Fusion proteins and methods of use thereof

Abstract

The present application provides a fusion protein having two polypeptides, each having an extracellular domain and an intracellular domain, where the two extracellular domains form a binding site for TGFβ and the two intracellular domains form the intracellular domain of the IL-23 receptor complex. The present application also provides a nucleic acid encoding the fusion protein, a vector comprising the nucleic acid, and an engineered cell expressing the fusion protein. Methods of producing the engineered cells and methods of treatment comprising administering the engineered cells are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application PCT / CN2021 / 087475, filed April 15, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file: Computer Readable Form (CRF) of the Sequence Listing (Filename: P11128-PCT.220415.Sequence Listing.TXT, Date Recorded: April 14, 2022, Size: 205,318 bytes) is incorporated herein by reference in its entirety.

[0003] Field of the Application The present application relates to fusion proteins capable of binding to TGFβ and mediating a pro-inflammatory signal. [Background technology]

[0004] Background of the Application Transforming growth factor beta (TGFβ) is a pleiotropic cytokine that participates in the tumor microenvironment as an immunosuppressive signaling molecule. TGFβ binds to the TGFβR1 and TGFβR2 serine / threonine kinase receptor complexes, resulting in receptor-mediated phosphorylation of the downstream transcription factors Smad2 and Smad3. Many tumors circumvent the cytostatic and antiproliferative effects of TGFβ by acquiring mutations in the TGFβ receptor and / or downstream Smad signaling proteins. TGFβ suppresses key molecules involved in the effector and cytolytic activity of T cells in vitro, including IFNγ secretion.

[0005] The disclosures of all publications, patents, patent applications, and published patent applications referenced herein are hereby incorporated by reference in their entireties. Summary of the Invention

[0006] Overview of the Application In one aspect, the present application provides a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising an intracellular domain of one of IL-12Rβ1 or IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of the other of TGFβR1 or TGFβR2, ii) a second transmembrane domain, and iii) a second intracellular domain comprising an intracellular domain of the other of IL-12Rβ1 or IL-23R. In some embodiments, the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of TGFβR1, TGFβR2, IL-12Rβ1, and IL-23R. In some embodiments, the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of IL-12Rβ1 or IL-23R.

[0007] In some embodiments of any of the fusion proteins described above, the fusion protein comprises: a) the first extracellular domain comprises the extracellular domain of TGFβR1, the first transmembrane domain comprises the transmembrane domain of TGFβR1, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR2, the second transmembrane domain comprises the transmembrane domain of TGFβR2, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; b) the first extracellular domain comprises the extracellular domain of TGFβR2, the first transmembrane domain comprises the transmembrane domain of TGFβR2, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR1, the second transmembrane domain comprises the transmembrane domain of TGFβR1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; c) the first extracellular domain comprises the extracellular domain of TGFβR1, the first transmembrane domain comprises the transmembrane domain of IL-23R, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR2, the second transmembrane domain comprises the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; or d) the first extracellular domain comprises the extracellular domain of TGFβR2, the first transmembrane domain comprises the transmembrane domain of IL-23R, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR1, the second transmembrane domain comprises the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1. Includes.

[0008] In some embodiments of any of the fusion proteins described above, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide.

[0009] In some embodiments of any of the fusion proteins described above, the first and second polypeptides are in a single polypeptide, and the first and second polypeptides are separated by a multicistronic element. In some embodiments, the multicistronic element comprises a 2A self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, or F2A. In some embodiments, the first polypeptide is N-terminal to the second polypeptide. In some embodiments, the first polypeptide is C-terminal to the second polypeptide. In some embodiments, the single polypeptide is, from the N-terminal to the C-terminal direction, a) a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of TGFβR1, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of TGFβR2, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1; b) a first extracellular domain comprising the extracellular domain of TGFβR2, a first transmembrane domain comprising the transmembrane domain of TGFβR2, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR1, a second transmembrane domain comprising the transmembrane domain of TGFβR1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1; c) a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of TGFβR1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of TGFβR2, and a second intracellular domain comprising the intracellular domain of IL-23R; d) a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-23R, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1; e) a first extracellular domain comprising the extracellular domain of TGFβR2, a first transmembrane domain comprising the transmembrane domain of IL-23R, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR1, a second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1; or f) a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-12Rβ1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-23R, and a second intracellular domain comprising the intracellular domain of IL-23R. Includes.

[0010] In some embodiments of any of the fusion proteins described above, the first and second extracellular domains form a binding site for TGFβ, and the first and second intracellular domains form an intracellular domain of an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.

[0011] In some embodiments of any of the fusion proteins described above, the transmembrane domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:5, or a functional variant having at least about 90% sequence identity; and / or the transmembrane domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:6, or a functional variant having at least about 90% sequence identity.

[0012] In some embodiments of any of the fusion proteins described above, the transmembrane domain of IL-23R comprises the amino acid sequence of SEQ ID NO:7, or a functional variant having at least about 90% sequence identity; and / or the transmembrane domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant having at least about 90% sequence identity.

[0013] In some embodiments of any of the fusion proteins described above, the extracellular domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:3, or a functional variant having at least about 90% sequence identity.

[0014] In some embodiments of any of the fusion proteins described above, the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO: 4, or a functional variant having at least about 90% sequence identity.

[0015] In some embodiments of any of the fusion proteins described above, the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO: 15, or a functional variant having at least about 90% sequence identity.

[0016] In some embodiments of any of the fusion proteins described above, the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO: 16, or a functional variant having at least about 90% sequence identity.

[0017] In some embodiments of any of the fusion proteins described above, the fusion protein comprises: a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:5, iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:6, and iii) a second intracellular domain comprising SEQ ID NO:16.

[0018] In some embodiments of any of the fusion proteins described above, the fusion protein comprises: a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:6, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:5, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16.

[0019] In some embodiments of any of the fusion proteins described above, the fusion protein comprises: a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16.

[0020] In some embodiments according to any of the fusion proteins described above, the fusion protein comprises: a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16.

[0021] In some embodiments of any of the fusion proteins described above, the first polypeptide and / or the second polypeptide further comprises a linker between two domains, one of which is a transmembrane domain and the other of which is an extracellular domain or an intracellular domain. In some embodiments, the linker comprises a juxtamembrane sequence, and the juxtamembrane sequence and the transmembrane domain are derived from the same molecule. In some embodiments, the juxtamembrane region comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12.

[0022] In some embodiments of any of the fusion proteins described above, the first polypeptide comprises the amino acid sequence of SEQ ID NO:49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:50.

[0023] In some embodiments of any of the fusion proteins described above, the first polypeptide comprises the amino acid sequence of SEQ ID NO:51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:52.

[0024] In some embodiments of any of the fusion proteins described above, the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54.

[0025] In some embodiments of any of the fusion proteins described above, the first polypeptide comprises the amino acid sequence of SEQ ID NO:55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:56.

[0026] In some embodiments of any of the fusion proteins described above, the fusion protein comprises an amino acid sequence set forth in any of SEQ ID NOs:20-25.

[0027] The application provides, in another aspect, a nucleic acid comprising one or more nucleic acid sequences encoding any of the fusion proteins described above or portions thereof.

[0028] In another aspect, the present application provides a nucleic acid comprising a nucleic acid sequence shown in any of SEQ ID NOs:28-33.

[0029] In some embodiments according to any of the nucleic acids described above, the nucleic acid further comprises a second nucleic acid sequence encoding a functional foreign receptor, the functional foreign receptor comprising an extracellular ligand binding domain and optionally an intracellular signaling domain. In some embodiments, the functional foreign receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof. In some embodiments, the functional foreign receptor specifically recognizes a tumor antigen. In some embodiments, the nucleic acid sequence encoding the functional foreign receptor is upstream to at least one of one or more nucleic acid sequences encoding a fusion protein ("fusion protein nucleic acid sequence"), and optionally, the functional foreign receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a third nucleic acid sequence encoding a second multicistronic element. In some embodiments, the second multicistronic element comprises a 2A autocleaving peptide selected from the group consisting of T2A, P2A, E2A, or F2A. In some embodiments, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the functional foreign receptor are separated by a second multicistronic element.

[0030] The present application provides, in another aspect, a vector comprising any of the nucleic acids described above.

[0031] In another aspect, the present application provides an engineered cell comprising any of the fusion proteins, nucleic acids, or vectors described above. In some embodiments, the engineered cell further comprises a functional foreign receptor, the functional foreign receptor comprising an extracellular ligand binding domain and optionally an intracellular signaling domain. In some embodiments, the functional foreign receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof. In some embodiments, the functional foreign receptor specifically recognizes a tumor antigen. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and a combination thereof.

[0032] The present application, in another aspect, provides a pharmaceutical composition comprising any of the engineered cells described above.

[0033] In another aspect, the application provides a method of treating a disease or condition in an individual, comprising administering to the individual any of the fusion proteins or pharmaceutical compositions described herein. In some embodiments, the disease or condition involves immunosuppression. In some embodiments, diseased tissue has a higher expression level of TGFβ than the corresponding tissue in the individual without the disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the disease or condition is an infectious disease or a condition associated with an infectious disease. In some embodiments, the engineered cells in the pharmaceutical composition are allogeneic to the individual. In some embodiments, the engineered cells in the pharmaceutical composition are autologous to the individual. In some embodiments, the method further comprises administering a second therapy or a second agent.

[0034] In another aspect, the present application provides a method for reducing an immunosuppressive signal in a diseased tissue in an individual, comprising administering to the individual any of the fusion proteins or pharmaceutical compositions described herein. In some embodiments, reducing the immunosuppressive signal comprises reducing signaling through TGFβR. In some embodiments, the diseased tissue has a higher expression level of TGFβ than the corresponding tissue in the individual without the disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the disease or condition is an infectious disease or a condition associated with an infectious disease. In some embodiments, the engineered cells in the pharmaceutical composition are allogeneic to the individual. In some embodiments, the engineered cells in the pharmaceutical composition are autologous to the individual. In some embodiments, the method further comprises administering a second therapy or a second agent. [Brief description of the drawings]

[0035] [Figure 1A] A line drawing structure of the TGB23 fusion protein is shown. [Figure 1B] The polypeptide encoding the TGB23 fusion protein is shown. SP refers to the signal peptide. TM refers to the transmembrane domain. [Diagram 2] Expression levels of fusion proteins TGB23-1, TGB23-2, TGB23-3, TGB23-4, TGB23-5, TGB23-6, wtTGFβR, TGB23-14, and TGB23-15 are shown. UnT refers to T cells not transduced with any of the fusion proteins, and wtTGFβR refers to the wild type TGFβ receptor. [Diagram 3]3A-3B show cell viability (A) and cell number (B) of T cells expressing TGB23-1, TGB23-2, TGB23-3, TGB23-4, TGB23-5, TGB23-6, wtTGFβR, TGB23-14, or TGB23-15 treated with 20 ng / mL TGFβ1. UnT refers to T cells not transduced with any of the fusion proteins. [Figure 4] Shown are anti-GPC3 CAR expression levels and TGFβR2 expression levels in CAR-positive cells of UnT, huLIC19309b CAR-T cells, and huLIC19309bT CAR-T cells. UnT refers to T cells not transduced with a CAR. [Diagram 5] 1 shows the expression levels of pSmad2 in UnT, huLIC19309b CAR-T cells, and huLIC19309bT CAR-T cells treated with or without 20 ng / mL TGFβ1 for 4 hours. UnT refers to T cells not transduced with CAR. [Figure 6A] 1 shows the expression levels of pStat3 in UnT, huLIC19309b CAR-T cells, and huLIC19309bT CAR-T cells treated with or without 20 ng / mL TGFβ1 for 4 hours. UnT refers to T cells not transduced with a CAR. [Figure 6B] 1 shows the expression levels of pStat4 in UnT, huLIC19309b CAR-T cells, and huLIC19309bT CAR-T cells treated with or without 20 ng / mL TGFβ1 for 4 hours. UnT refers to T cells not transduced with a CAR. [Figure 7] 7A-7B show cell viability (A) and cell number (B) of UnT, huLIC19309b CAR-T cells, and huLIC19309bT CAR-T cells treated with 20 ng / mL TGFβ1. UnT refers to T cells not transduced with a CAR. [Figure 8]Figures 8A-8B show the expression levels of PD1 in CD4+ CAR+ T cells (A) and CD8+ CAR+ T cells (B) of the two treatment groups (huLIC19309b CAR-T group and huLIC19309bT CAR-T group) after co-culture with PLCPRF5 and 5 ng / mL TGFβ1 in a re-challenge assay. [Figure 9A] The CAR positivity rate after co-culture with PLCPRF5 in the re-challenge assay is shown. [Figure 9B] The CAR positivity rate after co-culture with PLCPRF5 in the re-challenge assay is shown. [Figure 9C] The cell numbers after co-culture with PLCPRF5 in the re-challenge assay are shown. [Figure 9D] The cell numbers after co-culture with PLCPRF5 in the re-challenge assay are shown. [Figure 10] 10A-10B show the in vitro cytotoxicity of huLIC19309b CAR-T cells and huLIC19309bT CAR-T cells against PLCPRF5 cells (A) and Hep3B2.1-7 cells (B) with an E / T ratio of 1:1 in a re-challenge assay. E / T refers to the effective cell to target cell ratio. [Figure 11] Figure 1 shows the in vitro real-time cytotoxicity of huLIC19309b and huLIC19309bT CAR-T cells against Hep3B2.1-7 cells with an E / T ratio of 1:20. E / T refers to the effective cell to target cell ratio. [Figure 12] 12A-12C show the antitumor effect of CAR-T cells in an NCG mouse xenograft model. NCG mice were inoculated subcutaneously with Hep3B2.1-7 cells and treated with CAR-T cells at a dose of 0.3 M and 0.8 M (intravenous). Mouse body weight (A), tumor volume (B), and percentage of human CD3+ T cells in peripheral blood (C) were assessed. [Figure 13]Figures 13A-13C show the expression of TCRαβ (A), CD20 CAR (B), and TGB23-6 (C) in UnT, LCAR-UL186S T cells, and LCAR-UL186S+TGB23-6 T cells by flow cytometry. Untransduced T cells (UnT) were used as a negative control. [Figure 14] Figure 1 shows the in vitro cytotoxicity of LCAR-UL186S T cells and LCAR-UL186S+TGB23-6 T cells against Raji cells with different effector-target ratios. E / T refers to the effector to target cell ratio. [Figure 15] 1 shows the proliferation of LCAR-UL186S and LCAR-UL186S+TGB23-6 T cells after multiple rounds of Raji stimulation. Cells were harvested on days 0, 4, 7, and 11, and CD5 expression was detected by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Application The present application provides a fusion protein of a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising an intracellular domain of one of IL-12Rβ1 or IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of the other of TGFβR1 or TGFβR2, ii) a second transmembrane domain, and iii) a second intracellular domain comprising an intracellular domain of the other of IL-12Rβ1 or IL-23R. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, and the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex. The present application also provides engineered cells comprising such fusion proteins or nucleic acids encoding such fusion proteins, and optionally the engineered cells also comprise a functional exogenous receptor (e.g., a CAR or an engineered TCR). Methods of producing or using such engineered cells are also provided.

