Synthetic receptors for conditional activation of immune cells

Abstract

Methods are provided for activating recombinant immune cells expressing a synthetic receptor comprising an intracellular domain derived from killer cell immunoglobulin-like receptor 4 (KIR2DL4). The synthetic receptors described herein allow recombinant immune cells to be used for the treatment of cancer or infectious diseases in subjects in need thereof while limiting the overactivation of immune responses associated with conventional recombinant cell-based therapies. In various embodiments, disclosed herein are methods for activating recombinant immune cells expressing a synthetic receptor comprising: (a) an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a sequence of 10-29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), the method comprising binding an antigen to the antigen binding domain, thereby activating the recombinant immune cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 208,580, filed June 9, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on May 31, 2022, is named SRT-001WO_SL.txt, and is 536,567 bytes in size. [Background technology]

[0003] Synthetic immune receptors and recombinant cells expressing them are an exciting technology for personalized treatment of conditions such as cancer and infectious diseases. So-called "adoptive cell therapy" involves introducing a synthetic receptor (often a "chimeric antigen receptor" or "CAR") that targets a selected antigen into immune cells to generate the recombinant immune cells (often referred to as "CAR-T" or "CAR-NK" cells, depending on the type of immune cell selected). After injection into a subject, the recombinant immune cells selectively target diseased cells or pathogens.

[0004] Although CAR-based therapies have shown promise, especially in the treatment of B-cell malignancies, such therapies are not without risks. A particular limitation is the toxicity associated with overactivation of the immune response (e.g., cytokine release syndrome, neurotoxicity, and anaphylaxis). Therefore, new technologies are needed to reduce the toxicity associated with CAR-based therapies. One approach to limit the toxicity associated with CARs is the modulation of intracellular signaling dynamics associated with CAR activation. Conventional CAR molecules contain the CD3-zeta (CD3z) intracellular signaling domain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), which activates immune cells, such as T cells or NK cells, upon antigen binding to the CAR. However, robust activation of CD3z signaling can lead to overactivation of the immune response and resulting toxicity. Overactivation of immune cells can also lead to immune exhaustion, where immune cells lose activity against targets as the immune response persists.

[0005] One alternative approach is to replace the intracellular signaling domain of CD3z with an alternative domain that has reduced toxicity upon activation. Killer cell immunoglobulin-like receptors (KIRs) are immune receptors expressed by NK cells. KIRs may contain ITAMs or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) to regulate immune cell activation. Thus, KIR-based synthetic receptors may provide a promising alternative to conventional CD3z-based CARs for the treatment of diseases with reduced toxicity and less potential for exhaustion. Additional approaches include tailoring responses by combining KIR domains or portions thereof with CD3z domains or portions thereof to generate recombinant immune cells. Summary of the Invention [Means for solving the problem]

[0006] In various embodiments, disclosed herein are methods for activating a recombinant immune cell expressing a synthetic receptor comprising: (a) an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a sequence of 10-29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), the method comprising binding an antigen to the antigen-binding domain, thereby activating the recombinant immune cell.

[0007] In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9. In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 12-16. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 12. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 13. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 14. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 15. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 16.

[0008] In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0009] In some embodiments, the synthetic receptor further comprises a hinge region disposed between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 4 or 5. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 5.

[0010] In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:31.In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0011] In some embodiments, the antigen binding domain comprises an antibody or a functional fragment thereof. In some embodiments, the antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the antigen binding domain comprises a minimal active antibody fragment. In some embodiments, the antigen binding domain comprises a single domain antibody. In some embodiments, the antigen binding domain comprises a single light chain variable domain. In some embodiments, the antigen binding domain comprises a single heavy chain variable domain. In some embodiments, the antigen binding domain comprises a nanobody.

[0012] In some embodiments, the recombinant immune cell is a T cell, a natural killer (NK) cell, or a natural killer-type T (NK-T) cell. In some embodiments, the recombinant immune cell is a T cell. In some embodiments, the recombinant immune cell is a NK cell. In some embodiments, the recombinant immune cell is a NK-T cell.

[0013] In some embodiments, the antigen is a tumor associated antigen. In some embodiments, the antigen is a whole protein or a fragment thereof selected from the list consisting of CD19, mesothelin, CD123, BCMA, GD2, CD30, GPC3, CD22, HER2, CD20, EGFR, Flt3, CD33, Muc-16, CS1, and tumor neoantigens. In some embodiments, the antigen is CD19 or a fragment thereof. In some embodiments, the antigen is mesothelin or a fragment thereof. In some embodiments, the antigen is CD123 or a fragment thereof. In some embodiments, the antigen is BCMA or a fragment thereof. In some embodiments, the antigen is GD2 or a fragment thereof. In some embodiments, the antigen is CD30 or a fragment thereof. In some embodiments, the antigen is GPC3 or a fragment thereof. In some embodiments, the antigen is CD22 or a fragment thereof. In some embodiments, the antigen is HER2 or a fragment thereof. In some embodiments, the antigen is CD20 or a fragment thereof. In some embodiments, the antigen is EGFR or a fragment thereof. In some embodiments, the antigen is Flt3 or a fragment thereof. In some embodiments, the antigen is CD33 or a fragment thereof. In some embodiments, the antigen is Muc-16 or a fragment thereof. In some embodiments, the antigen is CS1 or a fragment thereof. In some embodiments, the antigen is a tumor neo-antigen. In some embodiments, the antigen is derived from an infectious agent. In some embodiments, the infectious agent is a bacterium, a virus, a fungus, or a parasite.

[0014] Also disclosed herein, in various embodiments, is a recombinant immune cell comprising an expressed synthetic receptor, the receptor comprising (a) an antigen binding domain, a transmembrane domain, and an intracellular signaling domain that comprises, or consists of, a sequence of 10-29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), wherein the recombinant immune cell is activated by binding of a cognate antigen to the antigen binding domain.

[0015] In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9. In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 12-16. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 12. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 13. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 14. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 15. In some embodiments, the intracellular signaling domain further comprises a costimulatory domain comprising an amino acid of SEQ ID NO: 16.

[0016] In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0017] In some embodiments, the synthetic receptor further comprises a hinge region disposed between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 4 or 5. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 5.

[0018] In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:31.In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an antibody or a functional fragment thereof. In some embodiments, the antigen binding domain comprises a single chain variable fragment (scFv).

[0019] In some embodiments, the antigen binding domain comprises a minimal active antibody fragment. In some embodiments, the antigen binding domain comprises a single domain antibody. In some embodiments, the antigen binding domain comprises a single light chain variable domain. In some embodiments, the antigen binding domain comprises a single heavy chain variable domain. In some embodiments, the antigen binding domain comprises a nanobody.

[0020] In some embodiments, the recombinant immune cell is a T cell, a NK cell, or a NK-T cell. In some embodiments, the recombinant immune cell is a T cell. In some embodiments, the recombinant immune cell is a NK cell. In some embodiments, the recombinant immune cell is a NK-T cell.

[0021] Also disclosed herein, in various embodiments, are methods of treating a disease in a subject in need thereof, comprising administering to the subject a recombinant immune cell as disclosed herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the recombinant cell is autologous to the subject. In some embodiments, the recombinant cell is allogeneic to the subject.

[0022] In some embodiments, the disease is cancer. In some embodiments, the cancer is a hematopoietic malignancy. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the antigen is a whole protein or a fragment thereof selected from the list consisting of CD19, mesothelin, CD123, BCMA, GD2, CD30, GPC3, CD22, HER2, CD20, EGFR, Flt3, CD33, Muc-16, CS1, and tumor neoantigens. In some embodiments, the antigen is CD19 or a fragment thereof. In some embodiments, the antigen is mesothelin or a fragment thereof. In some embodiments, the antigen is CD123 or a fragment thereof. In some embodiments, the antigen is BCMA or a fragment thereof. In some embodiments, the antigen is GD2 or a fragment thereof. In some embodiments, the antigen is CD30 or a fragment thereof. In some embodiments, the antigen is GPC3 or a fragment thereof. In some embodiments, the antigen is CD22 or a fragment thereof. In some embodiments, the antigen is HER2 or a fragment thereof. In some embodiments, the antigen is CD20 or a fragment thereof. In some embodiments, the antigen is EGFR or a fragment thereof. In some embodiments, the antigen is Flt3 or a fragment thereof. In some embodiments, the antigen is CD33 or a fragment thereof. In some embodiments, the antigen is Muc-16 or a fragment thereof. In some embodiments, the antigen is CS1 or a fragment thereof. In some embodiments, the antigen is a tumor neoantigen.

[0023] In some embodiments, the disease is an infectious disease caused by an infectious agent, hi some embodiments, the infectious agent is a bacterium, a virus, a fungus, or a parasite.

