herein by reference in their entirety.

[0271] The present disclosure has several aspects, illustrated by the following non-limiting examples.

[0272] Example 1

[0273] According to various embodiments as described herein, experiments were conducted to evaluate the effect of the engineered chimeric antigen receptor (CAR) with complementary or relative function (e.g., activation and inhibition), which has a dimerization domain to promote their co-location in effector cells (e.g., immune synapse). As further described herein, in order to perform desired functions (e.g., inhibition of activation signals), it is important that some CARs (e.g., activation CAR and inhibitory CAR) are expressed in close proximity to each other. The data provided herein show that using a dimerization domain in the extracellular domain of CAR contributes to their co-location, which enhances their synergistic effect and / or competitive effect.

[0274] Specifically, experiments were performed to evaluate the efficacy of generating activating CAR (aCAR) and inhibitory CAR (iCAR) with CD94 / NKG2C or CD94 / NKG2A dimerization domains, and their ability to regulate effector cell function. Exemplary aCAR / iCAR architectures are shown in Table 6 below.

[0275] Table 6: Exemplary aCAR / iCAR architecture.

[0276]

[0277] 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 that is uniquely expressed in CEA-positive healthy cells but not expressed in tumor cells). NK cell efficacy was evaluated using an Incucyte-based and flow cytometry-based killing assay. Briefly, target cells were engineered to express fluorescent proteins (e.g., mKate or mCherry) with or without VSIG2. They were seeded at approximately 50,000 target cells per well. Target cell area was measured and quantified using an Incucyte live microscope. NK donor cells are engineered to express a specific aCAR / iCAR combination that is capable of dimerization via CD94 / NKG2C or CD94 / NKG2A dimerization domains (expressed via separate plasmids). NK cells are added to wells containing target cells at a specific ratio of effector cells to target cells (e.g., 1:2). Images of each well (including fluorescent images) are acquired every four hours using an Incucyte live microscope. Every 2-3 days, engineered NK cells are collected and added to freshly seeded target cells for another round of imaging and quantification. Fluorescent area is used to determine target cell growth, which is normalized to t=0. The killing of target cells that do not express VSIG2 by NK cells comprising aCAR / iCAR combinations (absence of aCAR inhibition of iCAR) and the killing of target cells that express VSIG2 by NK cells comprising aCAR / iCAR combinations (iCAR inhibition of aCAR) were evaluated.

[0278] Figures 1A-1E Representative data evaluating the efficacy of aCAR / iCAR dimerization are provided. Figure 1A Successful co-transduction of the indicated aCAR / iCAR heterodimerization combination in two NK donor cells was demonstrated (Experiment #2396). Figures 1B-1C This indicates that when target cells express VSIG2, target cell killing is reduced, indicating that iCAR successfully inhibits aCAR. The NKG2C-aCAR+CD94-iCAR combination significantly reduced the killing effect of target cells ( Figure 1C ).like Figures 1D-1E These data are also expressed as a percentage of target cells alone (transduced with fluorescent reporter gene only), as shown. Figure 1E The results showed that using NK cells from donor #13, the corresponding iCAR dimerization partners had particularly significant inhibition of aCAR signaling, regardless of which dimerization domain was present in the aCAR or iCAR.

[0279] Example 2

[0280] Experimental killing assays were performed using aCAR / iCAR constructs transduced with a common non-dimerized CAR as positive (with VSIG2) and negative (without VSIG2) controls. Using these constructs, experiments were performed to establish a baseline level of fluorescence per well based on the percentage of individual target cells to compare the effect of non-dimerized CAR ( Figure 2A ), CAR containing NKG2C dimerization domain ( Figure 2B ) and CAR containing CD94 dimerization domain ( Figure 2C ) for normalization.

[0281] like Figures 3A-3C As shown in Figure 2, three rounds of killing were performed for all indicated conditions. Target cells with VSIG2 are represented by hollow shapes, while target cells without VSIG2 are represented by solid shapes; and each color reflects the use of the same NK effector cells (with or without target cells expressing VSIG2). The data for the first round are shown in Figure 2. Figure 3A The second round of data is shown in Figure 3B The third round of data is shown in Figure 3C Together, these data indicate a general trend of more fluorescence in the group with iCAR expression in the presence of VSIG2+ target cells, indicating successful aCAR / iCAR dimerization and greater iCAR inhibition under various conditions.