[0037] The present application provides engineered cells comprising the fusion proteins described herein capable of converting TGFβ signals into IL-23R-mediated signals. As shown in more detail in the Examples, engineered T cells with such fusion proteins exhibit higher survival and proliferation upon exposure to TGFβ than corresponding T cells without the fusion proteins. Furthermore, engineered T cells expressing both the fusion proteins and a CAR targeting a tumor antigen exhibit higher cytotoxicity against tumor cells both in vitro and in animal tumor models. Increased proliferation and cytotoxicity of engineered T cells have also been shown to be associated with overall sustained continuous exposure to TGFβ for a total of more than 300 hours. These results provide strong evidence that fusion proteins and engineered cells containing these fusion proteins can effectively treat diseases or conditions associated with immune suppression, such as cancer. These highly beneficial effects are particularly striking given the current limited and inconsistent understanding of IL-23 signaling in cancer biology.

[0038] I. Definition The term "antibody" includes monoclonal antibodies (including full-length four chain antibodies with an immunoglobulin Fc region or full-length heavy chain-only antibodies), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments (e.g., Fab, F(ab') 2 , Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Antibodies contemplated herein include single domain antibodies (such as heavy chain only antibodies).

[0039] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). An sdAb alone is capable of binding to an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred to herein as "V HThe term "heavy chain only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain normally found in four chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs. Some V H H may also be known as nanobodies. Camelid sdAbs are among the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). The basic V H H has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0040] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy and light chain variable domains are respectively referred to as "V H " and "V L " These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding sites. Heavy-chain-only antibodies from camelid species have a single heavy-chain variable region, which is called the "V H It is called "H".

[0041] The term "variable" refers to the fact that certain segments of the variable domains differ significantly in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed across the entire span of the variable domains. Rather, variability is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three HVRs that form loops that connect and in some cases form part of the β-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and contribute to the formation of the antigen-binding site of antibodies with HVRs from the other chain (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0042] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0043] "Single-chain Fv," also abbreviated as "sFv" or "scFv," refers to Fvs in which the VFs are joined together into a single polypeptide chain.H Antibody domains and V L An sFv polypeptide is an antibody fragment that contains an antibody domain. H Domain and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding.

[0044] As used herein, the terms "specifically bind", "specifically recognize", or "specific for" refer to a measurable and reproducible interaction (such as binding between a target and an antigen binding protein (such as an antigen binding domain, a ligand, an engineered TCR, a CAR, or a chimeric receptor)) that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antigen binding protein that specifically binds to a target (which may be an epitope) is an antigen binding protein that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than the antigen binding protein binds to other targets. In some embodiments, the extent of binding of the antigen binding protein to an unrelated target is less than about 10% of the binding of the antigen binding protein to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, an antigen binding protein specifically binds to an epitope on a protein that is conserved among proteins from different species, hi some embodiments, specific binding can include, but does not require, exclusive binding.

[0045] The term "specificity" refers to the selective recognition of an antigen-binding protein (such as a CAR or an antibody) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecificity" as used herein indicates that an antigen-binding protein (such as a CAR or an antibody) has two or more antigen-binding sites, at least two of which bind different antigens. "Bispecificity" as used herein indicates that an antigen-binding protein (such as a CAR or an antibody) has two different antigen-binding specificities. The term "monospecificity" as used herein indicates that an antigen-binding protein (such as a CAR or an antibody) has one or more binding sites, each of which binds the same antigen.

[0046] The term "valency" as used herein refers to the presence of a defined number of binding sites in an antigen-binding protein (such as a CAR or antibody). For example, a natural antibody, or a full-length antibody, has two binding sites and is bivalent. Thus, the terms "trivalent", "tetravalent", "pentavalent", and "hexavalent" refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein (such as a CAR or antibody).

[0047] The term "extracellular domain" refers to a fragment or portion of a membrane receptor on the outside of a cell. The extracellular domain is or includes a ligand binding or recognition domain (e.g., the TGFβ binding domain of TGFβR1 and TGFβR2 in the present application).

[0048] The term "intracellular domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to carry out a specialized function. Usually, the entire intracellular domain can be used, but in some cases, it is not necessary to use the entire domain. In cases where a truncated portion of the intracellular domain is used, such a truncated portion can be used instead of the entire domain, so long as it transmits an effector function signal. The term intracellular domain is meant to encompass any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal.

[0049] The term "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to the plasma membrane of a cell. The TM domain can be from natural, synthetic, semi-synthetic, or recombinant sources. The transmembrane domain can be from the same receptor molecule as either the extracellular domain or the intracellular domain.

[0050] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may include a population of antibodies with all K447 residues removed, a population of antibodies without the K447 residue removed, and a population of antibodies with a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0051] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody, CAR) and its binding partner (e.g., antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen, or CAR and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this application. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0052] "Percent (%) sequence identity" and "homology" with respect to a peptide, polypeptide, or nucleic acid are defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in a particular peptide, polypeptide, or nucleic acid sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0053] "Chimeric antigen receptor" or "CAR" as used herein refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto immune effector cells (such as T cells). Some CARs are also known as "artificial T cell receptors", "chimeric T cell receptors", or "chimeric immune receptors". In some embodiments, a CAR comprises an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T" refers to a T cell expressing a CAR. "GPC3 CAR" refers to a CAR with an extracellular binding domain specific for GPC3. "Bi-epitope CAR" refers to a CAR with an extracellular binding domain specific for two different epitopes.

[0054] An "isolated" nucleic acid molecule (e.g., encoding a fusion protein, a CAR, or an engineered TCR) as described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form other than the form or setting in which they are found in nature. Thus, isolated nucleic acid molecules are distinguished from the nucleic acids encoding the polypeptides and antibodies herein that naturally occur in cells.

[0055] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; or a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0056] Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, in which case the nucleotide sequence encoding the protein may contain intron(s) in some versions.

[0057] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term encompasses vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0058] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0059] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results (including clinical results). For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms resulting from a disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or decelerating the progression of the disease, providing remission of the disease (partial or complete), reducing the dose of one or more other pharmaceuticals required to treat the disease, slowing the progression of the disease, increasing the quality of life, and / or prolonging survival. Reduction of the pathological consequences of cancer is also encompassed by "treatment". The methods of the present application contemplate any one or more of these aspects of treatment.

[0060] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0061] The term "effective amount" as used herein refers to an amount of an agent (such as an engineered T cell described herein, or a pharmaceutical composition thereof) sufficient to treat a specified disorder, condition, or disease (such as ameliorating, alleviating, reducing, and / or delaying one or more of the symptoms, e.g., cancer or infectious disease). With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or a decrease in the rate of tumor growth (such as inhibiting tumor growth), or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of an agent (e.g., engineered T cells) or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the appearance and / or recurrence of tumors; and / or (vii) relieve to some extent one or more symptoms associated with cancer. In the case of infectious diseases (such as viral infections), a therapeutically effective amount of the engineered T cells or compositions thereof described herein may reduce the number of cells infected with a pathogen; reduce the production or release of antigens derived from the pathogen; inhibit (i.e., slow to some extent, and preferably stop) the spread of the pathogen to non-infected cells; and / or relieve to some extent one or more symptoms associated with the infection. In some embodiments, a therapeutically effective amount is an amount that prolongs the survival of a patient.

[0062] As used herein, "delaying" the onset of a disease means suspending, preventing, slowing, retarding, stabilizing, and / or postponing the onset of a disease (e.g., cancer or infectious disease). This delay can be of variable length depending on the history of the disease and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can effectively encompass prevention, in that the individual does not develop the disease. A method that "delays" the onset of cancer is one that reduces the probability of the onset of the disease in a given time frame and / or reduces the extent of the disease in a given time frame when compared to not using the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The onset of cancer can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset can also refer to cancer progression, which may be initially undetectable, including emergence, recurrence, and onset.

[0063] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into that individual.

[0064] "Allogeneic" refers to a graft derived from a different individual of the same species. "Allogeneic T cells" refers to T cells from a donor that has a tissue human leukocyte antigen (HLA) type that matches the recipient. Typically, matching is accomplished based on variations at three or more loci of the HLA genes, with perfect matches at these loci being preferred. In some instances, the allogeneic transplant donor can be related (usually a very well HLA-matched sibling), syngeneic (the patient's identical "identical" twin), or unrelated (an unrelated donor who is found to have a very close degree of HLA match). HLA genes are classified into two categories, type I and type II. In general, mismatches of type I genes (i.e., HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. Mismatches of HLA type II genes (i.e., HLA-DR or HLA-DQB1) increase the risk of graft-versus-host disease (GvHD).

[0065] "Patient" as used herein includes any human suffering from a disease (e.g., cancer, viral infection). The terms "subject", "individual" and "patient" are used interchangeably herein. The term "donor subject" or "donor" as used herein refers to a subject whose cells are obtained for further in vitro manipulation. A donor subject may be a patient who is to be treated with a cell population generated by the methods described herein (i.e., an autologous donor), or an individual who provides a blood sample (e.g., a lymphocyte sample) to be used to treat a different individual or patient during the generation of a cell population generated by the methods described herein (i.e., an allogeneic donor). A subject who receives cells prepared by the methods may be referred to as a "recipient" or "recipient subject".

[0066] The term "T cell receptor" (or "TCR") refers to a heterodimeric receptor composed of an αβ or γδ chain that pair on the surface of a T cell. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition via complementarity determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the non-variable subunit of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with antigenic peptide-peptide complexes (pMHC) bound to proteins encoded by the major histocompatibility complex (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, 868 p.).

[0067] The term "stimulation" as used herein refers to a primary response induced by ligation of cell surface moieties. For example, in the context of receptors, such stimulation involves ligation of the receptor and subsequent signaling events. In the context of stimulation of T cells, such stimulation refers to ligation of T cell surface moieties (such as binding of the TCR / CD3 complex) in one embodiment inducing subsequent signaling events. Furthermore, the stimulatory event may activate the cell and upregulate or downregulate the expression or secretion of molecules (such as downregulation of TGF-β). Thus, ligation of cell surface moieties, even in the absence of a direct signaling event, may result in rearrangement of the cytoskeletal structure or coalescence of cell surface moieties, each of which may promote, modify, or alter subsequent cellular responses.

[0068] The term "activation" as used herein refers to the state of a cell after cell surface moiety ligation sufficient to induce significant biochemical or morphological changes. Within the context of T cells, such activation refers to the state of a T cell that has been stimulated sufficiently to induce cell proliferation. T cell activation may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, the term implies either upregulation or downregulation of certain physicochemical processes. The term "activated T cells" refers to T cells that are currently undergoing cell division, cytokine production, regulatory or cytolytic effector function performance and / or have recently undergone the process of "activation".

[0069] The term "functional foreign receptor" as used herein refers to a foreign receptor (e.g., a CAR, an engineered receptor, an engineered TCR, or a TCR coupled to an antibody (ACTR)) that retains its biological activity after being introduced into a T cell or a fusion protein-expressing T cell described herein. Biological activity includes, but is not limited to, the ability of the foreign receptor in specifically binding to a molecule (e.g., a cancer antigen, or an antibody for ACTR) and appropriately transmitting a downstream signal (such as inducing cell proliferation, cytokine production, and / or performance of a regulatory or cytolytic effector function).

[0070] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0071] Reference herein to "about" a value or parameter encompasses (and describes) a variation about that value or parameter per se. For example, a description that refers to "about X" includes the description of "X."

[0072] As used herein, a reference to "is not" a value or parameter generally means and describes "other than" the value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancers other than type X.

[0073] The term "about X to Y" used in this specification has the same meaning as "about X to about Y".

[0074] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0075] II. Fusion Proteins of the Invention The present application provides, in one aspect, a fusion protein comprising: a) a first polypeptide comprising i) a first extracellular domain comprising the extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising the intracellular domain of one of IL-12Rβ1 or IL-23R; and b) a second polypeptide comprising i) a second extracellular domain comprising the extracellular domain of the other of TGFβR1 or TGFβR2, ii) a second transmembrane domain, and iii) a second intracellular domain comprising the intracellular domain of the other of IL-12Rβ1 or IL-23R.