[0024] Also provided herein, in various embodiments, is a method of activating a recombinant immune cell expressing a synthetic receptor, the synthetic receptor comprising (a) a means for binding an antigen of interest, (b) a transmembrane domain, and (c) an intracellular signaling domain comprising a sequence of 10-29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), the method comprising binding the antigen of interest, thereby activating the recombinant immune cell.

[0025] Also provided herein, in various embodiments, is a recombinant immune cell comprising an expressed synthetic receptor, the receptor comprising (a) a means for binding an antigen of interest, (b) a transmembrane domain, and (c) an intracellular signaling domain that comprises, or consists of, a sequence of 10-29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), wherein the recombinant immune cell is activated by binding of a cognate antigen to the antigen binding domain. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows activation of Jurkat T receptor cells expressing recombinant receptors without (-Raji) or with (+Raji) exposure to cancer cells. Data are presented as scatter plots with flow cytometry analysis with mCherry, a surrogate measure of receptor transgene expression on the X-axis, and mCitrine, a gene expression reporter of T cell activation on the Y-axis. A shows cells expressing a synthetic receptor without an antigen binding domain. B shows cells expressing a control anti-CD19 CAR containing the CD3z intracellular signaling domain (ICD). C shows cells expressing a synthetic receptor containing the full-length KIR2DL4 ICD. Quadrants indicate gates showing cells expressing recombinant receptors (right two quadrants) and activated cells (top two quadrants). [Diagram 2]Bar graph showing log 2-fold change in mCitrine reporter expression as a measure of T cell activation (left) and diagram of synthetic receptor constructs showing N- and / or C-terminal truncations of the KIR2DL4 ICD (right). [Diagram 3] A bar graph of the log2 fold change (log2FC) of activation potential on the Y-axis is shown. The X-axis shows synthetic receptors including an anti-CD19 positive control CAR with a CD3z ICD, a negative control CAR without an ICD (no ICD CAR). Additional constructs expressed (1-5) include full-length KIR2DL4 transmembrane domain (TM) and ICD (#1), KIR2DL4 TM with KIR2DL4 ICD truncation A (#2), KIR2DL4 TM with KIR2DL4 ICD ITIM T37A Y38F substitution (#3), KIR2DL4 TM without an ICD (#4), and CD8a TM with KIR2DL4 ICD (#5). Error bars represent standard error. [Figure 4A] Figure 1 shows transduction and functional evaluation of primary T cells expressing the indicated recombinant receptors with CD19 scFv. Transduction efficiency was measured using FITC-conjugated CD19 protein and reported as MFI. Cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells. [Figure 4B] Figure 4 shows transduction and functional assessment of primary T cells expressing the indicated recombinant receptors with CD19 scFv. Figure 4 shows the readout of a short-term luciferase-based cytotoxicity assay for the transduced T cells of Figure 4A exposed to Raji target cells. Cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells. [Figure 4C] Figure 4 shows transduction and functional assessment of primary T cells expressing the indicated recombinant receptors with CD19 scFv. Figure 4 shows the readout of a repeat luciferase-based cytotoxicity assay for the transduced T cells of Figure 4A exposed to Raji target cells. Cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells. [Diagram 5]Figure 5 shows transduction and functional assessment of primary NK cells expressing the indicated recombinant receptors with CD19 scFv. A shows transduction efficiency measured using FITC-conjugated CD19 protein and reported as MFI. B shows measurement of CD107a in transduced NK cells from Figure 5A activated with Raji target cells. [Figure 6] Figure 1 shows activation of Jurkat T receptor cells expressing recombinant receptors modified to express mCitrine as a marker of NFAT-NFkB gene activation and represented with CD19 or MSLN scFv. A shows Jurkat T cell activation after exposure to Raji target cells expressing CD19. B shows Jurkat T cell activation after exposure to K562 target cells expressing MSLN. The percentage of cells expressing mCitrine is measured by flow cytometry. [Figure 7] Figure 1 shows transduction and functional assessment of primary T cells expressing the indicated recombinant receptors with BCMA scFv. A shows transduction efficiency as measured by expression of mScarlet fluorescent reporter protein. B shows cytotoxicity of K562-BCMA+ target cells induced by transduced T cells from A after 24 hours of incubation. The cytotoxic capacity of these CAR T cells was assessed by flow cytometry. [Figure 8] Figure 1 shows the transduction and functional evaluation of primary T cells expressing the indicated recombinant receptors with MSLN scFv. A shows the transduction efficiency as measured by the expression of mScarlet fluorescent reporter protein. B shows the cytotoxicity of K562-MSLN+ target cells induced by transduced T cells from A after 24 hours of incubation. The cytotoxic capacity of these CAR T cells was evaluated by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0028] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context requires otherwise.

[0029] The term "antigen" refers to a molecule (e.g., a peptide or protein) or an immunologically active fragment thereof that can elicit an immune response. Peptide antigens are typically presented to immune cells, such as T lymphocytes (also called T cells), by antigen-presenting cells (APCs).

[0030] As used herein, "antigen-specific" refers to an immune response generated in a host that is specific for a given antigen. This term includes responses to antigens that are recognized by antibodies that can bind to the antigen of interest with high affinity, and responses to antigens by T cell receptors (TCRs) that recognize and bind to complexes that contain MHC molecules and short peptides that are degradation products of the antigen of interest.

[0031] The term "fusion peptide or protein" or "fused peptide or protein" refers to a recombinant protein that contains two or more proteins or peptides expressed in the same amino acid chain in sequence. Two or more protein or peptide nucleic acid coding sequences can be expressed sequentially in a single open reading frame of a vector or expression plasmid. Thus, the resulting peptide or protein contains a single amino acid chain with two or more proteins of interest connected via end-to-end fusion at the N-terminus or C-terminus. As used herein, "delaying disease onset" means to postpone, prevent, delay, block, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be of different duration depending on the disease history and / or individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, can be delayed.

[0032] An "effective amount" of an agent, e.g., cells or pharmaceutical composition, in the context of administration, refers to an amount effective, at dosages / amounts and for periods of time necessary, to achieve the desired result, such as a therapeutic or prophylactic result.

[0033] The term percent "identity" or "sequence identity" with respect to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have the same specified percentage of nucleotide or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the entire length of the two sequences being compared.

[0034] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on the designated program parameters.

[0035] Optimal alignment of sequences for comparison can be performed, for example, by the local identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for identity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0036] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available via the website of the National Center for Biotechnology Information.

[0037] The term "pharmaceutical composition" refers to a preparation that is in a form such that the biological activity of the active ingredient contained in the preparation is effective, and does not contain any additional components that are unacceptably toxic to the subject to which the formulation will be administered.

[0038] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0039] "Preventing" as used herein includes providing prophylaxis against the occurrence or recurrence of a disease in a subject susceptible to the disease, but not yet diagnosed with the disease, hi some embodiments, the cells and compositions provided are used to delay the onset of the disease or to slow the progression of the disease.

[0040] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

[0041] As used herein, the term "recombinant" refers to a non-naturally occurring protein, cell, or organism. The term is used interchangeably with "engineered" or "modified."

[0042] As used herein, the terms "subject" or "patient" are used interchangeably and refer to an organism to which recombinant immune cells expressing a synthetic receptor are administered. The subject may be a mammalian subject, e.g., a human subject. In some embodiments, the subject has a disease, condition, or disorder. In some embodiments, the disease, condition, or disorder is characterized by the presence of a particular antigen that can be targeted by the synthetic receptor.

[0043] As used herein, to "inhibit" a function or activity is to reduce a function or activity when compared to the same conditions excluding the condition or parameter of interest, or alternatively, when compared to another condition. For example, a recombinant immune cell that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the recombinant immune cell.

[0044] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to the complete or partial alleviation or reduction of a disease or condition or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or primary palliation of the disease, and improvement of remission or prognosis. The term does not imply complete cure of the disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0045] A "therapeutically effective amount" of an agent, e.g., a cell or pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treating a disease, condition, or disorder of interest, and / or for a pharmacokinetic or pharmacodynamic effect. A therapeutically effective amount may vary depending on factors such as the condition, age, sex, and weight of the subject, as well as the population of cells administered. In some embodiments, the methods provided include administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount.

[0046] II. Synthetic Receptors Disclosed herein are recombinant immune cells and methods of activating recombinant immune cells by activating synthetic receptors. A "synthetic receptor" is a fusion protein that contains moieties from multiple different proteins that trigger a response when bound by a ligand. In some embodiments, the synthetic receptor is a chimeric antigen receptor (CAR). A "chimeric antigen receptor" is a synthetic receptor that mimics the activity of a TCR and can induce antigen-specific immune cell activation upon binding to an antigen. CARs recognize cell surface antigens independent of human leukocyte antigens (HLA) and use one or more signaling molecules to activate genetically modified immune cells for killing, proliferation, and cytokine production (Jena et al., 2010). CARs can be used to confer the specificity of monoclonal antibodies to immune cells, thereby allowing large numbers of specific immune cells to be generated, for example, for use in adoptive cell therapy. In some embodiments, CARs include a targeting domain that includes an intracellular signaling domain, a hinge domain, a transmembrane domain, and a ligand-binding region. The specificity of other CAR designs can be derived from the ligand of the receptor (e.g., a peptide) or from pattern recognition receptors such as Dectin. In some cases, molecules can be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and gene products that conditionally eliminate T cells upon addition of a cytokine receptor.