[0282] In addition, three rounds of killing tests were conducted for some conditions. Figures 4A-4C Provides representative data for testing conditions involving CARs containing a CD94 dimerization domain; and Figures 5A-5C Representative data for testing conditions involving CARs containing an NKG2C dimerization domain are provided. Figures 6A-6C Representative data for controls are provided ( Figure 6A : Non-dimerized CAR and no virus control; Figure 6B : Single transduction control; Figure 6C : Non-dimeric CAR conditions (second and third rounds are shown).

[0283] A final round of killing assays was performed to compare the CD94 dimerization domain-containing aCAR + NKG2C dimerization domain-containing iCAR pairing with a non-dimerizing CAR and a virus-free control; data are available at Figures 7A-7C Available from Figure 7A (After two rounds of killing) and Figure 7BBoth data (after three rounds of killing) showed that in the presence of VSIG2+ target cells, fluorescence increased in the group with a CAR containing a CD94 dimerization domain + an iCAR containing a NKG2C dimerization domain pairing, indicating successful aCAR / iCAR dimerization and iCAR inhibition. Figure 7C Represented in the form of a bar graph Figure 7B data.

[0284] 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 listings.

[0285] Table 7: Nucleic acid and amino acid sequences.

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

Claims

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, optionally wherein the first dimerization domain or the second dimerization domain is selected from the group consisting of CD94, NKG2A and NKG2C.

2. The chimeric protein system according to claim 1 or 2, wherein The first dimerization domain comprises a dimerization domain from CD94, and the second dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C, optionally, 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.

3. The chimeric protein system according to claim 1 or 2, wherein The first dimerization domain comprises a dimerization domain from NKG2A or a dimerization domain from NKG2C, and the second dimerization domain comprises a dimerization domain from CD94, optionally, 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.

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

5. The chimeric protein system according to 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 includes 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.

7. The chimeric protein system according to claim 13, wherein The antigen binding domain binds to an antigen expressed on a cancer cell, 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, optionally wherein the antigen binding domain is specific for carcinoembryonic antigen (CEA), mesothelin, Axl, GPC3, FLT3, CD33, TROP2, MUCl, M UC16, 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, RAGE 1, 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, ephrin B2, CD22, insulin growth factor (IGF)-1, IGF-11, IGF-1 receptor, NKG2D, BCMA (CD269, TNFRSF 17), Claudin18.2, B7-H3, or Rorl.

8. The chimeric protein system according to any one of claims 1 to 16, wherein 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 from a LAT mutant, a BTLA transmembrane domain, a CDS transmembrane domain, a CD28 transmembrane domain, a CD3ζ 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. , LAIR1 transmembrane domain, GRB-2 transmembrane domain, Dok-1 transmembrane domain, Dok-2 transmembrane domain, SLAP1 transmembrane domain, SLAP2 transmembrane domain, CD200R transmembrane domain, SIRPα 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 chimeric protein system according to any one of claims 1 to 17, wherein The first polypeptide and / or the second polypeptide further comprises one or more intracellular signaling domains, optionally 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 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a β-actin ... 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 structure domain and an EAT-2 intracellular signaling domain, optionally wherein 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 signaling domain, a GITR intracellular signaling domain, a 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, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceR1g intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling 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. The chimeric protein system according to 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. An engineered polynucleotide encoding the first polynucleotide and / or the second polynucleotide of any one of claims 1 to 23.

13. An expression vector comprising the engineered polynucleotide of claim 24.

14. An engineered cell comprising the engineered polynucleotide of claim 25, the vector of claim 25, or the first polypeptide and / or the second polypeptide of any one of claims 1 to 24.

15. The engineered cell according to claim 26, wherein The cells are selected from the group consisting of: T cells, CD4+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphocytes, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, ESC-derived cells and iPSC-derived cells, optionally, wherein the cells are engineered to express effector molecules, optionally, wherein the engineered cells of claim 28, wherein the cells are natural killer (NK) cells, optionally, wherein the cells are allogeneic or autologous.

16. A pharmaceutical composition comprising the engineered cell according to any one of claims 26 to 31, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

17. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26 to 31, optionally wherein, The cancer includes 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. A method of enhancing immune cell-mediated killing of cancer cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26 to 31, optionally wherein, The cancer comprises a solid tumor, 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. A method for reducing off-target killing of healthy cells in a subject, the method comprising administering to the subject a therapeutically effective dose of the composition of claim 32 or the cell of any one of claims 26 to 31, wherein: The subject has been diagnosed with cancer, optionally wherein the cancer comprises a solid tumor, 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.

Images