[0076] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide including: i) a first extracellular domain including an extracellular domain of TGFβR1; ii) a first transmembrane domain including a transmembrane domain of TGFβR1; iii) a first intracellular domain including an intracellular domain of IL-23R; and b) a second polypeptide including: i) a second extracellular domain including an extracellular domain of TGFβR2; ii) a second transmembrane domain including a transmembrane domain of TGFβR2; and iii) a second intracellular domain including an intracellular domain of IL-12Rβ1. In some embodiments, the extracellular domain of TGFβR1 includes an amino acid sequence of SEQ ID NO: 3, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:4, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO:15, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:16, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:5, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the transmembrane domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:6, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR1 or TGFβR2 is a mutated or truncated extracellular domain of TGFβR1 or TGFβR2. In some embodiments, the intracellular domain of IL-12Rβ1 or IL-23R is a mutated or truncated intracellular domain of IL-12Rβ1 or IL-23R. In some embodiments, the mutant or truncated intracellular domain of IL-23R has the amino acid sequence of SEQ ID NO: 17 or 18, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the truncated intracellular domain of IL-12Rβ1 has the amino acid sequence of SEQ ID NO: 19, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is a signal peptide from TGFβR1 or TGFβR2, and optionally, the signal peptide and the extracellular domain in the polypeptide are derived from the same molecule. In some embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.In some embodiments, the first polypeptide and / or the second polypeptide further comprises a juxtamembrane peptide between the transmembrane domain and the intracellular domain. In some embodiments, the juxtamembrane peptide has a length of about 3 to about 40 amino acids. In some embodiments, the juxtamembrane peptide is derived from the juxtamembrane region of TGFβR1, TGFβR2, IL-23R, or IL-12Rβ1. In some embodiments, the juxtamembrane peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12. In some embodiments, a signal mediated via the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0077] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide including: i) a first extracellular domain including an extracellular domain of TGFβR2; ii) a first transmembrane domain including a transmembrane domain of TGFβR2; iii) a first intracellular domain including an intracellular domain of IL-23R; and b) a second polypeptide including: i) a second extracellular domain including an extracellular domain of TGFβR1; ii) a second transmembrane domain including a transmembrane domain of TGFβR1; and iii) a second intracellular domain including an intracellular domain of IL-12Rβ1. In some embodiments, the extracellular domain of TGFβR1 includes an amino acid sequence of SEQ ID NO: 3, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:4, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO:15, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:16, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:5, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the transmembrane domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:6, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR1 or TGFβR2 is a mutated or truncated extracellular domain of TGFβR1 or TGFβR2. In some embodiments, the intracellular domain of IL-12Rβ1 or IL-23R is a mutated or truncated intracellular domain of IL-12Rβ1 or IL-23R. In some embodiments, the mutant or truncated intracellular domain of IL-23R has the amino acid sequence of SEQ ID NO: 17 or 18, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the truncated intracellular domain of IL-12Rβ1 has the amino acid sequence of SEQ ID NO: 19, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is a signal peptide from TGFβR1 or TGFβR2, and optionally, the signal peptide and the extracellular domain in the polypeptide are derived from the same molecule. In some embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.In some embodiments, the first polypeptide and / or the second polypeptide further comprises a juxtamembrane peptide between the transmembrane domain and the intracellular domain. In some embodiments, the juxtamembrane peptide has a length of about 3 to about 40 amino acids. In some embodiments, the juxtamembrane peptide is derived from the juxtamembrane region of TGFβR1, TGFβR2, IL-23R, or IL-12Rβ1. In some embodiments, the juxtamembrane peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12. In some embodiments, a signal mediated via the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0078] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR1; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR2; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the extracellular domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:3, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:4, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO:15, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:16, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of IL-23R comprises the amino acid sequence of SEQ ID NO:7, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the transmembrane domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR1 or TGFβR2 is a mutated or truncated extracellular domain of TGFβR1 or TGFβR2. In some embodiments, the intracellular domain of IL-12Rβ1 or IL-23R is a mutated or truncated intracellular domain of IL-12Rβ1 or IL-23R. In some embodiments, the mutant or truncated intracellular domain of IL-23R has the amino acid sequence of SEQ ID NO: 17 or 18, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the truncated intracellular domain of IL-12Rβ1 has the amino acid sequence of SEQ ID NO: 19, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is a signal peptide from TGFβR1 or TGFβR2, and optionally, the signal peptide and the extracellular domain in the polypeptide are derived from the same molecule. In some embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.In some embodiments, the first polypeptide and / or the second polypeptide further comprises a juxtamembrane peptide between the extracellular domain and the transmembrane domain. In some embodiments, the juxtamembrane peptide has a length of about 3 to about 40 amino acids. In some embodiments, the juxtamembrane peptide is derived from the juxtamembrane region of TGFβR1, TGFβR2, IL-23R, or IL-12Rβ1. In some embodiments, the juxtamembrane peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12. In some embodiments, a signal mediated via the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0079] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the extracellular domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO: 3, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:4, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO:15, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:16, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of IL-23R comprises the amino acid sequence of SEQ ID NO:7, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the transmembrane domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the extracellular domain of TGFβR1 or TGFβR2 is a mutated or truncated extracellular domain of TGFβR1 or TGFβR2. In some embodiments, the intracellular domain of IL-12Rβ1 or IL-23R is a mutated or truncated intracellular domain of IL-12Rβ1 or IL-23R. In some embodiments, the mutant or truncated intracellular domain of IL-23R has the amino acid sequence of SEQ ID NO: 17 or 18, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the truncated intracellular domain of IL-12Rβ1 has the amino acid sequence of SEQ ID NO: 19, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is a signal peptide from TGFβR1 or TGFβR2, and optionally, the signal peptide and the extracellular domain in the polypeptide are derived from the same molecule. In some embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.In some embodiments, the first polypeptide further comprises a first juxtamembrane sequence between the first extracellular domain and the first transmembrane domain. In some embodiments, the second polypeptide further comprises a second juxtamembrane sequence between the second extracellular domain and the second transmembrane domain. The juxtamembrane sequence may act as a transition between two functional domains to provide flexibility and / or promote longevity of the fusion protein or signaling. In some embodiments, the first or second juxtamembrane domain and the first or second transmembrane domain in the first and / or second polypeptide are derived from the same molecule. In some embodiments, the first and / or second juxtamembrane peptide is derived from the juxtamembrane region of TGFβR1, TGFβR2, IL-23R, or IL-12Rβ1. In some embodiments, the first and / or second juxtamembrane domain has a length of about 3 to about 40 amino acids. In some embodiments, the juxtamembrane peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12. In some embodiments, the first juxtamembrane peptide is derived from IL-23R (e.g., SEQ ID NO: 11) and / or the second proximal peptide is derived from IL-12Rβ1 (e.g., SEQ ID NO: 12). In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0080] In some embodiments, a) a first polypeptide comprising: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:5 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:15 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:6 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and iii) a second intracellular domain comprising SEQ ID NO:16 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first transmembrane domain and the first intracellular domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO:9, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second transmembrane domain and the second intracellular domain, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 10, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0081] In some embodiments, a first polypeptide comprises: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:6 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:15 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:5 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first transmembrane domain and the first intracellular domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 10, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second transmembrane domain and the second intracellular domain, and optionally the linker comprises the amino acid sequence of SEQ ID NO:9, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0082] In some embodiments, a first polypeptide comprises: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:7 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:15 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 11, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 12, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0083] In some embodiments, a first polypeptide comprises: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:7 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:15 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 11, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 12, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0084] In some embodiments, a) a first polypeptide comprising: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:7 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:17 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; Fusion proteins are provided that comprise: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 11, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 12, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0085] In some embodiments, a first polypeptide comprises: i) a first extracellular domain comprising an amino acid sequence of SEQ ID NO:3 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a first transmembrane domain comprising an amino acid sequence of SEQ ID NO:7 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a first intracellular domain comprising an amino acid sequence of SEQ ID NO:18 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:19 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 11, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, and optionally the linker comprises an amino acid sequence of SEQ ID NO: 12, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0086] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:5, iii) a first linker comprising the amino acid sequence of SEQ ID NO:9, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:6, iii) a second linker comprising the amino acid sequence of SEQ ID NO:10, and iv) a second intracellular domain comprising SEQ ID NO:16. In some embodiments, a signal mediated via the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0087] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO: 4, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO: 6, iii) a first linker comprising the amino acid sequence of SEQ ID NO: 10, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO: 3, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO: 5, iii) a second linker comprising the amino acid sequence of SEQ ID NO: 9, and iv) a second intracellular domain comprising SEQ ID NO: 16. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0088] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first linker comprising the amino acid sequence of SEQ ID NO:11, iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second linker comprising the amino acid sequence of SEQ ID NO:12, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iv) a second intracellular domain comprising SEQ ID NO:16. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0089] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO: 4, ii) a first linker comprising the amino acid sequence of SEQ ID NO: 11, iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO: 7, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO: 3, ii) a second linker comprising the amino acid sequence of SEQ ID NO: 12, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO: 8, and iv) a second intracellular domain comprising SEQ ID NO: 16. In some embodiments, a signal mediated via the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0090] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a first linker comprising the amino acid sequence of SEQ ID NO:11; iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:17; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a second linker comprising the amino acid sequence of SEQ ID NO:12; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8; and iv) a second intracellular domain comprising SEQ ID NO:16.

[0091] In some embodiments, a fusion protein is provided that comprises: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a first linker comprising the amino acid sequence of SEQ ID NO:11; iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:18; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a second linker comprising the amino acid sequence of SEQ ID NO:12; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8; and iv) a second intracellular domain comprising SEQ ID NO:19.

[0092] In some embodiments, a fusion protein is provided that includes a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 49 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 50 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0093] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 50 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 49 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0094] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 51 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 52 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0095] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0096] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 55 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 56 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0097] In some embodiments, a fusion protein is provided that includes: a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 56 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 55 or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0098] In some embodiments, a fusion protein is provided that includes an amino acid sequence set forth in any of SEQ ID NOs:20-27 (e.g., SEQ ID NOs:20-25), or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0099] In some embodiments, the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is a signal peptide from TGFβR1 or TGFβR2, and optionally, the signal peptide and the extracellular domain in the polypeptide are from the same molecule. In some embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for TGFβ, and the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling through the IL-23 receptor complex.

[0100] In some embodiments, the first polypeptide and the second polypeptide are in a single polypeptide, and the first polypeptide and the second polypeptide are separated by a multicistronic element. In some embodiments, the multicistronic element comprises a 2A self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, or F2A. In some embodiments, the first polypeptide is N-terminal to the second polypeptide. In some embodiments, the first polypeptide is C-terminal to the second polypeptide.

[0101] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of TGFβR1, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of TGFβR2, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0102] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR2, a first transmembrane domain comprising the transmembrane domain of TGFβR2, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR1, a second transmembrane domain comprising the transmembrane domain of TGFβR1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0103] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of TGFβR1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of TGFβR2, and a second intracellular domain comprising the intracellular domain of IL-23R. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0104] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-23R, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0105] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR2, a first transmembrane domain comprising the transmembrane domain of IL-23R, a first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR1, a second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0106] In some embodiments, a fusion protein is provided that comprises a single polypeptide comprising, from N-terminal to C-terminal, the following: a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-12Rβ1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-23R, and a second intracellular domain comprising the intracellular domain of IL-23R. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transduced from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0107] In some embodiments, fusion proteins are provided that are encoded by a nucleic acid comprising a nucleic acid sequence set forth in any of SEQ ID NOs:28-35 (e.g., SEQ ID NOs:28-33), or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0108] TGFβ signaling, TGFβR1 and TGFβR2 TGF-β (including three isoforms) is a potent, pleiotropic cytokine that regulates mammalian development, differentiation, and homeostasis in nearly all cell types and tissues. Each of the three TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) is initially synthesized as a 75 kDa homodimer known as pro-TGF-β. Pro-TGF-β is then cleaved in the Golgi to form mature TGF-β homodimers. These 25 kDa homodimers interact with latency-associated proteins to form small latent complexes. In the endoplasmic reticulum, single latent TGF-β binding proteins form disulfide bonds with TGF-β homodimers to form large latent complexes, allowing targeted transport to the extracellular matrix. After transport, the large latent complex interacts with fibronectin fibers and heparin sulfate proteoglycans on the cell membrane. Finally, the large latent complex localizes to fibrillin-rich microfibrils in the extracellular matrix where it is stored until it is activated. There, latent TGF-β is stored and remains biologically unavailable until it is activated. Latent TGF-β is activated by multiple factors, including proteases, thrombospondin 1, reactive oxygen species, and integrins. These factors release mature TGF-β by liberating it from the microfibril-associated large latent complex. This occurs through release from latency-associated proteins, degradation of latent TGF-β binding proteins, or modification of the latent complex conformation.

[0109] Once the ligand is activated, TGF-β signaling is mediated through SMAD and non-SMAD pathways to regulate transcription, translation, microRNA biogenesis, protein synthesis, and post-translational modifications. Although the downstream effects of TGF-β are highly context-dependent, its signaling is at least partially conserved in many cell types. In the canonical pathway, TGF-β ligands bind to the type 2 TGF-β receptor (TGFβR2), which recruits the type 1 TGF-β receptor (TGFβR1). These receptors dimerize and autophosphorylate on serine / threonine residues, allowing phosphorylation of SMAD2 and SMAD3 by TGFβR1. Here, activated SMAD proteins dissociate from SMAD anchor for receptor activation (SARA) proteins, hetero-oligomerize with SMAD4, translocate to the nucleus, and interact with a myriad of transcriptional coregulators and other factors to mediate the expression or repression of target genes.

[0110] Several natural isoforms of human TGFβR1 or TGFβR2 proteins are known. The domains or regions of TGFβR1 or TGFβR2 described herein (such as the extracellular domain, transmembrane domain, or juxtamembrane region of TGFβR1 or TGFβR2) encompass the corresponding domains or regions derived from natural isoforms of TGFβR1 or TGFβR2. In some embodiments, the corresponding domains or regions derived from natural isoforms of TGFβR1 or TGFβR2 have the intended effect corresponding to any of TGB23-1, TGB23-2, TGB23-3, TGB23-4, TGB23-5, and TGB23-6. For exemplary naturally occurring isoform sequences of TGFβR1, see NCBI Accession Nos. P36897.1 (SEQ ID NO:66), CAF02096.2 (SEQ ID NO:67), CAF02097.1 (SEQ ID NO:68), and AAH71181.1 (SEQ ID NO:69). For exemplary naturally occurring isoform sequences of TGFβR2, see NCBI Accession Nos. P37173.2 (SEQ ID NO:70), Q62312.1 (SEQ ID NO:71), the amino acid sequence of SEQ ID NO:72, and ABK42378.1 (SEQ ID NO:88).