[0047] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described, for example, in International Patent Application Publication Nos. WO2000 / 14257, WO2013 / 126726, WO2012 / 129514, WO2014 / 031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002 / 131960, US2013 / 287748, US2013 / 0 Nos. 149337, 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, as well as those described in European Patent Application Nos. EP 2537416 and / or Sadelain. et al.,Cancer Discov.2013 April;3(4):388-398, Davila et al.(2013)PLoS ONE 8(4):e61338, Turtle et al.,Curr.Opin.Immunol.,2012 October;24(5):633-39, Wu et al.,Cancer,2012 March 18(2):160-75.

[0048] IIa. Intracellular signaling domains The targeting domain is generally linked to one or more intracellular signaling components, such as, in the case of CARs, a signaling component that mimics activation via an antigen receptor complex, such as a TCR complex, and / or a signal via another cell surface receptor. Thus, in some embodiments, the targeting (e.g., antigen binding) and transmembrane domains are linked to one or more intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the targeting domain. In one embodiment, one of the domains in the receptor is used, such as the transmembrane domain that naturally associates with the CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins and to minimize interactions with other members of the receptor complex.

[0049] In some embodiments, the intracellular signaling domain comprises one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ITIMs are protein domains found on the intracellular cytoplasmic domains of many immune receptors. ITIMs can inhibit cell activation by immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the intracellular signaling domains that comprise one or more ITIMs do not comprise ITAMs.

[0050] In various embodiments, the intracellular signaling domain of the synthetic receptors disclosed herein comprises the amino acid sequence of the intracellular signaling domain of KIR2DL4. Exemplary intracellular domains of, or derived from, Killer Cell Immunoglobulin-Like Receptor 2DL4 (KIR2DL4) are provided in Table 1. In various embodiments, the synthetic receptor comprises an intracellular signaling domain comprising the amino acid sequence provided in Table 1. [Table 1]

[0051] In some embodiments, the intracellular signaling domain comprises or consists of an amino acid sequence corresponding to the sequence of amino acids 263-377 of full-length KIR2DL4 (SEQ ID NO:6). In some embodiments, the intracellular signaling domain comprises the amino acid substitutions T37A and / or Y38F within the amino acid sequence of SEQ ID NO:6. In embodiments where the length of the intracellular signaling domain is less than the length of SEQ ID NO:6, the percent identity is defined with respect to the identity of the signaling domain to SEQ ID NO:6 over the length of the intracellular signaling domain.

[0052] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO:6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO:6.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 6. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO: 6.

[0053] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO:7.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 7. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO: 7.

[0054] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO:8.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 8. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO:8.

[0055] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO:9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO:9.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 9. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO: 9.

[0056] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO: 10.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO: 10.

[0057] In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 70% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 75% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 80% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 85% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 90% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 95% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 96% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 97% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 98% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99% identical to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.5% identical to SEQ ID NO:11.In some embodiments, the intracellular signaling domain comprises or consists of a segment having an amino acid sequence at least 99.9% identical to SEQ ID NO: 11. In some embodiments, the intracellular signaling domain comprises or consists of a segment having the amino acid sequence of SEQ ID NO:11.

[0058] In some embodiments, the intracellular signaling domain comprises or consists of a sequence of 10-29 amino acids. In some embodiments, the intracellular signaling domain comprises or consists of a sequence corresponding to, or having the essential percent identity to, amino acid sequence 263-272 of full-length KIR2DL4. In some embodiments, the percent identity is 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%. In some embodiments, the intracellular signaling domain comprises or consists of a sequence corresponding to, or having the essential percent identity to, amino acid sequence 263-291 of full-length KIR2DL4. In some embodiments, the percent identity is 90%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%.

[0059] KIR KIRs are a large family of receptors present on specific subsets of lymphocytes, including NK cells. KIR nomenclature is based on the number of extracellular domains (KIR2D or KIR3D) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). Within humans, the presence or absence of a given KIR is variable from one NK cell to another within an NK population present in a single individual. Within the human population, there are also relatively high levels of polymorphism of KIR molecules, with certain KIR molecules present in some but not all individuals. Certain KIR gene products cause stimulation of lymphocyte activity when bound to the appropriate ligand. All identified stimulatory KIRs have short cytoplasmic tails with charged transmembrane residues that associate with adaptor molecules with ITAMs, other than other KIR gene products that are inhibitory in nature. All identified inhibitory KIRs have long cytoplasmic tails and appear to interact with different subsets of HLA antigens, depending on the KIR subtype. Inhibitory KIRs exhibit one or more ITIMs in their cytoplasmic portion that recruit phosphatases. Known inhibitory KIR receptors include members of the KIR2DL and KIR3DL subfamilies. KIR receptors with two Ig domains (KIR2D) recognize epitopes shared by HLA-C allotypes: KIR2DL2 (formerly p58.2), or the closely related gene product KIR2DL3, recognizes an epitope shared by group 2 HLA-C allotypes (Cw1, 3, 7, and 8), while KIR2DL1 (p58.1) recognizes an epitope shared by reverse group 1 HLA-C allotypes (Cw2, 4, 5, and 6). Recognition by KIR2DL1 is determined by the presence of a Lys residue at position 80 of the HLA-C allele. Recognition by KIR2DL2 and KIR2DL3 is determined by the presence of an Asn residue at position 80. Importantly, the majority of HLA-C alleles have either an Asn or Lys residue at position 80. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4 alleles.Finally, a homodimer of a molecule containing three Ig domains, KIR3DL2 (p140), recognizes HLA-A3 and HLA-A11.

[0060] Inhibitory KIRs and other class I inhibitory receptors (Moretta et al., 1997; Valiante et al., 1997a; Lanier, 1998) can be coexpressed by NK cells, but in any given individual's NK repertoire there will be cells expressing a single KIR, and therefore the corresponding NK cells will only be blocked by cells expressing a particular set of class I alleles.

[0061] Costimulatory domain In various embodiments, the intracellular signaling domain further comprises a costimulatory domain. The costimulatory domain can provide additional signaling input to support immune cell activation in response to activation of the synthetic receptor. Further details regarding the function of the costimulatory domain are provided in Weinkove et al., Clin Transl Immunology. 2019;8(5):e1049.

[0062] In some embodiments, the costimulatory domain comprises an intracellular component of the TCR complex, such as, for example, a TCR CD3 chain that mediates T cell activation and cytotoxicity, such as the CD3 zeta (CD3z) chain. In some embodiments, the costimulatory domain comprises an intracellular component of an immune costimulatory molecule, such as CD28, 4-1BB, DAP10, or DAP12. Exemplary costimulatory domains are provided in Table 2. In various embodiments, the synthetic receptor comprises a costimulatory domain comprising an amino acid sequence provided in Table 2. [Table 2]

[0063] In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 96% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 97% identical to SEQ ID NO:12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 12. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.9% identical to SEQ ID NO: 12. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 12.

[0064] In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.9% identical to SEQ ID NO: 13. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 13.

[0065] In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 96% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 97% identical to SEQ ID NO:14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 14. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.9% identical to SEQ ID NO: 14. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 14.

[0066] In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 96% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 97% identical to SEQ ID NO:15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 15. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 99.9% identical to SEQ ID NO: 15. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 15.

[0067] IIb. Transmembrane domain In various embodiments, the synthetic receptor comprises a transmembrane domain. In some embodiments, the targeting domain and / or hinge domain are linked to one or more transmembrane domains. In some embodiments, the transmembrane domain comprises one or more positively charged amino acid residues. Exemplary transmembrane domains that may be used in the synthetic receptors disclosed herein are provided in Table 3. In various embodiments, the synthetic receptor comprises a transmembrane domain comprising an amino acid sequence provided in Table 3. [Table 3]

[0068] In some embodiments, the transmembrane domain comprises or consists of a sequence similar or identical to the sequence of the transmembrane domain of KIR2DL4, or a functional fragment thereof. In some embodiments, the transmembrane domain comprises or consists of a sequence similar or identical to the sequence of the transmembrane domain of CD8a. In some embodiments, the transmembrane domain comprises or consists of a sequence similar or identical to the sequence of the transmembrane domain of CD28.

[0069] In some embodiments, the transmembrane domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 70% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 75% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 80% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 85% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 95% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain comprises a segment having the amino acid sequence of SEQ ID NO:1.

[0070] In some embodiments, the transmembrane domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 70% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 75% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 85% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 90% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 95% identical to SEQ ID NO:2. In some embodiments, the transmembrane domain comprises a segment having the amino acid sequence of SEQ ID NO:2.

[0071] In some embodiments, the transmembrane domain comprises or consists of a segment having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 70% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 75% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 85% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 90% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having an amino acid sequence at least 95% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain comprises a segment having the amino acid sequence of SEQ ID NO:3.

[0072] In some embodiments, the recombinant receptor, such as a CAR, further comprises a spacer that may be, or may include at least a portion of, an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region, or a variant or modified version thereof. In some aspects, the portion of the constant region functions as a spacer region between the antigen recognition component, e.g., the scFv, and the transmembrane domain. The spacer may be of a length that results in increased responsiveness of the cell after antigen binding compared to the absence of the spacer. In some examples, the spacer is 12 amino acids long, about 12 amino acids long, or about 12 amino acids long or less. Exemplary spacers include spacers having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between any of the endpoints of the recited ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to a CH2 and CH3 domain, or an IgG4 hinge linked to a CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Application Publication No. WO2014 / 031687, U.S. Patent No. 8,822,647, or published application US2014 / 0271635.

[0073] IIc. Targeting Domains In various embodiments, the synthetic receptor comprises a targeting domain. The targeting domain is capable of binding to a ligand of interest and inducing a conformational change that results in receptor activation. In some embodiments, the targeting domain is an antigen-binding domain. The antigen-binding domain is capable of binding to an extracellular antigen with high specificity. In some embodiments, the antigen-binding domain comprises an antibody or a functional fragment thereof. As used herein, the term "antigen-binding domain" is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, as well as other moieties that are capable of specifically binding to a target antigen.

[0074] In various embodiments, the antigen binding domain is derived from an immunoglobulin. In some embodiments, the antigen binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the antigen binding site is a single domain (single domain antibody or sdAb), e.g., an antigen binding site derived from a camelid antibody, e.g., a camelid antibody (V NAR ), or a nanobody (V H H). The antigen binding domain may be in any suitable format, including antigen binding fragments (Fab), F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments (including single chain variable fragments (scFv)), and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. Antigen binding domains may also include engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and multispecific, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, heteroconjugate antibodies such as tandem di-scFv, tandem tri-scFv.

[0075] In various embodiments, the antigen-binding site comprises a set of complementarity determining regions (CDRs). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and there are four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

[0076] The exact amino acid sequence boundaries of a given CDR can be determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (the “Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (the “Contact” numbering scheme), Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January;27(1):55-77 (the "IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 June.8;309(3):657-70, (the "Aho" numbering scheme).

[0077] The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes numbering is based on the most common antibody region sequence length, with insertions accommodated by an insertion letter, e.g., "30a", and deletions found in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0078] In various embodiments, the antigen-binding domain is not derived from an immunoglobulin. Non-limiting examples of non-immunoglobulin antigen-binding domains include affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, peptide aptamers, designed ankyrin repeat proteins (DARPins), monobodies, nanoCLAMPs, endogenous ligands or fragments thereof, small molecules, sugars, lipids, peptides, and any other suitable moiety that specifically and / or selectively binds to a target antigen of interest.

[0079] The present invention may include any suitable antigen binding domain that currently exists or that may be developed in the future.

[0080] A non-limiting list of exemplary antigen binding domains suitable for the synthetic receptors disclosed herein are provided as SEQ ID NOs: 35-277. Table 4 identifies the target antigens bound by each antigen binding domain provided in SEQ ID NOs: 35-277. In various embodiments, the synthetic receptor comprises an antigen binding domain comprising an amino acid sequence provided in Table 4. [Table 4]

[0081] In some embodiments, the antigen-binding domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 99.5% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 99.9% identical to an amino acid sequence selected from any one of SEQ ID NOs: 35-277. In some embodiments, the antigen-binding domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-277.

[0082] In some embodiments, the targeting domain binds to a regulatory domain of another protein inside the cell or on the cell surface, hi some embodiments, the targeting domain is intracellular and binds to the intracellular domain of a receptor.

[0083] IId. Hinge domain In various embodiments, the synthetic receptor comprises a hinge domain. The hinge domain can link the extracellular portion (e.g., targeting domain) of the synthetic receptor to the transmembrane portion and the intracellular portion. In addition to its structural role within the protein, the hinge domain can affect the activity of the synthetic receptor. Exemplary hinge domains that can be used in the synthetic receptors disclosed herein are provided in Table 5. In various embodiments, the synthetic receptor comprises a hinge domain comprising an amino acid sequence provided in Table 5. [Table 5]

[0084] In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:4 or 5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4 or 5. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0085] In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO:4.

[0086] In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO:5.

[0087] IIe. Full-length receptors and receptor fragments Table 6 shows exemplary sequences of fragments of synthetic receptors that can be operably linked to any targeting domain described herein or known in the art. In some embodiments, the synthetic receptor comprises, from N-terminus to C-terminus, a targeting domain, a hinge, a transmembrane domain (TM), and an intracellular signaling domain (ICD). In various embodiments, the synthetic receptor comprises an amino acid sequence provided in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0088] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-34.

[0089] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:17. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:17.

[0090] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:18.

[0091] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:19. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:19.

[0092] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:20.

[0093] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:21.

[0094] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:22.

[0095] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:23.

[0096] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:24. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:24.

[0097] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:25. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:25.

[0098] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:26.

[0099] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:27.

[0100] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:28. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:28.

[0101] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:29.

[0102] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:30.

[0103] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:31.

[0104] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:32.

[0105] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:33.

[0106] In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the synthetic receptor comprises the amino acid sequence of SEQ ID NO:34.

[0107] III. Recombinant immune cells Disclosed herein are activated recombinant immune cells expressing synthetic receptors and methods for their activation. An "activated immune cell" is an immune cell that has encountered an antigen presented by an "antigen presenting cell" (or "APC"). The APC presents the antigen to the immune cell via major histocompatibility complex (MHC) proteins. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC loci code for two major classes of MHC membrane molecules, termed class I and class II MHC. "Professional APCs" (e.g., dendritic cells, mononuclear phagocytes, or B cells) express MHC class II, while non-professional APCs, which can be any nucleated cell that is not a "professional APC," express MHC class I. Upon encountering an APC, the immune cell enters an "activated state," and the activated immune cell can directly induce cell death in a target cell or promote the function of other immune cells (e.g., by releasing cytokines into the extracellular environment). An activated immune cell is contrasted with an "inactive immune cell." Inactive immune cells do not affect target cells or other immune cells. Inactive immune cells may be "naive immune cells", meaning that the immune cells have never encountered an antigen. Inactive immune cells may also be previously active immune cells that have been inactivated (e.g., by encountering "regulatory" immune cells).

[0108] IIIa. Types of immune cells T cells In some embodiments, the recombinant immune cell can be a T cell. T cells are typically activated by effective interaction between the T cell receptor (TCR) complex and the major histocompatibility (MHC) antigen complex on the surface of a target cell. In some cases, immune cell activation against a target cell requires costimulation. For example, T cell activation can require stimulation by both interaction between the TCR and the MHC antigen complex (signal 1) and interaction between one or more co-receptors and one or more target cell antigens (signal 2). Failure to provide signal 2 can result in T cell anergy / tolerance or apoptosis (Macian et al, Curr Opin Immunol, 16(2):209-216, 2004; Pardigon et al, J Immunol, 164(9):4493-4499, 2000; Ward, Biochem J, 318(Pt 2):361-377, 1996; Zhong et al, Mol Ther, 18(2):413-420, 2010).

[0109] CD8 + T cells In some embodiments, the T cells are CD8 positive "cytotoxic" T cells. Cytotoxic T cells recognize and kill target cells that display peptide fragments presented on the cell surface in the context of MHC-I molecules. Cytotoxic T cells store preformed cytotoxins in lytic granules that fuse with the membrane of target cells. CD8+ cytotoxic T cells further express Fas ligand (FasL), which induces apoptosis in Fas-expressing target cells.

[0110] CD4 + T cells In some embodiments, the T cells are CD4 positive "T helper" (T H ) cells. H T cells function to regulate B cell proliferation and B cell responses. H CD4 cells play an important role in humoral immunity and immunopathology. + T helper cells are H 1 and T HThey differentiate into one of two types of cells, both of which express CD4 and recognize peptide fragments that are processed in intracellular vesicles and presented on the cell surface in the context of MHC-II molecules. H T cells can directly or indirectly activate several other immune cells, including macrophages and B cells, thereby promoting more efficient destruction and clearance of intracellular microorganisms. H T cells may also be involved in pathways that lead to the activation of CD8 cytotoxic T cells (e.g., dendritic cell "licensing"). H T cells stimulate the differentiation of B cells and promote the production of antibodies and other effector molecules of the humoral immune response. H In response to antigenic stimulation and the cytokine environment, T cells H 1 or T H T helper cells, which are initially activated by antigen in the presence of IL-12, can differentiate into T H 1 cells, but those activated in the presence of IL-4 develop primarily into T H Precursor T helper cells develop into T H 1 or T H It may take several cell divisions before one is able to synthesize cytokines that represent either of the two pathways. H 1 and T H The two cell phenotypes differ from each other in the early activation signaling pathways, particularly in the role of TCR-associated protein tyrosine kinases. TCR and its downstream protein tyrosine kinases, such as Fyn, p56(Ick), and ZAP-70, mediate T cell activation and proliferation. H 1 and T H 2 It is involved in cell development and differentiation.