[0111] In some embodiments, the TGFβR1 described herein comprises an amino acid sequence set forth in any of SEQ ID NOs: 66-69, or a variant (e.g., a functional variant) having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0112] In some embodiments, the TGFβR2 described herein comprises an amino acid sequence set forth in any of SEQ ID NOs: 70-72 and 88, or a variant (e.g., a functional variant) having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0113] Also encompassed herein are variants or portions thereof (e.g., domains, e.g., extracellular domain, transmembrane domain) of TGFβR1 or TGFβR2. In some embodiments, TGFβR1 and / or TGFβR2 also have any one or more (e.g., 2, 3, 4, 5, or more) modifications (e.g., substitutions, truncations, deletions, additions, such as any of the modifications described herein) in the extracellular domain and / or transmembrane domain. In some embodiments, the variant or modified TGFβR1 and / or TGFβR2 binds to TGFβ in a manner equivalent to or increased over the native isoforms of TGFβR1 and / or TGFβR2 (e.g., the canonical sequences of TGFβR1 and / or TGFβR2 (e.g., SEQ ID NO: 66 or 70)). In some embodiments, the variant or modified TGFβR1 and / or TGFβR2 transduce TGFβ signaling in a manner equivalent to or increased than naturally occurring isoforms of TGFβR1 and / or TGFβR2 (e.g., the canonical sequences of TGFβR1 and / or TGFβR2 (e.g., SEQ ID NO: 66 or 70)).

[0114] In some embodiments, TGFβR1 has a deletion of amino acids at positions 24-26 according to the numbering set forth in SEQ ID NO: 66. In some embodiments, TGFβR1 has an insertion of an alanine at position 26 according to the numbering set forth in SEQ ID NO: 66. In some embodiments, TGFβR1 has an I139V substitution according to the numbering set forth in SEQ ID NO: 66. In some embodiments, TGFβR1 has a V153I substitution according to the numbering set forth in SEQ ID NO: 66.

[0115] In some embodiments, TGFβR2 has an M36V substitution according to the numbering set forth in SEQ ID NO: 70. In some embodiments, TGFβR2 has a C61R substitution according to the numbering set forth in SEQ ID NO: 70. In some embodiments, TGFβR2 has an I73V substitution according to the numbering set forth in SEQ ID NO: 70. In some embodiments, TGFβR2 has an R190H substitution according to the numbering set forth in SEQ ID NO: 70. In some embodiments, TGFβR2 has a V191I substitution according to the numbering set forth in SEQ ID NO: 70.

[0116] IL-23 signaling, IL23R and IL-12Rβ1 Interleukin 23 (IL-23) is a proinflammatory cytokine involved in the resolution of infections caused by certain extracellular pathogens. In addition, several reports have established a firm link between excess IL-23 production and the development of inflammatory diseases in mouse models of experimental autoimmune encephalomyelitis (EAE), psoriasis, and inflammatory bowel disease. See, for example, Cua et al. Nature. 2003; 421: 744-748. Furthermore, increased levels of IL-23 have been found in biopsies from patients with Crohn's disease, ulcerative colitis, and psoriasis.

[0117] The role of IL-23 in tumor biology remains puzzling. See, for example, Yan et al., Cold Spring Harb Perspect Biol. 2018 Jul 2; 10(7): a028530. Meanwhile, various studies seem to suggest that IL-23 promotes tumor growth and metastasis. A seminal paper by Langowski et al. (Nature 442: 461-465, 2006) provided the first demonstration that mice lacking IL-23p19 were resistant to DMBA / TPA-induced skin papillomas, and this resistance correlated with a significant increase in skin-infiltrating CD8+ T cells and a reduction in IL-17A, matrix metallopeptidase 9 (MMP9), CD31, granulocytes (Gr-1+) and macrophages (CD11b+, F4 / 80+). A study by Teng et al. (Proc Natl Acad Sci 107:8328-8333,2010) further confirmed the resistance phenotype of these mice to DMBA / TPA-induced skin papillomas and highlighted their resistance to MCA-induced fibrosarcomas. Furthermore, this study showed that IL-23 not only suppressed the antitumor function of T cells, as first demonstrated by Langowski et al., but also suppressed the anti-metastatic function of NK cells (Teng et al.2010). In two other de novo mouse models of colon carcinogenesis (CPC-APC, ApcMin / +), both IL-23 and IL-17A had tumor-promoting effects, as loss of IL-23 / IL-23R / IL-17R or blockade of IL-23R / IL-17A resulted in reduced tumor burden (Wu et al. Nat Med 15:1016-1022, 2009; Grivennikov et al., Nature 491:254-258, 2012). Meanwhile, studies also suggest that IL-23 may have tumor-suppressing effects when overexpressed in different tumor cell lines and transplanted into mice (e.g., Ngiow et al., Trends Immunol 34:548-555, 2013).

[0118] IL-23 consists of a subunit called p40 (interleukin-12 subunit beta, Uniprot ID: P29460) and a subunit called p19 (interleukin-23 subunit alpha, Uniprot ID Q9NPF7) (Oppmann et al. Immunity 13:715-725(2000)). Subunit p40 is a common subunit shared by interleukin-12 (i.e., IL-12). IL-12 and IL-23 have been suggested to play contradictory roles in cancer. See, for example, Yan et al., Cold Spring Harb Perspect Biol. 2018 Jul 2;10(7):a028530. The antitumor and antimetastatic effects of IL-12 are thought to be mediated by IFN-γ activation of STAT4, whereas the mechanism of action of IL-23 has not been fully elucidated.

[0119] IL-23 binds to and signals through a heterodimeric receptor complex (i.e., the IL-23 receptor) composed of IL-12Rβ1 and IL-23R (Uniprot ID: Q5VWK5) subunits. IL-12Rβ1 is also part of the IL-12 receptor, which is a heterodimeric receptor formed by IL-12Rβ1 (Uniprot ID: P42701) and IL-12Rβ2 (Uniprot ID: Q99665), whereas IL-23R is unique to the IL-23 receptor complex. IL-23R pairs with IL-12Rβ1 to confer IL-23 responsiveness to cells expressing both subunits. Inhibition of either IL-12Rβ1 or IL-23R will result in blocking the IL-23 response of cells expressing both receptor chains (Parham, C. et al. J Immunol 168:5699-5708 (2002)).

[0120] IL-23R associates with JAK2 and STAT3 in a ligand-dependent manner. IL-12Rβ1 directly interacts with Tyk2. IL-23 binding to the IL-23 receptor causes phosphorylation and activation of JAK-STAT signaling molecules: JAK2, Tyk2, and STAT1, STAT3, STAT4, STAT5. The most important STAT induced by IL-23 is STAT3. IL-23-induced activation of STAT3 leads to direct binding of phosphorylated STAT3 to the IL-17 and IL-17F promoters. STAT3 also upregulates the expression of RORγ, a Th17-specific transcriptional regulator important for the expression of two members of the interleukin-17 family (IL-17 and IL-17F). IL-23-induced JAK2 activation induces the PI3K cat class IA / AKT(PKB) and NF-kB p50 / p65 pathways, which are required for IL-17 production. AKT(PKB) can activate both NF-kB p50 / p65 (IKKα / IKK / I-kB(cat) pathway) and STAT3 (via an undefined mechanism).

[0121] Multiple naturally occurring isoforms of human IL-23R or IL-12Rβ1 proteins are known. The domains or regions of IL-23R or IL-12Rβ1 described herein (such as the transmembrane domain, juxtamembrane domain, or intracellular domain of IL-23R or IL-12Rβ1) encompass the corresponding domains or regions from various naturally occurring isoforms of IL-23R or IL-12Rβ1. In some embodiments, the corresponding domains or regions from naturally occurring isoforms of IL-23R or IL-12Rβ1 have an intended effect equivalent to any of TGB23-1, TGB23-2, TGB23-3, TGB23-4, TGB23-5, and TGB23-6. For exemplary IL-23R native isoform sequences, see NCBI Accession Nos. AAM44229.1 (SEQ ID NO:73), AAY18347.1 (SEQ ID NO:74), AAY18349.1 (SEQ ID NO:75), and the amino acid sequences set forth in SEQ ID NOs:76 and 77. For exemplary IL-12Rβ1 native isoform sequences, see NCBI Accession No. CAC10446.1 (SEQ ID NO:78) and UniProt P42701-2 (SEQ ID NO:79).

[0122] In some embodiments, the IL-23R described herein comprises an amino acid sequence set forth in any of SEQ ID NOs:73-77, or a variant (e.g., a functional variant) having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0123] In some embodiments, the IL-12Rβ1 described herein comprises an amino acid sequence set forth in any of SEQ ID NOs:78-79, or a variant (e.g., a functional variant) having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0124] Also encompassed herein are variants or portions thereof (e.g., domains, such as transmembrane or intracellular domains) of IL-23R or IL-12Rβ1. In some embodiments, IL-23R and / or IL-12Rβ1 also have any one or more (e.g., 2, 3, 4, 5, or more) modifications (e.g., substitutions, truncations, deletions, additions, such as any of the modifications described herein) in the transmembrane and / or intracellular domains. In some embodiments, the variant or modified IL-23R and / or IL-12Rβ1 transduces signaling through IL-23R / IL-12Rβ1 in a manner equivalent to or increased over native isoforms of IL-23R and / or IL-12Rβ1 (e.g., the canonical sequences of IL-23R and / or IL-12Rβ1 (e.g., SEQ ID NO: 73 or 78)).

[0125] In some embodiments, the IL-23R has any one or more substitutions selected from the group consisting of R381Q, V475A, N481D, and S581R substitutions according to the numbering set forth in SEQ ID NO:73.

[0126] In some embodiments, the nucleotides encoding IL-23R have a modification in the 3' untranslated region of the IL-23R gene as described in Zwiers et al. See, e.g., the rs10889677 variant discussed in Zwiers et al., The Journal of Immunology. 2012;188:1573-1577.

[0127] Transmembrane domain In some embodiments, the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of TGFβR1, TGFβR2, IL-12Rβ1, and IL-23R. In some embodiments, the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of IL-12Rβ1 or IL-23R. In some embodiments, the transmembrane domain and the extracellular domain in the first and / or second polypeptide are derived from the same molecule. In some embodiments, the transmembrane domain and the intracellular domain in the first and / or second polypeptide are derived from the same molecule.

[0128] In some embodiments, the transmembrane domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:5, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:6, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of IL-23R comprises the amino acid sequence of SEQ ID NO:7, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the transmembrane domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0129] Linker (e.g. juxtamembrane sequence) In some embodiments, the first polypeptide or the second polypeptide further comprises a linker (e.g., juxtamembrane sequence) between two domains, one of which is a transmembrane domain and the other domain is an extracellular domain or an intracellular domain. Without being bound by theory, the linker (e.g., juxtamembrane sequence) may act as a transition between two functional domains to provide flexibility and / or promote the longevity of the fusion protein or signaling. In some embodiments, the linker (e.g., juxtamembrane sequence) is between the extracellular domain and the transmembrane domain. In some embodiments, the linker (e.g., juxtamembrane sequence) is between the transmembrane domain and the intracellular domain.

[0130] Exemplary Juxtamembrane Sequences In some embodiments, the linker is a juxtamembrane sequence derived from a juxtamembrane region from a transmembrane molecule, hi some embodiments, the juxtamembrane sequence and the transmembrane domain are derived from the same molecule.

[0131] In some embodiments, the juxtamembrane sequence between the extracellular domain and the transmembrane domain is a truncated C-terminal sequence of the extracellular domain derived from the same molecule as the transmembrane domain. In some embodiments, the juxtamembrane sequence between the transmembrane domain and the intracellular domain is a truncated N-terminal sequence of the intracellular domain derived from the same molecule as the transmembrane domain. In some embodiments, the juxtamembrane sequence has a length of about 2 amino acids to about 50 amino acids, such as about 3 amino acids to about 40 amino acids. In some embodiments, the juxtamembrane sequence comprises an amino acid sequence set forth in any of SEQ ID NOs: 9-12. "Sequence of IL-23R extracellular domain near the membrane", "sequence of IL-12Rβ1 extracellular domain near the membrane", "sequence of TGFβR1 intracellular domain near the membrane", and "sequence of TGFβR2 intracellular domain near the membrane" refer to different juxtamembrane sequences used in exemplary embodiments.

[0132] Other Exemplary Linkers Exemplary linkers include any of the peptide or non-peptide linkers described below. In some embodiments, the linker (such as a peptide linker) contains flexible residues (such as glycine and serine) so that adjacent domains can move freely relative to one another.

[0133] Exemplary linkers include any of the peptide or non-peptide linkers described below. In some embodiments, the linker (such as a peptide linker) contains flexible residues (such as glycine and serine) so that adjacent domains can move freely relative to one another.

[0134] Peptide Linker In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker has a length of about 1 to about 50, about 2 to about 40, about 3 to about 30, or about 4 to about 20 amino acids. In some embodiments, the linker is a GS linker.

[0135] The essential technical feature of such a peptide linker is that said peptide linker does not contain any polymerization activity. The characteristics of peptide linkers, including the absence of promotion of secondary structures, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A particularly preferred amino acid in the context of "peptide linker" is Gly. Furthermore, peptide linkers that do not promote any secondary structures are also preferred. The linkage of domains to each other can be provided, for example, by genetic engineering. Methods for preparing fused, operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994; or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0136] The peptide linker may be a stable linker, which is not cleavable by proteases, particularly by matrix metalloproteinases (MMPs).

[0137] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO:61), glycine-serine polymers (e.g., (GS) n (SEQ ID NO:62), (GSGGS) n (SEQ ID NO:63), (GGGGS) n (SEQ ID NO: 64), and (GGGS)n (including SEQ ID NO: 65), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to act as neutral tethers between components. Glycine has more significant access to the φψ space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that the design of fusion proteins may include all or part of a flexible linker, such that the linker includes a flexible linker portion and one or more portions that confer less flexible structure to provide the desired fusion protein structure. In some embodiments, the linker is a GS linker.

[0138] In some embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:57.