[0111] T H 1 and T H Both cells express CD4 and recognize peptide fragments that are processed in intracellular vesicles and presented on the cell surface in the context of MHC-II molecules. HT cells can activate several other immune cells, including macrophages and B cells, thereby promoting more efficient destruction and clearance of intracellular microorganisms. H 1 cells also express CD8 + They may be involved in pathways that lead to the activation of cytotoxic T cells (e.g., dendritic cell "licensing"). H 2 cells stimulate the differentiation of B cells and promote the production of antibodies and other effector molecules of the humoral immune response.

[0112] T H 17 cells are pro-inflammatory T cells that express IL-17. H A subset of cells. H 17 cells are T H 1 and T H It is developmentally distinct from the 2-cell type. H Cell T H The signaling pathway that induces differentiation into 17 cells is T reg Inhibits differentiation.

[0113] Follicular T helper cells (T FH ) is CD4 + Follicular T helper cells are a subset of cells that help cognate B cells form and maintain germinal center (GC) responses and are essential for developing humoral immune responses. These cells are broadly defined by their expression of the chemokine receptor CXCR5, which guides follicular B cells through a gradient of the chemokine CXCL131. T FH The cells also express the transcription factor Bcl6 (which represses Blimp-1 / Prdm1) and high levels of the costimulatory receptor ICOS, both of which are important for their differentiation and maintenance. FH T cells secrete large amounts of IL-21, which supports GC formation, isotype switching, and plasma cell formation. FH The cells can be found in secondary lymphoid organs in humans and mice. FH The cells are also present in peripheral blood and are found at elevated levels in individuals with autoantibodies.

[0114] NK-T cells In some embodiments, the T cells are natural killer T ("NK-T") cells. Natural killer T cells represent a subset of T lymphocytes with unique properties. NK-T cells, unlike functionally differentiated conventional αβ T cells, share properties of both natural killer cells and T cells, and upon stimulation with a ligand, become T H Type 1 and T H NK-T cells can rapidly generate both type 2 and type 3 responses (innate immunity). Paradoxically, activation of NK-T cells can lead to either suppression or stimulation of immune responses. For example, T H 1 Cytokine production is thought to promote cellular immunity with antitumor, antiviral / antibacterial, and adjuvant activities; however, T H 2The production of cytokines is thought to suppress autoimmune diseases and promote antibody production.

[0115] Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds to autologous and foreign lipids and glycolipids. The TCR of NK-T cells can recognize glycolipid antigens presented (chaperoned) by CD1 d molecules. The primary response of NK-T cells is the rapid secretion of cytokines, including IL-4, IFN-γ, and IL-10, after stimulation, thus influencing diverse immune responses and pathogenic processes. NK-T cells can be homogeneous or heterogeneous populations. In one exemplary implementation, the population can be "non-invariant NK-T cells," which can include, for example, human and mouse bone marrow and human liver T cell populations, which are CD1d-reactive non-invariant T cells that express diverse TCRs and can also produce large amounts of IL-4 and IFN-γ. The best-known subset of CD1d-dependent NK-T cells expresses the invariant TCR-alpha (TCR-t) chain. These are referred to as type I or invariant NK-T cells (iNK-T cells). These cells are conserved between humans (Vα24i NK-T cells) and mice (Vα14i NK-T cells) and are involved in many immunological processes.

[0116] γδ T cells CD4 + and CD8 + The main population, including the subset, expresses receptors composed of alpha and beta chains. A small subset expresses TCRs made of gamma and delta chains. In some embodiments, the T cells are gamma delta (γδ) T cells. Gamma delta (γδ) T cells constitute 3-10% of circulating lymphocytes, and the Vδ2+ subset constitutes 75% of γδ-T cells in the blood. Vδ2+ cells recognize non-peptide epitopes and do not require antigen presentation by MHC or HLA. The majority of Vδ2+ T cells also express the Vγ9 chain and are stimulated by exposure to mevalonate and 5-carbon pyrophosphate compounds that are intermediates in the non-mevalonate sterol / isoprenoid synthesis pathway. Responses to isopentenyl pyrophosphate (5-carbon) are universal among healthy humans. Another subset of γδ-T cells, Vδ+, constitutes a much smaller percentage of T cells circulating in the blood, while Vδ+1 cells are commonly found in epithelial mucosa and skin.

[0117] In general, γδ-T cells have several functions, including killing tumor cells and pathogen-infected cells. Stimulation through their unique TCR, which consists of two glycoprotein chains, γ and δ, improves the capacity for cytotoxicity, cytokine secretion, and other effector functions. The TCR of γδ-T cells has unique specificity, and the cells themselves arise at high clonal frequency, allowing for rapid innate-like responses against tumors and pathogens.

[0118] NK cells In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subpopulation of lymphocytes involved in non-conventional immunity. Characteristics and biological properties of NK cells include expression of surface antigens including CD16, CD56, and / or CD57, absence of alpha / beta or gamma / delta TCR complexes on the cell surface, ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activation of specific cytolytic enzymes, ability to kill tumor cells or other diseased cells that express NK activating receptor ligands, ability to release cytokines that stimulate or inhibit immune responses, and ability to undergo multiple cell divisions to produce daughter cells with similar biological properties to the parent cell. In the context of the present invention, "active" NK cells refer to biologically active NK cells, more specifically, NK cells that have the ability to lyse target cells. For example, "active" NK cells are capable of killing cells that express NK activating receptor ligands and do not express "self" MHC / HLA antigens (KIR-mismatched cells).

[0119] NK cells are negatively regulated by major histocompatibility complex (MHC) class I specific inhibitory receptors (Karre et al., 1986; Ohlen et al., 1989). These specific receptors bind to polymorphic determinants of MHC class I molecules or HLA present on other cells and inhibit NK cell lysis.

[0120] IIIb. Immune cell isolation Immune cells can be isolated using any suitable method known in the art. For example, T cells can be isolated from human peripheral blood mononuclear cells (PBMCs), PBMCs collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. NK cells can be isolated from human PBMCs or umbilical cord blood. Immune cells can also be differentiated from stem or progenitor cells, such as, for example, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or hematopoietic progenitor cells (HPCs). In some embodiments, the immune cells are primary cells isolated from a patient prior to manipulation. In some embodiments, the immune cells are derived from established cell lines expanded ex vivo. Non-limiting examples of cell lines for use in the present invention include NK-70 NK cells and Jurkat T cells.

[0121] In some embodiments, the isolated immune cells are autologous. "Autologous" cells are administered to the same subject from which they were isolated. In some embodiments, the isolated immune cells are allogeneic. "Allogeneic" (or "xenogeneic") cells are administered to a subject different from the one from which they were isolated. The isolated immune cells can be expanded ex vivo for periods ranging from one or more days to several weeks or more.

[0122] IIIc. Expression of synthetic receptors in immune cells The synthetic receptors disclosed herein can be made using methods known to those of skill in the art. In some embodiments, the isolated immune cells contain a nucleic acid encoding the synthetic receptor sequence described herein and are known as "recombinant" cells. In some embodiments, the nucleic acid is an RNA molecule that is translated into a synthetic receptor protein. In some embodiments, the nucleic acid is a DNA molecule that encodes the synthetic receptor protein (i.e., the DNA is transcribed into mRNA and the mRNA is translated into the synthetic receptor protein). In some embodiments, the nucleic acid sequence further comprises a "signal sequence" that localizes the synthetic receptor to the surface of the cell. In some embodiments, the signal sequence can be an endogenous signal sequence of the N-terminal component of the synthetic receptor. In some embodiments, the signal sequence can be a different signal sequence than the signal sequence of the synthetic receptor. However, the signal sequence selected should be compatible with the secretory pathway of the immune cell so that the receptor is presented on the cell surface.

[0123] In some embodiments, nucleic acid is delivered to immune cells using a vector that contains the nucleic acid sequence that codes for synthetic receptor.Nucleic acid vector can be designed to deliver desired gene to immune cells under the control of regulated eukaryotic promoter, such as MNDU3 promoter, CMV promoter, EF1 alpha promoter, or ubiquitin promoter.Vector can also contain selectable marker, if not for other reasons, to facilitate their manipulation in vitro.