[0139] In some embodiments, the peptide linker comprises an IgG hinge region, such as a human IgG1 hinge region. In some embodiments, the peptide linker comprises an IgG hinge region, such as a human IgG1 hinge region. In some embodiments, the peptide linker comprises a modified sequence derived from an IgG hinge region, such as a human IgG1 hinge region.

[0140] III. Nucleic Acids and Vectors The application also provides nucleic acids encoding any of the fusion proteins described herein, or portions thereof.

[0141] In some embodiments, the nucleic acid comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 28-35, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the nucleic acid comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 28-33, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0142] In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding a functional foreign receptor (such as any of the functional foreign receptors described in the "Functional Foreign Receptors" section). In some embodiments, the functional foreign receptor comprises an extracellular ligand binding domain and optionally an intracellular signaling domain. In some embodiments, the functional foreign receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof. In some embodiments, the functional foreign receptor specifically recognizes a tumor antigen. In some embodiments, the functional foreign receptor comprises a CAR. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 36, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 89, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0143] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0144] In some embodiments, the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 38, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0145] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0146] In some embodiments, a nucleic acid sequence encoding a functional exogenous receptor is upstream to at least one of one or more nucleic acid sequences encoding a fusion protein (a "fusion protein nucleic acid sequence"), and optionally, the functional exogenous receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a third nucleic acid sequence encoding a second multicistronic element.

[0147] In some embodiments, a nucleic acid sequence encoding a functional exogenous receptor is downstream to at least one of one or more nucleic acid sequences encoding a fusion protein (a "fusion protein nucleic acid sequence"), and optionally, the functional exogenous receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a third nucleic acid sequence encoding a second multicistronic element.

[0148] In some embodiments, the second multicistronic element comprises a 2A self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, or F2A.

[0149] vector The present application provides vectors for cloning and expressing any one of the fusion proteins and / or functional foreign receptors (e.g., engineered TCR or CAR) described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells (such as mammalian cells). In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, and derivatives thereof. In some embodiments, the vector is a lentiviral vector. See, e.g., Example 2. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as other virology and molecular biology manuals.

[0150] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying fusion protein coding sequences and / or self-inactivating lentiviral vectors carrying foreign receptors (e.g., engineered TCR, CAR) can be packaged by protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration and propagation of the transgene in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0151] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon (such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system). In some embodiments, the vector is a polymer-based non-viral vector, such as poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly(ethyleneimine) (PEI), and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector (such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN)). In some embodiments, the vector is a peptide-based gene non-viral vector (such as poly-L-lysine). Any of the known non-viral vectors suitable for genome editing can be used to introduce nucleic acids encoding fusion proteins and / or nucleic acids encoding foreign receptors (such as engineered TCRs, CARs) into engineered immune effector cells (such as T cells). See, e.g., Yin H. et al. Nature Rev. Genetics (2014) 15:521-555; Aronovich EL et al. "The Sleeping Beauty transposon system: a non-viral vector for gene therapy." Hum. Mol. Genet. (2011) R1:R14-20; and Zhao S. et al. "PiggyBac transposon vectors: the tools of the human gene editing." Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference.In some embodiments, any one or more of the fusion proteins and / or nucleic acids encoding exogenous receptors (e.g., engineered TCRs, CARs) described herein are introduced into engineered immune effector cells (e.g., T cells) by physical methods, including but not limited to electroporation, sonoporation, photoporation, magnetofection, hydroporation.

[0152] In some embodiments, a vector (e.g., a viral vector such as a lentiviral vector) comprises any one of the nucleic acids encoding the fusion protein and / or exogenous receptor (e.g., engineered TCR, CAR) described herein. The nucleic acid may be cloned into the vector using any molecular cloning method known in the art, including, for example, using a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any of the promoters known in the art may be used in the present invention. Promoters may be broadly categorized as constitutive or regulatable (such as inducible) promoters.

[0153] promoter In some embodiments, the nucleic acid encoding the fusion protein and / or the foreign receptor (e.g., engineered TCR, CAR) described herein is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary promoters contemplated herein include, but are not limited to, cytomegalovirus immediate early promoter (CMV), human elongation factor 1 alpha (hEF1 alpha), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled with CMV early enhancer (CAGG), Rous sarcoma virus (RSV) promoter, polyoma enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, and "myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site replacement (MND)" promoter. The efficiency of such constitutive promoters for driving transgene expression has been widely compared in a large number of studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to drive CAR expression in primary human T cells and concluded that the hEF1α promoter not only induced the highest level of transgene expression, but also was optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8):1453-1464(2009)). In some embodiments, the nucleic acid encoding the fusion protein and / or the exogenous receptor (e.g., engineered TCR, CAR) described herein) is operably linked to the hEF1α promoter or the PGK promoter.

[0154] In some embodiments, the promoter is selected from the group consisting of EF-1 promoter, CMV IE gene promoter, EF-la promoter, ubiquitin C promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, simian virus 40 (SV40) promoter, cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-alpha), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken β-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), β-actin promoter (β-ACT), simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site replacement (MND) promoter, NFAT promoter, TETON® promoter, and NFκB promoter.

[0155] In some embodiments, the nucleic acid encoding the fusion protein and / or the exogenous receptor (e.g., engineered TCR, CAR) described herein is operably linked to an inducible promoter. An inducible promoter belongs to the category of regulatable promoters. An inducible promoter can be induced by one or more conditions (such as a physical condition, the microenvironment of the engineered immune effector cell (e.g., T cell), or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducer), or a combination thereof). In some embodiments, the induction condition does not induce expression of an endogenous gene in the engineered mammalian cell and / or in the subject receiving the pharmaceutical composition. In some embodiments, the induction condition is selected from the group consisting of an inducer, irradiation (ionizing radiation, light, etc.), temperature (heat, etc.), redox conditions, tumor environment, and activation conditions of the engineered mammalian cell. In some embodiments, the inducible promoter can be an NFAT promoter, a TETON® promoter, or an NFκB promoter.

[0156] In some embodiments, the vector also contains a selectable marker gene or reporter gene to select cells expressing the fusion protein and / or the foreign receptor described herein (e.g., engineered TCR, CAR) from a population of host cells transfected via the vector (e.g., lentiviral vector). Both the selectable marker and the reporter gene can be adjacent to appropriate regulatory sequences to allow expression in the host cell. For example, the vector can contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid sequence.

[0157] Multicistronic elements In some embodiments, the vector comprises more than one nucleic acid encoding a fusion protein and / or a foreign receptor (e.g., engineered TCR, CAR) as described herein. In some embodiments, the vector (e.g., a viral vector such as a lentiviral vector) comprises a first nucleic acid encoding a fusion protein and a second nucleic acid encoding a functional foreign receptor (e.g., engineered TCR, CAR) comprising an extracellular ligand binding domain and an optional intracellular signaling domain, the first nucleic acid being operably linked to the second nucleic acid via a third nucleic acid encoding a multicistronic element. In some embodiments, the third nucleic acid encoding a multicistronic element is an internal ribosome entry site (IRES). An IRES is an RNA element that allows translation initiation in a cap-independent manner. In some embodiments, the multicistronic element comprises (e.g., is) a self-cleaving 2A peptide (P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, etc.). In some embodiments, the multicistronic element is a P2A peptide comprising the amino acid sequence of SEQ ID NO: 48 or 60. In some embodiments, the multicistronic element comprises a peptide linker (such as a flexible linker) as described above in the "Peptide Linkers" section of "II. Fusion Proteins." In some embodiments, the flexible linking sequence is (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) n and wherein n is an integer of at least 1. In some embodiments, the third nucleic acid encodes a selectable marker (such as LNGFR). In some embodiments, the third nucleic acid encodes a multicistronic element comprising one or more types of linking sequences described herein, such as a self-cleaving 2A peptide (e.g., P2A) followed by a flexible linker of Gly-Ser (e.g., (GGGS) 3 ), or a self-cleaving 2A peptide (e.g., P2A) followed by a selectable marker (e.g., LNGFR, etc.).

[0158] Thus, in some embodiments, a vector (e.g., a viral vector such as a lentiviral vector) is provided that comprises a first nucleic acid encoding a fusion protein (such as any of those fusion proteins described herein). In some embodiments, the fusion protein comprises a) a first polypeptide comprising i) a first extracellular domain comprising the extracellular domain of TGFβR2, ii) a first transmembrane domain comprising the transmembrane domain of IL-23R, and iii) a first intracellular domain comprising the intracellular domain of IL-23R; and b) a second polypeptide comprising i) a second extracellular domain comprising the extracellular domain of TGFβR1, ii) a second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and iii) a second intracellular domain comprising the intracellular domain of IL-12Rβ1.

[0159] In some embodiments, the vector (e.g., a viral vector such as a lentiviral vector) further comprises a second nucleic acid encoding a functional exogenous receptor (e.g., an engineered TCR, CAR) comprising an extracellular ligand binding domain and an optional intracellular signaling domain. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to different promoters.

[0160] In some embodiments, a vector (e.g., a viral vector, such as a lentiviral vector) is provided that includes a first nucleic acid encoding a fusion protein (such as any of the fusion proteins described herein) and a second nucleic acid encoding a functional foreign receptor (e.g., an engineered TCR, CAR) comprising an extracellular ligand binding domain and an intracellular signaling domain, wherein the first nucleic acid and the second nucleic acid are operably linked to different promoters (e.g., EF1-α and a promoter different from EF1-α). In some embodiments, the first nucleic acid is upstream of the second nucleic acid. In some embodiments, the first nucleic acid is downstream of the second nucleic acid.

[0161] In some embodiments, a vector (e.g., a viral vector such as a lentiviral vector) is provided that includes, from upstream to downstream, a promoter (e.g., EF1-α), a first nucleic acid encoding a functional foreign receptor (e.g., an engineered TCR, CAR) comprising an extracellular ligand binding domain and an intracellular signaling domain, optionally a second promoter, and a second nucleic acid encoding a fusion protein. In some embodiments, the fusion protein includes a) a first polypeptide including i) a first extracellular domain including an extracellular domain of TGFβR2, ii) a first transmembrane domain including a transmembrane domain of IL-23R, and iii) a first intracellular domain including an intracellular domain of IL-23R; and b) a second polypeptide including i) a second extracellular domain including an extracellular domain of TGFβR1, ii) a second transmembrane domain including a transmembrane domain of IL-12Rβ1, and iii) a second intracellular domain including an intracellular domain of IL-12Rβ1.

[0162] Functional exogenous receptors Exemplary functional foreign receptors described herein may include an extracellular ligand-binding domain and, optionally, an intracellular signaling domain. In some embodiments, the functional foreign receptor is an engineered TCR (e.g., an engineered TCR that specifically recognizes a tumor antigen). In some embodiments, the functional foreign receptor is a non-TCR receptor. In some embodiments, the non-TCR receptor is a chimeric antigen receptor (CAR) that includes: (a) an extracellular ligand-binding domain that includes one or more (e.g., any one of 1, 2, 3, 4, 5, 6, or more) binding moieties that specifically recognize an antigen (e.g., a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the extracellular ligand-binding domain of the CAR includes one or more (e.g., any one of 1, 2, 3, 4, 5, 6, or more) binding moieties that include an antigen-binding fragment (e.g., an sdAb (e.g., an sdAb that targets a tumor antigen)) (hereinafter referred to as "anti-antigen CAR"). In some embodiments, the extracellular ligand-binding domain of the CAR comprises one or more binding moieties comprising at least one domain derived from the extracellular domain of a ligand or receptor (hereinafter also referred to as "ligand / receptor CAR"), where the ligand or receptor is a cell surface antigen. In some embodiments, the receptor is derived from an Fc-binding domain (such as the extracellular domain of an Fc receptor (e.g., FcγR)). A CAR comprising an extracellular ligand-binding domain comprising one or more binding moieties comprising an Fc-binding domain is hereinafter also referred to as an "antibody-coupled T cell receptor" (ACTR). In some embodiments, when the Fc-containing protein is administered to or co-expressed in an ACTR-T cell, the Fc-containing protein confers binding specificity of the ACTR-expressing T cell to an antigen as described herein. In some embodiments, the Fc-containing protein is an Fc-containing antibody (e.g., a full-length antibody) or an Fc fusion protein (such as an antigen-binding fragment-Fc fusion protein, an Fc receptor / ligand fusion protein, an Fc fusion protein comprising the variable region of a TCR fused to the Fc region of immunoglobulin G (IgG) ("TCR-Fc fusion protein"), etc.).The ACTR / Fc-containing protein system is hereafter referred to as "anti-antigen ACTR."

[0163] antigen The extracellular ligand-binding domain of the functional foreign receptor described herein may specifically recognize any antigen on a target cell. In some embodiments, the antigen is a cell surface molecule. In some embodiments, the antigen acts as a cell surface marker on a target cell associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. In some embodiments, the extracellular ligand-binding domain specifically recognizes a single tumor antigen. In some embodiments, the extracellular ligand-binding domain specifically recognizes one or more epitopes of a single tumor antigen. In some embodiments, the extracellular ligand-binding domain specifically recognizes two or more tumor antigens. Tumors express multiple proteins that may serve as target antigens for immune responses, particularly T-cell mediated immune responses. The antigen specifically recognized by the extracellular ligand-binding domain may be an antigen on a single diseased cell, or an antigen expressed on different cells that each contribute to the disease. The antigen specifically recognized by the extracellular ligand-binding domain may be directly or indirectly involved in the disease.

[0164] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. The choice of targeted antigen of the present invention will depend on the particular type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigens, BCMA (B cell maturation antigen), carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut These include hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), glypican 3 (GPC3), claudin 18.2, PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0165] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a large number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens (such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer). Other target molecules belong to the group of transformation-associated molecules (such as oncogene HER2 / nerve sheath / ErbB-2). Yet another group of target antigens are onco-fetal antigens (such as carcinoembryonic antigen (CEA)). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens (such as CD19, CD20, and CD37) are other candidates for target antigens in B-cell lymphomas.

[0166] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells of the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed in normal cells under conditions that do not successfully induce a state of immune tolerance to the antigen. Expression of antigens in tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed in normal cells during fetal development when the immune system is immature and not able to respond, or TAA can be an antigen that is normally present at very low levels in normal cells, but is expressed at much higher levels in tumor cells.