[0124] The nucleic acid or nucleic acid vector can be delivered into the isolated immune cells using any suitable method known to those skilled in the art. In one non-limiting example, a nucleic acid vector containing a nucleic acid sequence encoding a synthetic receptor can be designed to produce a virus. The virus can be produced using a packaging cell line and then contacted with the isolated immune cells to cause the isolated immune cells to express the synthetic receptor protein. Non-limiting examples of suitable viruses include retroviruses, lentiviruses, and adenoviruses. In another non-limiting example, the nucleic acid or nucleic acid vector can be delivered into the isolated immune cells using a transfection reagent. The transfection reagent allows the nucleic acid to penetrate the cell membrane where it is transcribed and / or translated to produce the synthetic receptor. Non-limiting examples of transfection reagents include cationic polymers (e.g., polyethyleneimine), cationic lipids (e.g., lipofectamine 2000), and other reagents such as FuGENE (Fugent, LLC., Madison, WI, USA) or calcium phosphate.

[0125] Immune cells used in the present invention may be expanded prior to administration to a subject using any suitable method known to one of skill in the art. In some embodiments, the immune cells are expanded prior to expression of the synthetic receptor. In some embodiments, the immune cells are expanded after expression of the synthetic receptor. In some embodiments, expansion of the recombinant immune cells occurs following their activation.

[0126] IIId. Activation of recombinant immune cells Recombinant immune cells expressing the synthetic receptors described herein can be activated using any suitable method known to those skilled in the art. For example, recombinant immune cells can be contacted with antigen-presenting cells (APCs). In some embodiments, recombinant immune cells can be activated ex vivo, for example, by contacting them with APCs that present the antigen recognized by the expressed synthetic receptor. In some embodiments, naive recombinant immune cells can be administered to a subject to be activated in vivo.

[0127] Professional antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of MHC class II proteins. In contrast, all nucleated vertebrate cells express MHC class-I molecules and can also function as APCs. APCs internalize antigens and re-express some of the antigen along with MHC molecules on their outer cell membrane. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is referred to as the HLA complex, and in mice, the H-2 complex.

[0128] IV. Treatment with Recombinant Immune Cells IVa. Subjects and diseases As detailed herein, the CAR immune cells of the embodiments can be used to treat a wide range of diseases and conditions in a subject. Essentially any disease with specific or enhanced expression of a particular antigen can be treated by targeting CAR cells to the antigen. For example, infectious diseases and cancers can be treated with the methods and / or compositions of the present invention. These include cancers such as primary, metastatic, recurrent, sensitive-to-therapy, and refractory-to-therapy cancers (e.g., chemo-refractory cancers). The cancers can be blood, lung, brain, colon, prostate, breast, liver, kidney, stomach, cervix, ovary, testis, pituitary, esophagus, spleen, skin, bone, and the like (e.g., B-cell lymphoma or melanoma). For cancer treatment, CAR cells typically target cancer cell antigens (also known as tumor-associated antigens (TAA)).

[0129] IVb. Tumor antigens In some embodiments, the targeting domain of the synthetic receptor (which may be referred to as the antigen binding domain) is designed to target a tumor-associated antigen. The TAA may be of any type, so long as it is expressed on the cell surface of the tumor cells. Non-limiting exemplary embodiments of tumor-associated antigens include CD19, mesothelin, CD123, BCMA, GD2, CD30, GPC3, CD22, HER2, HER3, CD20, EGFR, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, c-Met, AKT, epithelial tumor antigen, melanoma-associated antigen, Flt3, CD33, Muc-16, CS1, and tumor-associated neoantigens. A "neoantigen" is a mutant protein that is present in some or all cells of a tumor, but not in non-tumor cells.

[0130] In some embodiments, intracellular tumor-associated antigens, such as HA-1, survivin, WT1, and p53, may be targeted. This can be achieved, for example, by synthetic receptors expressed on universal T cells that recognize processed peptides written from intracellular tumor-associated antigens in the context of HLA. In addition, universal T cells can be genetically modified to express T cell receptor pairs that recognize intracellular processed tumor-associated antigens in the context of HLA.

[0131] In some embodiments, the synthetic receptor can be co-expressed with a membrane-bound cytokine to improve persistence when the amount of tumor-associated antigen is low. For example, a CAR can be co-expressed with membrane-bound IL-15.

[0132] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematopoietic malignancy. In some embodiments, the cancer is a cancer including, but not limited to, epithelial neoplasms, squamous cell neoplasms, squamous cell carcinomas, basal cell neoplasms, basal cell carcinomas, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (adenomas), adenomas, adenocarcinomas, gastritis plastica insulinomas, glucagonomas, gastrinomas, VIP-producing tumors, cholangiocarcinomas, hepatocellular carcinomas, adenoid cystic carcinomas, carcinoid tumors of the appendix, prolactinomas, oncocytomas, Hurthle cell adenomas, renal cell carcinomas, Grabitz tumors, multiple endocrine tumors, endometrioid adenomas, skin and adnexal neoplasms, mucoepidermoid neoplasms, cystic , mucinous and serous neoplasms, cystadenoma, pseudomyxoma peritonei, ductal, lobular and myeloid neoplasms, acinar cell neoplasms, composite epithelial neoplasms, Warthin's tumor, thymoma, specialized sex gland neoplasms, sex cord-stromal tumors, theca cell tumors, granulosa cell tumors, androgenic tumors, Sertoli-Leydig cell carcinoma, glomus tumors, paragangliomas, pheochromocytoma, glomus tumors, nevi and melanomas, melanocytic nevi, malignant melanoma, melanoma, nodular melanoma, dysplastic nevi, lentigo maligna, superficial spreading melanoma, malignant acral lentigo melanoma, Askin tumor, botryoid sarcoma, cartilage Sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, germinoma, dysgerminoma, seminoma, non-germinomatous germ cell tumor, embryonal carcinoma, germinal sinus tumor, choriomas, teratomas, polyembryoma, gonadoblastoma, nephroblastoma, medulloblastoma, retinoblastoma and / or sarcomas, including sarcomas of the lip, pharynx, hypopharynx, tongue, salivary gland, stomach, adenocarcinoma, thyroid (medullary and papillary thyroid), kidney, renal parenchymal, cervical, uterine, endometrial, choriocarcinoma, testicular, bladder, melanoma, glioblastoma, astrocytic tumor, brain tumors such as meningioma, medulloblastoma, and peripheral neuroectodermal tumors, gallbladder, lung, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, and plasmacytoma.Hematopoietic malignancies include chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (also called Malt lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell These include, but are not limited to, large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia / lymphoma, extranodal NK / T cell lymphoma, nasal type, enteropathy type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30 positive T cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified anaplastic large cell lymphoma, classical Hodgkin lymphoma (nodular sclerosis, mixed cytology, lymphocyte rich, lymphocyte depleted or not depleted), and nodular lymphocyte predominant Hodgkin lymphoma.

[0133] IVc. Infectious disease antigen In some embodiments, the targeting domain of the synthetic receptor is designed to target an antigen from an organism (i.e., an infectious agent) that causes an infectious disease. In some embodiments, the infectious agent is a bacterium, a virus, a fungus, or a parasite. In some embodiments, the infectious disease is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. In some embodiments, the infectious agent is a bacterium. Examples of bacteria that cause infections include Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Enterobacter sakazakii, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi and Salmonella typhimurium, Shigella sonnei, StaphylococcusExamples of bacteria that may be present include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, Yersinia enterocolitica, and Yersinia pestis.

[0134] In some embodiments, infectious agent is virus.The example of virus that causes infection includes but is not limited to Adenoviridae, Picornoviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Coronaviridae, Papillomaviridae, Reoviridae, Hepaviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomaviridae, Pneumoviridae, Filoviridae, Parvoviridae, Poxviridae, Rhabdoviridae, Togaviridae.

[0135] In some embodiments, the infectious agent is a fungus. Examples of fungi that cause infection include, but are not limited to, Aspergillus, Candida, Cryptococcus, Fusarium, Histoplasma, Basidiobolus, Conidiobolus, Pneumocystis, Mucor, Rhizopus, Absidia, Cunninghamella, Trichophyton, Microsporum, Epidermophyton, and Stachybotrys.

[0136] In some embodiments, the infectious agent is a parasite (eg, a protozoan, an amoeba). Examples of parasites causing infections include, but are not limited to, Acanthamoeba, Balamuthia mandrillaris, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Dientamoeba fragilis, Endolimax nana, Entamoeba dispar, Entamoeba hartmanni, Entamoeba histolytica, Entamoeba coli, Entamoeba moshkovskii, Giardia lamblia, Iodamoeba butschlii, Leishmania aethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Naegleria fowleri, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Sappinia diploidea, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi Taenia solium, Taenia saginata, Hymenolepis., Echinococcus granulosus, Echinococcus multilocularis, Multiceps multiceps, Schistosoma mansoni, Schistosoma japonicum, Fasciola hepatica, Ascaris, Enterobius, Filarioidea, Onchocerca, Rhabditis, Trichuris, Necator americanus, and Ancylostoma. .