[0167] Non-limiting examples of TSA or TAA antigens include: differentiation antigens (such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2) and tumor-specific multilineage antigens (such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5); overexpressed fetal antigens (such as CEA); overexpressed oncogenes and mutated tumor suppressor genes (such as p53, Ras, HER2 / neu); unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, etc.; and viral antigens (such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7). Other macromolecular protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0168] In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, claudin 18.2, glycolipid F77, PD-L1, PD-L2, and any combination thereof. In some embodiments, the antigen is GPC3. In some embodiments, the antigen is CD20.

[0169] In some embodiments, the antigen is a pathogen antigen, such as a fungal antigen, a viral antigen, or a bacterial antigen. In some embodiments, the fungal antigen is from the genus Aspergillus or Candida. In some embodiments, the viral antigen is from herpes simplex virus (HSV), respiratory syncytial virus (RSV), metapneumovirus (hMPV), rhinovirus, parainfluenza (PIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), JC virus (John Cunningham virus), BK virus, HIV, Zika virus, human coronavirus, norovirus, encephalitis virus, or Ebola.

[0170] In some embodiments, the cell surface antigen is a ligand or receptor. In some embodiments, the extracellular ligand binding domain comprises one or more binding moieties comprising at least one domain derived from the extracellular domain of a ligand or receptor, and the ligand or receptor is a cell surface antigen described herein. In some embodiments, the ligand or receptor is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, NKG2D, BCMA, APRIL, BAFF, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the ligand is derived from APRIL or BAFF, which can bind to BCMA. In some embodiments, the receptor is derived from an Fc binding domain (such as the extracellular domain of an Fc receptor). In some embodiments, the Fc receptor is an Fc gamma receptor (FcγR). In some embodiments, the FcγR is selected from the group consisting of CD16A (FcγRIIIa), CD16B (FcγRIIIb), CD64A, CD64B, CD64C, CD32A, and CD32B.

[0171] Chimeric antigen receptors (CARs) In some embodiments, the functional foreign receptor is a CAR that comprises: (a) an extracellular ligand binding domain that comprises one or more (such as any one of 1, 2, 3, 4, 5, 6, or more) binding moieties that specifically recognize an antigen (such as any of the antigens described above (e.g., GPC3); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, the one or more binding moieties are derived from one or more antibodies (e.g., full-length antibodies). In some embodiments, the one or more binding moieties are derived from camelid antibodies. In some embodiments, the one or more binding moieties are derived from human antibodies. In some embodiments, the one or more binding moieties are derived from camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibodies (scFv), bis-scFv, (scFv) 2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, nanobodies). In some embodiments, the one or more binding moieties are sdAbs (e.g., anti-GPC3 sdAb, anti-BCMA sdAb). In some embodiments, the extracellular ligand binding domain comprises two or more sdAbs linked together. In some embodiments, the one or more binding moieties are non-antibody binding proteins (such as polypeptide ligands, or engineered proteins that bind to antigens). In some embodiments, the one or more binding moieties comprise at least one domain derived from the extracellular domain of a ligand or receptor, wherein the ligand or receptor is a cell surface antigen. In some embodiments, the ligand or receptor is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, NKG2D, BCMA, APRIL, BAFF, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the ligand is derived from APRIL or BAFF, which can bind to BCMA. In some embodiments, the receptor is derived from an Fc binding domain (such as the extracellular domain of an Fc receptor). In some embodiments, the Fc receptor is an Fc gamma receptor (FcγR). In some embodiments, the FcγR is selected from the group consisting of CD16A (FcγRIIIa), CD16B (FcγRIIIb), CD64A, CD64B, CD64C, CD32A, and CD32B. In some embodiments, the CAR is monovalent and monospecific. In some embodiments, the CAR is multivalent (e.g., bispecific) and monospecific. In some embodiments, the CAR is multivalent (e.g., bivalent) and multispecific (e.g., bispecific).In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, claudin 18.2, glycolipid F77, PD-L1, PD-L2, and any combination thereof. In some embodiments, the antigen is GPC3. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3zeta, CD3epsilon, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137(4-1BB), CD152, CD154, and PD-1. In some embodiments, the transmembrane domain is derived from CD8alpha. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3zeta, CD3gamma, CD3epsilon, CD3delta, FcRgamma (FCER1G), FcRbeta (FcεRIb), CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain is derived from CD3zeta. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain.In some embodiments, the costimulatory signaling domain is selected from the group consisting of CARD11, CD2 (LFA-2), CD7, CD27, CD28, CD30, CD40, CD54 (ICAM-1), CD134 (OX40), CD137 (4-1BB), CD162 (SELPLG), CD258 (LIGHT), CD270 (HVEM, LIGHTR), CD276 (B7-H3), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), LFA-1 (lymphocyte function associated antigen-1), NKG2C, CDS, GITR, BAFF. The costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of R, NKp80 (KLRF1), CD160, CD19, CD4, IPO-3, BLAME (SLAMF8), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, CD150 (SLAMF1), CD152 (CTLA-4), CD223 (LAG3), CD273 (PD-L2), CD274 (PD-L1), DAP10, TRIM, ZAP70, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the costimulatory signaling domain comprises a cytoplasmic domain of CD137. In some embodiments, the CAR described herein further comprises a hinge domain located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the CAR comprises a polypeptide comprising, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular ligand binding domain (e.g., one or more sdAbs that specifically recognize a tumor antigen), a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.

[0172] In some embodiments, a CAR comprises a polypeptide comprising, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular ligand binding domain comprising one or more binding moieties targeting a tumor antigen (e.g., anti-GPC3 svFv or anti-CD20 scFv), a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, a CAR comprises a polypeptide comprising, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular ligand binding domain comprising one or more binding moieties targeting a tumor antigen (e.g., anti-GPC3 scFv or anti-CD20 scFv), a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.

[0173] In some embodiments, the CAR of the present application is a "BCMA-ligand CAR." In some embodiments, the CAR comprises a polypeptide comprising, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular ligand binding domain comprising one or more binding moieties comprising at least one domain derived from APRIL or BAFF, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the extracellular ligand binding domain comprises an APRIL domain. In some embodiments, the extracellular ligand binding domain comprises a BAFF domain. In some embodiments, the extracellular ligand binding domain comprises an APRIL domain and a BAFF domain.

[0174] In some embodiments, the CAR of the present application is an ACTR. Engineered T cells with ACTR can bind to an Fc-containing protein (such as a monoclonal antibody, e.g., an anti-BCMA antibody), which then acts as a bridge to the tumor cell. In some embodiments, the CAR comprises a polypeptide comprising, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular ligand binding domain comprising one or more binding moieties comprising an Fc binding domain (such as an Fc receptor, e.g., FcγR), a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the FcγR is selected from the group consisting of CD16A (FcγRIIIa), CD16B (FcγRIIIb), CD64A, CD64B, CD64C, CD32A, and CD32B.

[0175] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:36.

[0176] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:89.

[0177] Any CAR known in the art or developed by the inventors can be used to construct the CARs described herein, including those described in PCT / CN2020 / 112181, PCT / CN2020 / 112182, PCT / CN2017 / 096938, and PCT / CN2016 / 094408, the contents of which are incorporated herein by reference in their entireties. Exemplary structures of CARs are shown in Figures 15A-15D of PCT / CN2017 / 096938.

[0178] Multivalent and / or multispecific CARs In some embodiments, the CAR described herein is a multivalent CAR comprising: (a) an extracellular ligand binding domain comprising one or more (such as any one of 2, 3, 4, 5, 6, or more) binding moieties that specifically recognize an antigen (e.g., any of the antigens described herein); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, one or more of the binding moieties are antigen-binding fragments. In some embodiments, one or more of the binding moieties comprise a single domain antibody (e.g., an anti-GPC3 sdAb). In some embodiments, one or more of the binding moieties are derived from a camelid antibody. In some embodiments, one or more of the binding moieties are derived from a four-chain antibody. In some embodiments, one or more of the binding moieties are scFvs. In some embodiments, one or more of the binding moieties are derived from a human antibody. In some embodiments, one or more of the binding moieties are polypeptide ligands or other non-antibody polypeptides that specifically bind to an antigen. In some embodiments, the multivalent CAR is monospecific, i.e., the multivalent CAR targets a single antigen and contains two or more binding sites for a single antigen. In some embodiments, the multivalent CAR is multispecific, i.e., the multivalent CAR targets more than one antigen and contains two or more binding sites for at least one antigen. Binding moieties specific for the same antigen may bind to the same epitope of the antigen (i.e., a "monoepitopic CAR") or to different epitopes of the antigen (i.e., a "multiepitopic CAR" such as a bi-epitopic or tri-epitopic CAR). Binding sites specific for the same antigen may contain the same or different sdAbs.In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, claudin 18.2, glycolipid F77, PD-L1, PD-L2, and any combination thereof. In some embodiments, the antigen is GPC3.

[0179] In some embodiments, the CAR described herein is a multivalent (such as bivalent, trivalent, or higher valent) CAR that includes: (a) an extracellular ligand binding domain that includes a plurality (such as at least about any one of 2, 3, 4, 5, 6, or more) of binding moieties that specifically bind to an antigen (such as a tumor antigen (e.g., GPC3, CD20)); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the CAR described herein is a multivalent (such as bivalent, trivalent, or higher valent) CAR that includes: (a) an extracellular ligand binding domain that includes a plurality (such as at least about any one of 2, 3, 4, 5, 6, or more) of binding moieties that specifically bind to an antigen (such as a tumor antigen (e.g., GPC3, CD20)); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the CAR described herein is a multivalent (such as bivalent, trivalent, or higher valent) CAR comprising: (a) an extracellular ligand binding domain comprising multiple (such as at least about any one of 2, 3, 4, 5, 6, or more) sdAbs that specifically bind to an antigen (such as a tumor antigen (e.g., GPC3, CD20)); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the first epitope and the second epitope are different. In some embodiments, the first epitope and the second epitope are the same. In some embodiments, the first binding moiety is an sdAb and the second binding moiety is derived from a human antibody (e.g., scFv). In some embodiments, the multivalent CAR specifically binds to two different epitopes on the antigen. In some embodiments, the multivalent CAR specifically binds to three or more different epitopes on the antigen. In some embodiments, a CAR described herein is a multivalent (such as bivalent, trivalent, or higher valent) CAR comprising: (a) an extracellular ligand binding domain comprising a first sdAb that specifically binds to a first epitope of an antigen (such as a tumor antigen (e.g., GPC3, CD20)) and a second sdAb that specifically binds to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain.In some embodiments, the first epitope and the second epitope are different. In some embodiments, the first epitope and the second epitope are the same. In some embodiments, the CAR described herein is a multivalent (such as bivalent, trivalent, or higher) CAR comprising: (a) an extracellular ligand-binding domain comprising a first sdAb that specifically binds to a first antigen (such as a tumor antigen (e.g., GPC3, CD20)) and a second sdAb that specifically binds to a second antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, claudin 18.2, glycolipid F77, PD-L1, PD-L2, and any combination thereof. In some embodiments, the antigen is GPC3. In some embodiments, the antigen is CD20.

[0180] In some embodiments, the CAR described herein comprises: (a) a first sdAb that specifically binds to a first epitope of GPC3 ("anti-GPC3 sdAb1" or "anti-GPC3 V"); H H1") and a second sdAb that specifically binds to a second epitope of GPC3 ("anti-GPC3 sdAb2" or "anti-GPC3 V H (b) an extracellular ligand-binding domain comprising an extracellular ligand-binding domain (e.g., GPC3 sdAb1 and GPC3 sdAb2 "H2"); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-GPC3 sdAb1 and the anti-GPC3 sdAb2 are the same. In some embodiments, the anti-GPC3 sdAb1 and the anti-GPC3 sdAb2 are different.

[0181] Extracellular Ligand Binding Domain The extracellular ligand binding domain of the CAR described herein comprises one or more (such as any one of 1, 2, 3, 4, 5, 6, or more) binding moieties (sdAbs). In some embodiments, the one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, the one or more binding moieties are derived from a four-chain antibody. In some embodiments, the one or more binding moieties are derived from a camelid antibody. In some embodiments, the one or more binding moieties are derived from a human antibody. In some embodiments, the one or more binding moieties are derived from a camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain Fv antibody (scFv), bis-scFv, (scFv) 2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, nanobodies). In some embodiments, the one or more binding moieties are sdAbs (e.g., anti-BCMA sdAbs). In some embodiments, the one or more binding moieties are non-antibody binding proteins (such as polypeptide ligands, or engineered proteins that bind to antigens). In some embodiments, the one or more binding moieties comprise at least one domain derived from the extracellular domain of a ligand or receptor, and the ligand or receptor is a cell surface antigen. In some embodiments, the ligand or receptor is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, NKG2D, BCMA, APRIL, BAFF, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the ligand is derived from APRIL or BAFF, which can bind to BCMA. In some embodiments, the receptor is derived from an Fc binding domain (such as the extracellular domain of an Fc receptor). In some embodiments, the Fc receptor is an Fcγ receptor (FcγR). In some embodiments, the FcγR is selected from the group consisting of CD16A (FcγRIIIa), CD16B (FcγRIIIb), CD64A, CD64B, CD64C, CD32A, and CD32B. The binding moieties can be directly fused to each other via a peptide bond or a peptide linker.

[0182] In some embodiments, the extracellular ligand binding domain of the CAR is a scFv that targets a tumor antigen. In some embodiments, the extracellular ligand binding domain of the CAR is an anti-GPC3 scFv. In some embodiments, the anti-GPC3 scFv is a VFv having a sequence as set forth in any one of SEQ ID NOs: 80. H A heavy chain variable region (VH-CDR1, VH-CDR2, and VH-CDR3) including the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, respectively. Hb) a V having a sequence as set forth in any one of SEQ ID NOs: 81; L A light chain variable region (VL-CDR1, VL-CDR2, and VL-CDR3) including the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, respectively. L In some embodiments, the anti-GPC3 scFv comprises a heavy chain variable region (VH) comprising an amino acid sequence comprising VH-CDR1 as set forth in SEQ ID NO: 82, an amino acid sequence comprising VH-CDR2 as set forth in SEQ ID NO: 83, and an amino acid sequence comprising VH-CDR3 as set forth in SEQ ID NO: 84. H ) and b) a light chain variable region (VL-CDR1) comprising an amino acid sequence comprising VL-CDR2 as set forth in SEQ ID NO: 85, an amino acid sequence comprising VL-CDR3 as set forth in SEQ ID NO: 86, and an amino acid sequence comprising VL-CDR3 as set forth in SEQ ID NO: 87. L In some embodiments, the anti-GPC3 scFv comprises a heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 80, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. H and / or a light chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 81, or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. L ).

[0183] In some embodiments, the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO:58 or a variant having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO:58.

[0184] Single Domain Antibodies (sdAbs) In some embodiments, the CAR comprises an extracellular ligand binding domain that comprises one or more sdAbs. The sdAbs can be of the same or different origin and of the same or different size. Exemplary sdAbs include heavy chain only antibodies (e.g., V H H or V NAR ), a binding molecule that is naturally devoid of light chains, a single domain (V H or V L Such sdAbs include, but are not limited to, humanized heavy chain only antibodies, human sdAbs produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed by the inventors can be used to construct the CARs described herein, including the sdAbs described in PCT / CN2017 / 096938 and PCT / CN2016 / 094408, the contents of which are incorporated herein by reference in their entirety. Exemplary structures of CARs are shown in Figures 15A-15D of PCT / CN2017 / 096938. sdAbs may be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. Single domain antibodies contemplated herein also include naturally occurring sdAb molecules from species other than Camelidae and sharks.

[0185] In some embodiments, sdAbs are derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies that are devoid of light chains (also referred to herein as "heavy chain only antibodies"). Such single domain molecules are disclosed, for example, in WO94 / 04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For reasons of clarity, variable domains derived from heavy chain molecules that are naturally devoid of light chains are represented by the conventional V domains of four chain immunoglobulins. HIn order to distinguish it from H H. Such V H H molecules may be derived from antibodies made in Camelidae species (e.g. camel, llama, vicuna, dromedary, alpaca, guanaco). Other non-Camelidae species may produce heavy chain molecules that are naturally devoid of light chains, and such V H H is within the scope of this application.

[0186] V from Camelidae H H molecules are about 10 times smaller than IgG molecules. They are single polypeptides and can be very stable and resistant to extreme pH and temperature conditions. Furthermore, they can be resistant to the action of proteases, which is not the case for conventional four-chain antibodies. Furthermore, V H In vitro expression of H produces high yields of properly folded, functional V H In addition, antibodies generated in camelids may recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than camelids (see, for example, WO9749805). Thus, one or more V H Multispecific or multivalent CARs that contain H domains may interact more efficiently than multispecific or multivalent CARs that contain antigen-binding fragments derived from conventional four-chain antibodies. H Since H is known to bind into "unconventional" epitopes (such as cavities or grooves), such V H The affinity of H-containing CARs may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0187] In some embodiments, the sdAb is derived from the variable region of an immunoglobulin found in cartilaginous fish. For example, the sdAb may be derived from an immunoglobulin isotype known as the novel antigen receptor (NAR) found in the serum of sharks. Methods for producing single domain molecules derived from the variable region of the NAR ("IgNAR") are described in WO03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0188] In some embodiments, sdAbs are recombinant, CDR grafted, camelized, humanized, deimmunized, and / or in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues in the framework regions. Camelization involves altering the V H V from a conventional four-chain antibody (as occurs in nature) by one or more of the amino acid residues occurring at the corresponding position(s) in the H domain. H The term "camelization" refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a domain. Such camelization can be performed in a manner known per se and will be clear to the skilled person, for example on the basis of the further description herein. Such "camelization" substitutions are preferably H -V L Boundary amino acid positions and / or V H -V L Insertions are made at amino acid positions which lie at boundaries and / or at so-called Camelidae hallmark residues as defined herein (see, e.g., WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290, 1994; Davies and Riechmann Protein Engineering 9(6):531-537, 1996; Riechmann J. Mol. Biol. 259:957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth. 231:25-38, 1999).

[0189] In some embodiments, the sdAb is a human sdAb produced by a transgenic mouse or rat expressing a human heavy chain segment. See, e.g., US20090307787A1, US Patent Application No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity matured.

[0190] In some embodiments, a naturally occurring V H The H domain is the V domain of camelids. H The method may be obtained from a (naive or immune) library of H sequences. Such a method may or may not involve screening such a library with said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known per se. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, a (naive or immune) VH sequence may be obtained from a (naive or immune) library of H sequences. Such a method may or may not involve screening such a library with said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known per se. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. H Improved synthetic or semi-synthetic libraries derived from the H library can be used, e.g., by techniques such as random mutagenesis and / or CDR shuffling as described in WO00 / 43507, to generate (naive or immune) V H V obtained from the H library H H Library, etc.

[0191] In some embodiments, sdAbs are generated from traditional four chain antibodies, see e.g. EP0368684, Ward et al. (Nature 1989 Oct.12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003,21(11):484-490; WO06 / 030220; and WO06 / 003388.

[0192] The various binding moieties (sdAbs, ligand / receptor domains, etc.) in the multispecific or multivalent CARs described herein can be fused to each other via a peptide linker (such as any of the peptide linkers described in Section II). In some embodiments, the binding moieties (sdAbs, ligand / receptor domains, etc.) are fused directly to each other without a peptide linker. The peptide linkers connecting the different binding moieties (sdAbs, ligand / receptor domains, etc.) can be the same or different. The different domains of the CAR can also be fused to each other via a peptide linker.

[0193] Each peptide linker in a CAR may have the same or different length and / or sequence, depending on the structural and / or functional features of the sdAb and / or various domains (e.g., ligand / receptor domains). Each peptide linker may be selected and optimized independently. The length, degree of flexibility, and / or other properties of the peptide linker(s) used in a CAR may have some effect on properties including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not sterically interfere with each other. For example, in a multivalent or multispecific CAR described herein that includes sdAbs directed against a multimeric antigen, the length and flexibility of the peptide linker is preferably such that each sdAb in the multivalent CAR can bind to an antigenic determinant on each of the subunits of the multimer. In some embodiments, a short peptide linker may be placed between the transmembrane domain and the intracellular signaling domain of the CAR. In some embodiments, the peptide linker includes flexible residues (such as glycine and serine) so that adjacent domains can move freely relative to one another. For example, a glycine-serine doublet can be a suitable peptide linker.

[0194] The peptide linker can be any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids in length. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids in length, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 1 amino acid to about 100 amino acids. In some embodiments, the peptide linker comprises the amino acid sequence of any of SEQ ID NOs: 57 and 61-65.

[0195] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO1996 / 34103.

[0196] Transmembrane domain The CAR of the present application comprises a transmembrane domain that can be directly or indirectly fused to an extracellular ligand-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain that is compatible for use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane).

[0197] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of two or more alpha helices, a beta barrel, or other stable structure capable of passing through the phospholipid bilayer of a cell. In addition, transmembrane domains may also be classified, or alternatively, based on the transmembrane domain topology, including the number of passes the transmembrane domain makes across the membrane and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, whereas a multiple-pass membrane protein crosses the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins may be defined as type I, type II, or type III, depending on the ends of the membrane protein relative to the inside and outside of the cell and the topology of the membrane-spanning segment(s). Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have a single transmembrane domain, but are oriented such that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the location of the N- and C-termini.

[0198] In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multiple-pass membrane proteins may also be adapted for use in the CAR described herein. Multiple-pass membrane proteins may contain multiple (at least 2, 3, 4, 5, 6, 7, or more) alpha-helical or beta-sheet structures. Preferably, the N-terminus and C-terminus of the multiple-pass membrane protein are on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side.

[0199] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDIIa, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD1 60, CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CDIIa, LFA-1, I TGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3zeta, CD3epsilon, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137(4-1BB), CD152, CD154, and PD-1. In some embodiments, the transmembrane domain is derived from CD8alpha. In some embodiments, the transmembrane domain is derived from CD28.

[0200] The transmembrane domain for use in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha-helix or beta-sheet. In some embodiments, the protein segment is at least approximately 20 amino acids (e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids). Examples of synthetic transmembrane domains are known in the art, such as those in U.S. Patent No. 7,052,906B1 and PCT Publication No. WO2000 / 032776A2, the relevant disclosures of which are incorporated herein by reference.

[0201] The transmembrane domain may include a transmembrane region located C-terminal to the transmembrane domain and a cytoplasmic region. The cytoplasmic region of the transmembrane domain includes three or more amino acids, which in some embodiments help to orient the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain includes positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain includes the amino acids arginine, serine, and lysine.

[0202] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the CAR comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues (such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine). In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydrophobic or hydrophobic or hydrophilic character of a protein or a segment of a protein can be assessed by any method known in the art (e.g., Kyte and Doolittle hydrophobicity analysis).

[0203] Intracellular signaling domain The CAR of the present application comprises an intracellular signaling domain. The intracellular signaling domain is involved in the activation of at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity (including secretion of cytokines). The term "cytoplasmic signaling domain" therefore refers to a portion of a protein that transmits an effector function signal and directs the cell to carry out a specialized function. Usually, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. In cases where a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used instead of the intact chain, so long as it transmits the effector function signal. The term cytoplasmic signaling domain is therefore meant to encompass any truncated portion of the cytoplasmic signaling domain that is sufficient to transmit an effector function signal.

[0204] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. An "ITAM" as used herein is a conserved protein motif that is generally present in the tails of signaling molecules expressed in many immune cells. The motif may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs in signaling molecules are important for intracellular signal transduction, which is at least partially mediated by phosphorylation of tyrosine residues in the ITAM and subsequent activation of the signaling molecule. ITAMs can also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3zeta, CD3gamma, CD3epsilon, CD3delta, FcRgamma (FCER1G), FcRbeta (FcεRIb), CD5, CD22, CD79a, CD79b, CD66d, FcgammaRIIa, DAP10, and DAP12.

[0205] In some embodiments, the primary intracellular signaling domain is derived from CD3zeta. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3zeta. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3zeta.

[0206] Costimulatory Signaling Domain Many immune effector cells (e.g., T cells) require costimulation in addition to stimulating antigen-specific signals to promote cell proliferation, differentiation, and survival, and to activate cell effector functions. In some embodiments, the CAR comprises at least one costimulatory signaling domain. The term "costimulatory signaling domain" as used herein refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response (such as an effector function). The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein, which transmits a signal and regulates responses mediated by immune cells (such as T cells, NK cells, macrophages, neutrophils, or eosinophils). The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as, but not limited to, proliferation and survival) by the immune cell.

[0207] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domain (such as two copies of the costimulatory signaling domain of CD28 or CD137 (4-1BB)). In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins (such as any two or more costimulatory proteins described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3zeta) and one or more costimulatory signaling domains (such as 4-1BB). In some embodiments, one or more costimulatory signaling domains and a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains may be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains may provide additive or synergistic stimulatory effects.

[0208] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be adapted for use in the CARs described herein. The type(s) of costimulatory signaling domain are selected based on factors such as the type of immune effector cell on which the effector molecule is expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of costimulatory signaling domains for use in CARs can be the cytoplasmic signaling domains of costimulatory proteins, such as members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B ... R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B);Members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and other costimulatory molecules (CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1) and NKG2C, etc.;

[0209] In some embodiments, one or more costimulatory signaling domains are selected from the group consisting of CARD11, CD2 (LFA-2), CD7, CD27, CD28, CD30, CD40, CD54 (ICAM-1), CD134 (OX40), CD137 (4-1BB), CD162 (SELPLG), CD258 (LIGHT), CD270 (HVEM, LIGHTR), CD276 (B7-H3), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), LFA-1 (lymphocyte function associated antigen-1), NKG2C, CDS, GITR, The co-stimulatory molecule is selected from the group consisting of BAFFR, NKp80 (KLRF1), CD160, CD19, CD4, IPO-3, BLAME (SLAMF8), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, CD150 (SLAMF1), CD152 (CTLA-4), CD223 (LAG3), CD273 (PD-L2), CD274 (PD-L1), DAP10, TRIM, ZAP70, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the one or more costimulatory signaling domains are derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0210] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises a costimulatory signaling domain derived from 4-1BB (CD137). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3zeta and the costimulatory signaling domain of 4-1BB.

[0211] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3zeta and the costimulatory signaling domain of CD28.

[0212] In some embodiments, the intracellular signaling domain in a CAR of the present application comprises a costimulatory signaling domain of CD28 and a costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3zeta, a costimulatory signaling domain of CD28, and a costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises a polypeptide that comprises, from N-terminus to C-terminus, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and a cytoplasmic signaling domain of CD3zeta.

[0213] Variants of any of the costimulatory signaling domains described herein are also within the scope of the present disclosure, such that the costimulatory signaling domain is capable of regulating the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue variations compared to the wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues in the costimulatory signaling domain may result in increased signaling and enhanced stimulation of immune response compared to a costimulatory signaling domain that does not comprise a mutation. Mutation of amino acid residues in the costimulatory signaling domain may result in decreased signaling and reduced stimulation of immune response compared to a costimulatory signaling domain that does not comprise a mutation.

[0214] Hinge The CAR of the present application may comprise a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, which may allow protein flexibility and movement of one or both of the domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule may be used.

[0215] The hinge domain can contain about 10-100 amino acids (e.g., any one of about 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids). In some embodiments, the hinge domain can be any one of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0216] In some embodiments, the hinge domain is a naturally occurring protein hinge domain. Any protein hinge domain known in the art to contain a hinge domain is suitable for use in the CAR described herein. In some embodiments, the hinge domain is at least a portion of a naturally occurring protein hinge domain, conferring flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α.