[0137] IVd. Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are provided herein that comprise recombinant immune cells that express synthetic receptors. Pharmaceutical compositions generally include one or more of any pharma- ceutically acceptable carriers or excipients. In some embodiments, the compositions include at least one additional therapeutic agent.

[0138] In various embodiments, the choice of carrier is determined, in part, by the particular cell and / or method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, for example, buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, and the like. Examples of suitable surfactants include, but are not limited to, proteins such as albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, counterion forming salts such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0139] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known to those skilled in the art. Exemplary methods are described in detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0140] The formulation or composition may contain two or more active ingredients useful for the particular indication, disease, or condition being treated, which have complementary activities to the active recombinant cells, preferably binding molecules or cells, and where the activities of each do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition may further comprise, for example, a chemotherapeutic agent (e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.), an antibiotic agent (e.g., penicillin, cephalosporin, polymyxin, rifamycin, lipiarmycin, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, etc.), an antiviral agent (e.g., abacavir, adefovir, baloxavir ... Other pharmaceutically active agents or drugs, such as marboxil, cobicistat, doravirine, efavirenz, etravirine, imiquimod, methisazone, nitazoxanide, oseltamivir, peramivir, remdesivir, umifenovir, valacyclovir, valganciclovir, zanamivir, etc.), antifungal agents (e.g., polyenes, azoles, arylamines, echinocandins, etc.), or antiparasitic agents (e.g., nitazoxanide, malarsoprol, eflornithine, metronidazole, tinidazole, miltefosine, mebendazole, pyrantel pamoate, albendazole, praziquantel, rifampin, etc.). In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharma- ceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and from organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, e.g., p-toluenesulfonic acid.

[0141] In some embodiments, the pharmaceutical composition contains the active recombinant immune cells in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or more, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other administration regimes may be useful and can be determined. The desired dose can be delivered by a single bolus administration of the composition, multiple boluses of the composition, or continuous infusion administration of the composition.

[0142] In some embodiments, the composition is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0143] Sterile injectable solutions can be prepared by incorporating the binding molecule in a suitable carrier, diluent, or solvent, such as a mixture with excipients such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, etc., depending on the desired preparation. Standard texts can be consulted in some embodiments to prepare suitable preparations.

[0144] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0145] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0146] IVe. Medication and Administration Also provided herein, in various embodiments, are methods and uses of active recombinant cells expressing synthetic receptors. Such methods and uses include, for example, therapeutic methods and uses involving administration of the cells or compositions comprising the cells to a subject having a disease, condition, or disorder. In some embodiments, the cells and / or compositions are administered in an effective amount to effect treatment of the disease or disorder. Uses include such methods and treatments, as well as the use of the cells in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the methods are carried out by administering the cells or compositions comprising the cells to a subject having or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0147] Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T cell therapy methods are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., U.S. Patent No. 4,690,915 to Rosenberg et al., Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933, Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9, Davila et al. (2013) PLoS ONE 8(4):e61338.

[0148] Recombinant cells may be administered using standard administration techniques, formulations, and / or devices. Formulations and devices such as syringes and vials for storing and administering the compositions are provided. Administration of cells may be autologous or xenogeneic. For example, immunoresponsive cells or precursors may be obtained from one subject and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) may be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells), the therapeutic composition is generally formulated in a unit dose injectable form (solution, suspension, emulsion).

[0149] In some embodiments, in the context of recombinant cells expressing a synthetic receptor, a subject is administered a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, or a range defined by any two of the foregoing values). 0 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the aforesaid values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges, and / or such number of cells per kilogram of the subject's body weight are administered.

[0150] In some embodiments, the recombinant cells are administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell population is administered to a subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery. EXAMPLES

[0151] Example 1 - Activation of T cells by KIR2DL4-based synthetic receptors A chimeric antigen receptor (CAR) containing the transmembrane (TM) and intracellular signaling domain (ICD) of KIR2DL, together with a negative control CAR without the antigen-binding domain and a positive control CAR with the CD3z signaling domain, was transduced into four Jurkat T cells using lentiviral vectors. The transduced cells were then cultured for 2 days before a portion of the cells was harvested and stimulated by co-culturing with naturally CD19-expressing Raji cells at a 3:1 ratio (Raji:Jurkat). Both unstimulated and stimulated cells were then cultured for an additional 24 hours before flow cytometry analysis. Expression of the integrated lentiviral transgene-encoded receptor was measured as the fluorescence of the mCherry protein expressed from the same vector. Immune activation was measured as the fluorescence of the NFAT-NFkB gene reporter, which upon immune stimulation leads to the expression of the mCitrine-PEST short-lived fluorescent protein.

[0152] As shown in Figure 1A, cells expressing a CAR lacking the antigen-binding domain were not activated by exposure to CD19-expressing cells. Cells expressing a positive control CD3z-containing CAR were robustly activated after exposure to Raji cells (Figure 1B). Finally, cells expressing a KIR2DL-based CAR were also robustly activated after exposure to Raji cells. These results indicate that KIR2DL4-based CARs can promote antigen-specific activation of T cells.

[0153] Example 2 - Characterization of the KIR2DL4 intracellular signaling domain To determine the portions of the KIR2DL4 ICD that are necessary and / or sufficient for immune cell activation, 9 C-terminal, 7 N-terminal, and 6 N- and C-terminal KIR2DL4 ICD truncation mutants were generated based on predicted sequence features by progressively removing 6-20 amino acids. The ITIM motif in KIR2DL4 (VTYAQL) is present at positions 36-41 of the full-length ICD (SEQ ID NO:2). Further mutants, including synthetic receptors containing the truncation mutants, were then transduced into Jurkat T cells via lentiviral vectors. These transduced cells were then cultured for 2 days before a portion of the cells was harvested and mixed with native CD19-expressing Raji cells at a 3:1 ratio (Raji:Jurkat). Both unstimulated and stimulated cells were cultured for an additional 24 hours before flow cytometry analysis.

[0154] As shown in Figure 2, a synthetic receptor containing a C-terminal truncation mutant (C9: "truncated A" or "#2CAR") containing the first 29AA of the ICD had a strong ability to activate T cells. Furthermore, a CAR containing a KIR2DL4 ICD lacking the first 10 amino acids resulted in a reduced ability to induce T cell activation (N1). These data indicate that the combination of some parts of the ICD 10-29 amino acid long N-terminal region is essential for the immune activation function of the synthetic receptor.

[0155] The essential parts of the KIR2DL4-based synthetic receptors are further characterized. A negative control CAR lacking an ICD (No ICD CAR), a positive control CAR with a CD3z ICD, and multiple variants of the synthetic receptor, including a CAR with full-length KIR2DL4 TM-ICD (#1CAR), KIR2DL4 TM with KIR2DL4 ICD truncation A (#2CAR), KIR2DL4 TM with KIR2DL4 ICD ITIM substitution mutant (#3CAR), KIR2DL4 TM without an ICD (#4CAR), and CD8a TM with KIR2DL4 ICD (#5CAR). These synthetic receptors were then transduced into Jurkat T cells via lentiviral vectors. The transduced cells were then cultured for 2 days before being stimulated by co-culturing with naturally CD19-expressing Raji cells at a ratio of 3:1 (Raji:Jurkat). The stimulated cells were cultured for an additional 24 hours before FACS analysis. 100,000 each of the highest (positive) and lowest activated cells were isolated and next generation sequencing was used to identify the construct contained in these cells. The proportion of constructs in the positive and negative populations of cells is expressed as log2 fold change. Experiments were performed in three independent replicates.

[0156] As shown in Figure 3, synthetic receptors containing the KIR2DL4 TM domain and either the full-length KIR2DL4 ICD, the first 29 amino acids, or the full-length ICD with mutations in the ITIMs have a strong ability to activate T cells. Completely removing the ICD and leaving only the KIR2DL4 TM domain (#4CAR) is not sufficient to induce any activity. Interestingly, removing the KIR2DL4 TM and replacing it with CD8a TM (#5) completely eliminates activity even in the presence of the full-length KIR2DL4 ICD.

[0157] In separate experiments, primary T cells were mock treated (untransduced) or transduced with the indicated chimeric receptors. These constructs were cloned with CD19 scFv. At the end of production, transduction efficiency was measured with FITC-conjugated CD19 protein and reported as MFI. Transduced T cells were then exposed to luciferase-expressing Raji cells at a 1:1 effector-to-target ratio to measure cytotoxicity (Figure 4A). Short-term cytotoxicity is measured after 24 hours (Figure 4B). Repetitive cytotoxicity is measured by two consecutive rounds of Raji cell exposure every 48 hours (Figure 4C). In both cases, cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells.

[0158] Primary NK cells were mock treated (non-transduced) or transduced with the indicated chimeric receptors. These constructs were cloned with CD19 scFv. At the end of production, transduction efficiency was measured with FITC-conjugated CD19 protein and is reported as MFI. Transduced NK cells were then exposed to Raji cells at a 1:1 effector-to-target ratio for 1 h to induce functional degranulation (Figure 5A). Degranulation is measured by flow cytometry-based detection of CD107a on the cell surface (Figure 5B).

[0159] Taken together, these results indicate that the KIR2DL4 transmembrane domain as well as the first 10-29 amino acids are required for immune cell activation by KIR2DL4-based synthetic receptors. These results also demonstrate that synthetic receptors containing this domain can support short-term and sustained immune cell activation.

[0160] Example 3 - Combination of KIR2DL4-based synthetic receptor activation and costimulatory domains Engineered Jurkat T cells were mock treated (non-transduced) or transduced with lentiviral vectors encoding two positive control CARs with either CD28 (28z) or 4-1BB (41BBz) costimulatory domains, or with different chimeric receptors containing the KIR2DL4 domain. The receptors include: KIR2DL4 TM with C8 truncated [KIR2DL4(C8)] ICD domain (#6CAR, SEQ ID NO: 32), WT KIR2DL4 TM with KIR2DL4(C8) and 4-1BB ICD domain (#7CAR; SEQ ID NO: 33), and WT KIR2DL4 TM with KIR2DL4(C8) and DAP10 ICD domain (#8CAR; SEQ ID NO: 44). These constructs were cloned with CD19 or MSLN scFv.

[0161] In one experiment, the transduced cells were cultured for 2 days and then a portion of the cells was harvested and mixed with naturally CD19-expressing Raji cells or MSLN-expressing K562 cells at a 1:1 effector-to-target cell ratio for 24 h. Immune activation was measured by flow cytometry as the fluorescence of the NFAT-NFkB gene reporter, which upon immune stimulation leads to the expression of the mCitrine-PEST short-lived fluorescent protein. Data are presented as the percentage of cells expressing mCitrine as measured by flow cytometry. Figure 6A shows the CD19 + Figure 6B shows the activation of CD19-CAR immune cells against target cells. + Shows activation of MSLN-CAR immune cells against target cells.

[0162] In separate experiments, primary T cells were mock treated (untransduced) or transduced with the indicated chimeric receptors. These constructs were cloned with BCMA scFv. At the end of production, transduction efficiency is measured by expression of mScarlet fluorescent reporter protein (Figure 7A). Transduced T cells are then exposed to BCMA-expressing K562 cells at an effector-to-target ratio of 5:1 for 24 hours to measure cytotoxicity by flow cytometry. Cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells (Figure 7B).

[0163] In separate experiments, primary T cells were mock treated (untransduced) or transduced with the indicated chimeric receptors. These constructs were cloned with MSLN scFv. At the end of production, transduction efficiency is measured by expression of mScarlet fluorescent reporter protein (Figure 8A). Transduced T cells are then exposed to MSLN-expressing K562 cells at an effector-to-target ratio of 5:1 for 24 hours to measure cytotoxicity by flow cytometry. Cytotoxicity was calculated by normalizing the killing in each condition to wells containing only target cells (Figure 8B).

[0164] These results indicate that costimulatory domains such as CD3z, 4-1BB, or DAP10 are compatible with KIR2DL4-based synthetic receptors and can be used to support immune cell activation.

[0165] Incorporation by Reference The entire disclosures of each of the patent documents and scientific articles referred to herein are incorporated by reference for all purposes.

[0166] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

**Claim 1** A method of activating a recombinant immune cell expressing a synthetic receptor, wherein the receptor comprises (a) an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain comprising a sequence of 10 to 29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), the method comprising binding an antigen to the antigen-binding domain, thereby activating the recombinant immune cell. **Claim 2** The method according to claim 1, wherein the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9. **Claim 3** The method according to claim 1, wherein the intracellular signaling domain further comprises a co-stimulatory domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 12 to 16. **Claim 4** The method according to claim 1, wherein the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3. **Claim 5** The method according to claim 1, wherein the synthetic receptor further comprises a hinge region inserted between the antigen-binding domain and the transmembrane domain. **Claim 6** The method according to claim 5, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 4 or 5. **Claim 7** The method according to claim 1, wherein the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NOs: 17 to 34. **Claim 8** The method according to any one of claims 1 to 7, wherein the antigen-binding domain comprises an antibody or a functional fragment thereof. **Claim 9** The method according to any one of claims 1 to 7, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv), a minimal active antibody fragment, a single-domain antibody, a single light-chain variable domain, a single heavy-chain variable domain, or a nanobody. **Claim 10** The method according to any one of claims 1 to 7, wherein the recombinant immune cell is a T cell, a natural killer (NK) cell, or a natural killer T (NK-T) cell. **Claim 11** The method according to any one of claims 1 to 7, wherein the antigen is a tumor-associated antigen. **Claim 12** The method according to any one of claims 1 to 7, wherein the antigen is an entire protein or a fragment thereof selected from the list consisting of CD19, mesothelin, CD123, BCMA, GD2, CD30, GPC3, CD22, HER2, CD20, EGFR, Flt3, CD33, Muc-16, CS1, and tumor neoantigen.

13. The method according to any one of claims 1 to 7, wherein the antigen is derived from an infectious agent.

14. The method according to claim 13, wherein the infectious agent is a bacterium, a virus, a fungus, or a parasite.

15. A recombinant immune cell comprising an expressed synthetic receptor, wherein the receptor comprises (a) an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain comprising or consisting of a sequence of 10 to 29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), The recombinant immune cell, wherein the recombinant immune cell is activated by binding a cognate antigen to the antigen-binding domain.

16. The recombinant immune cell according to claim 15, wherein the intracellular signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9.

17. The recombinant immune cell according to claim 15, wherein the intracellular signaling domain further comprises an amino acid sequence selected from any one of SEQ ID NOs: 12 to 16.

18. The recombinant immune cell according to claim 15, wherein the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, or 3.

19. The recombinant immune cell according to claim 15, wherein the synthetic receptor further comprises a hinge region inserted between the antigen-binding domain and the transmembrane domain.

20. The recombinant immune cell according to claim 19, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 4 or 5.

21. The recombinant immune cell according to claim 15, wherein the synthetic receptor comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NOs: 17 to 34.

22. The recombinant immune cell according to claim 15, further comprising the cognate antigen bound to the antigen-binding domain, and the cell is in an active state.

23. The recombinant immune cell according to claim 15, wherein the antigen-binding domain comprises an antibody or a functional fragment thereof.

24. The recombinant immune cell according to claim 15, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv), a minimal active antibody fragment, a single-domain antibody, a single light-chain variable domain, a single light-chain variable domain, or a nanobody.

25. The recombinant immune cell according to claim 15, wherein the recombinant immune cell is a T cell, an NK cell, or an NK-T cell.

26. A pharmaceutical composition for use in the treatment of a disease in a subject, the pharmaceutical composition comprising the recombinant immune cell according to any one of claims 15 to 25.

27. The pharmaceutical composition according to claim 26, wherein the subject is a mammal.

28. The pharmaceutical composition according to claim 26, wherein the subject is a human.

29. The pharmaceutical composition according to claim 26, wherein the recombinant cell is autologous to the subject.

30. The pharmaceutical composition according to claim 26, wherein the recombinant cell is allogeneic to the subject.

31. The pharmaceutical composition according to any one of claims 26, wherein the disease is cancer.

32. The pharmaceutical composition according to claim 31, wherein the cancer is a hematological malignancy or a solid tumor.

33. The pharmaceutical composition according to claim 31, wherein the antigen is a whole protein or a fragment thereof selected from the list consisting of CD19, mesothelin, CD123, BCMA, GD2, CD30, GPC3, CD22, HER2, CD20, EGFR, Flt3, CD33, Muc-16, CS1, and tumor neoantigen.

34. The pharmaceutical composition according to claim 26, wherein the disease is an infection caused by an infectious agent.

35. The pharmaceutical composition according to claim 34, wherein the infectious agent is a bacterium, a virus, a fungus, or a parasite.

36. A method of activating a recombinant immune cell expressing a synthetic receptor, the receptor comprising (a) means for binding to an antigen of interest, (b) a transmembrane domain, and (c) an intracellular signaling domain comprising a sequence of 10 to 29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4). The method, comprising binding the receptor to the antigen of interest, thereby activating the recombinant immune cell. **Claim 37** A recombinant immune cell comprising an expressed synthetic receptor, wherein the receptor comprises (a) means for binding an antigen of interest, (b) a transmembrane domain, and (c) an intracellular signaling domain comprising or consisting of a sequence of 10 to 29 amino acids derived from killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), The recombinant immune cell, wherein the recombinant immune cell is activated by binding the antigen of interest to the synthetic receptor.

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