[0217] The hinge domain of an antibody (such as an IgG antibody, an IgA antibody, an IgM antibody, an IgE antibody, or an IgD antibody) is also compatible for use in the pH-dependent chimeric receptor system described herein. In some embodiments, the hinge domain is a hinge domain connecting constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises an antibody hinge domain and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises an antibody hinge domain and an antibody CH3 constant region. In some embodiments, the hinge domain comprises an antibody hinge domain and antibody CH2 and CH3 constant regions. In some embodiments, the antibody is an IgG antibody, an IgA antibody, an IgM antibody, an IgE antibody, or an IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

[0218] Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker such as a (GxS)n linker, where x and n can independently be integers from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0219] Signal peptide The CAR of the present application may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide will target the effector molecule to the secretory pathway of the cell and allow incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including naturally occurring protein signal sequences or synthetic non-naturally occurring signal sequences, that are compatible for use in the CARs described herein will be apparent to one of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α.

[0220] ACTR is a chimeric protein that combines an Fc receptor (CD16) with a signaling domain (4-1BB / CD3ζ). Engineered T cells bearing ACTR can bind to monoclonal antibodies, which then act as a bridge to tumor cells.

[0221] In some embodiments, the functional foreign receptor is a chimeric receptor comprising: (a) an extracellular ligand-binding domain that comprises at least one domain derived from the extracellular domain of a ligand or receptor, where the ligand or receptor is a cell surface antigen (e.g., NKG2D, BCMA, IL-3, IL-13); (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0222] In some embodiments, the extracellular ligand binding domain comprises at least one domain derived from a ligand of BCMA (e.g., APRIL, BAFF). In some embodiments, the extracellular ligand binding domain comprises an antigen binding fragment (e.g., an sdAb) that specifically recognizes one or more epitopes of BCMA.

[0223] Manipulated TCR In some embodiments, the functional foreign receptor is an engineered TCR (e.g., an engineered TCR that specifically recognizes a tumor antigen, or a tumor antigen-MHC complex) that comprises an extracellular ligand-binding domain comprising Va and Vβ derived from a wild-type TCR, where the Va and Vβ together specifically recognize an antigen (e.g., a tumor antigen, such as any of the antigens described herein), and the Va, Vβ, or both comprise mutations in one or more CDRs compared to the wild-type TCR. In some embodiments, the mutations lead to amino acid substitutions (such as conservative amino acid substitutions). In some embodiments, the engineered TCR binds to the same cognate peptide-MHC as bound by the wild-type TCR. In some embodiments, the engineered TCR binds to the same cognate peptide-MHC with a higher affinity compared to that bound by the wild-type TCR. In some embodiments, the engineered TCR binds to the same cognate peptide-MHC with a lower affinity compared to that bound by the wild-type TCR. In some embodiments, the engineered TCR binds to a non-cognate peptide-MHC that is not bound by the wild-type TCR. In some embodiments, the engineered TCR is a single chain TCR (scTCR). In some embodiments, the engineered TCR is a dimeric TCR (dTCR). In some embodiments, the wild-type TCR binds HLA-A2. In some embodiments, the engineered TCR further comprises an intracellular signaling domain (such as a primary intracellular signaling domain derived from CD3ζ).

[0224] In some embodiments, the engineered TCR comprises an extracellular ligand binding domain comprising Vα and Vβ derived from a wild-type TCR, the Vα and Vβ together specifically recognize a tumor antigen or a tumor antigen-MHC complex, and the Vα, Vβ, or both comprise mutations in one or more CDRs compared to the wild-type TCR. In some embodiments, the engineered anti-tumor antigen TCR has a higher binding affinity to a tumor antigen than the wild-type anti-tumor antigen TCR. In some embodiments, the engineered TCR further comprises an intracellular signaling domain (such as a primary intracellular signaling domain derived from CD3ζ).

[0225] IV. Engineered Cells The present application provides engineered cells that contain any of the fusion proteins, vectors, and / or nucleic acids described herein.

[0226] In some embodiments, the engineered cell comprises a) a fusion protein (such as any of the fusion proteins described herein) and b) a functional exogenous receptor (such as any of the functional exogenous receptors described herein). In some embodiments, both the fusion protein and the functional exogenous receptor are expressed on the surface of the engineered cell.

[0227] In some embodiments, engineered cells (e.g., immune cells, e.g., T cells, e.g., CD8 T cells) are provided that contain (or express) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising the intracellular domain of one of IL-12Rβ1 or IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising the extracellular domain of the other of TGFβR1 or TGFβR2, ii) a second transmembrane domain, and iii) a second intracellular domain comprising the intracellular domain of the other of IL-12Rβ1 or IL-23R. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0228] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR1; ii) a first transmembrane domain comprising a transmembrane domain of TGFβR1; iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR2; ii) a second transmembrane domain comprising a transmembrane domain of TGFβR2; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell.

[0229] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of TGFβR2; iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of TGFβR1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell.

[0230] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR1; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR2; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0231] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0232] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:5; iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:6; and iii) a second intracellular domain comprising SEQ ID NO:16. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first transmembrane domain and the first intracellular domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second transmembrane domain and the second intracellular domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0233] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:6; and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:5; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first transmembrane domain and the first intracellular domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second transmembrane domain and the second intracellular domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell.In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0234] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell.In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0235] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8; and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the first polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the first extracellular domain and the first transmembrane domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the second polypeptide further comprises a linker (e.g., a juxtamembrane sequence) between the second extracellular domain and the second transmembrane domain, optionally, the linker comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell.In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0236] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first linker comprising the amino acid sequence of SEQ ID NO:11, iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second linker comprising the amino acid sequence of SEQ ID NO:12, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iv) a second intracellular domain comprising SEQ ID NO:16. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0237] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: 1) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising an intracellular domain of one of IL-12Rβ1 or IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising the extracellular domain of the other of TGFβR1 or TGFβR2, ii) a second transmembrane domain, and iii) a second intracellular domain comprising the intracellular domain of the other of IL-12Rβ1 or IL-23R; and 2) a functional exogenous receptor comprising an extracellular ligand binding domain and optionally an intracellular signaling domain. In some embodiments, the functional foreign receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, a signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0238] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a functional foreign receptor, including a) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1; and 2) a chimeric antigen receptor (e.g., a chimeric antigen receptor that recognizes a tumor antigen, e.g., an anti-GPC3 CAR, e.g., an anti-CD20 CAR). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0239] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: 1) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1; and 2) a functional foreign receptor comprising an engineered TCR (e.g., an engineered TCR that recognizes a tumor antigen). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0240] In some embodiments, the engineered cell (e.g., immune cell, e.g., T cell, e.g., CD8 T cell) comprises (or expresses) a fusion protein comprising: 1) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR2; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR1; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1; and 2) a functional foreign receptor comprising a T cell antigen coupler (TAC). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0241] In some embodiments, an engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a fusion protein comprising: 1) a first polypeptide comprising, from N-terminal to C-terminal direction, i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first linker comprising the amino acid sequence of SEQ ID NO:11, iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction, i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second linker comprising the amino acid sequence of SEQ ID NO:12, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iv) a second intracellular domain comprising SEQ ID NO:16; and 2) a functional foreign receptor comprising a chimeric antigen receptor (e.g., a chimeric antigen receptor that recognizes a tumor antigen, e.g., an anti-GPC3 CAR, e.g., an anti-CD20 CAR). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0242] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a functional foreign receptor, comprising: 1) a fusion protein comprising: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a first linker comprising the amino acid sequence of SEQ ID NO:11; iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a second linker comprising the amino acid sequence of SEQ ID NO:12; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8; and iv) a second intracellular domain comprising SEQ ID NO:16; and 2) an engineered TCR (e.g., an engineered TCR that recognizes a tumor antigen). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0243] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises (or expresses) a functional foreign receptor comprising: 1) a fusion protein comprising: a) a first polypeptide comprising, from N-terminal to C-terminal direction: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4; ii) a first linker comprising the amino acid sequence of SEQ ID NO:11; iii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; and iv) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3; ii) a second linker comprising the amino acid sequence of SEQ ID NO:12; ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8; and iv) a second intracellular domain comprising SEQ ID NO:16; and 2) a T cell antigen coupler (TAC). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0244] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises a nucleic acid (or a vector comprising a nucleic acid) comprising a nucleic acid sequence as set forth in any of SEQ ID NOs: 28-33 (e.g., SEQ ID NOs: 31-33, e.g., SEQ ID NO: 33). In some embodiments, the engineered cell further comprises a functional exogenous receptor (e.g., a CAR, e.g., an engineered TCR) or a nucleic acid sequence encoding same. In some embodiments, the nucleic acid comprising a nucleic acid sequence as set forth in any of SEQ ID NOs: 28-33 further comprises a nucleic acid sequence encoding a functional exogenous receptor (e.g., a CAR, e.g., an engineered TCR). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR-deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0245] In some embodiments, the engineered cell (e.g., an immune cell, e.g., a T cell, e.g., a CD8 T cell) comprises a nucleic acid comprising: 1) a nucleic acid sequence set forth in SEQ ID NO: 33; and 2) a nucleic acid sequence encoding a CAR that recognizes a tumor antigen (e.g., an anti-GPC3 CAR, e.g., an anti-CD20 CAR, e.g., a CAR comprising the amino acid sequence of SEQ ID NO: 36 or 89). In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cell is a γδ T cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is an endogenous TCR deficient T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the signal mediated through the IL-23 receptor complex intracellular domain is transmitted from the extracellular domain to the intracellular domain of the fusion protein upon binding of TGFβ.

[0246] V. Methods for Producing Engineered Cells One aspect of the invention provides a method of producing any of the engineered T cells described above. The method generally comprises introducing into a cell (e.g., referred to herein as a T cell "progenitor cell") a nucleic acid comprising a nucleic acid sequence encoding any of the fusion proteins described herein. In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding a functional foreign receptor (such as an engineered TCR, CAR, or ACTR as described herein). In some embodiments, the method further comprises introducing into the progenitor cell a second nucleic acid comprising a nucleic acid sequence encoding a functional foreign receptor (such as an engineered TCR, CAR, or ACTR as described herein). In some embodiments, the progenitor cell is an immune cell. In some embodiments, the progenitor cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof.

[0247] In some embodiments, the progenitor cells (e.g., progenitor T cells) are derived from blood, bone marrow, lymph, or lymphoid organs and are cells of the immune system (such as cells of innate or adaptive immunity). In some embodiments, the cells are human cells.

[0248] In some embodiments, the precursors (e.g., precursor T cells) are derived from a cell line (e.g., a T cell line). The cells, in some embodiments, are obtained from a xenogeneic source (e.g., from mouse, rat, non-human primate, and pig).

[0249] In some embodiments, the precursor T cells are CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cells are natural killer T (NKT) cells. In some embodiments, the precursor T cells are engineered T cells, such as with any of the functional foreign receptors described herein (e.g., engineered TCRs or CARs). In some embodiments, the precursor T cells produce IL-2, TNF, and / or TNF upon expression of a functional foreign receptor described herein (e.g., engineered TCRs, CARs) and binding to a target cell (such as a tumor cell expressing a tumor antigen (e.g., GPC3)). In some embodiments, the CD8+ T cells lyse antigen-specific target cells upon expression of a functional foreign receptor described herein (e.g., engineered TCRs, CARs) and binding to a target cell.

[0250] In some embodiments, progenitor cells (eg, progenitor T cells) are differentiated from stem cells (such as hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells).

[0251] In some embodiments, the fusion protein and / or functional foreign receptor (e.g., engineered TCR, CAR) is introduced into the progenitor cell (e.g., progenitor T cell) by transfection with any one of the nucleic acids or vectors (e.g., non-viral vectors and viral vectors such as lentiviral vectors) described herein. In some embodiments, the functional foreign receptor (e.g., engineered TCR, CAR) is introduced into the progenitor cell (e.g., progenitor T cell) by inserting the protein into the cell membrane while passing the cells through a microfluidic system (such as CELL SQUEEZE®) (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0252] Methods for introducing a vector (e.g., a viral vector) or an isolated nucleic acid into a mammalian cell are known in the art. The vectors described herein can be transferred into a cell (e.g., a T cell) by physical, chemical, or biological methods.

[0253] Physical methods for introducing a vector (e.g., a viral vector) into a cell (e.g., a T cell) include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, a vector (e.g., a viral vector) is introduced into a cell by electroporation.

[0254] Biological methods for introducing vectors into cells (e.g., T cells) include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0255] Chemical means for introducing vectors (e.g., viral vectors) into cells (e.g., T cells) include colloidal dispersion systems (such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vehicle in vitro is a liposome (e.g., artificial membrane vesicle).

[0256] In some embodiments, RNA molecules encoding any of the fusion proteins and / or functional foreign receptors (e.g., engineered TCRs, CARs) described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into cells (e.g., T cells) via known methods (such as mRNA electroporation). See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035.

[0257] In some embodiments, the transduced or transfected cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected cells are cultured and expanded for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected cells are further evaluated or screened to select engineered mammalian cells.

[0258] Reporter genes can be used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that code for a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is manifested by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0259] Other methods for confirming the presence of nucleic acids encoding any of the fusion proteins and / or functional exogenous receptors (e.g., engineered TCRs, CARs) described herein in engineered cells (e.g., engineered T cells) include, for example, molecular biological assays well known to those of skill in the art (such as Southern and Northern blotting, RT-PCR and PCR); biochemical assays (such as detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blots), fluorescence activated cell sorting (FACS), or magnetic activated cell sorting (MACS)) (see also the Examples section).

[0260] Thus, in some embodiments, a method of producing an engineered cell (e.g., an engineered T cell, an allogeneic T cell, or an endogenous TCR-deficient T cell) is provided, comprising introducing into a progenitor cell a nucleic acid comprising a nucleic acid sequence encoding a fusion protein described herein. In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding any of a functional exogenous receptor (e.g., an engineered TCR, a CAR) comprising an extracellular ligand binding domain and an intracellular signaling domain. In some embodiments, the progenitor cell comprises a second nucleic acid encoding a functional exogenous receptor (e.g., an engineered TCR, a CAR) comprising an extracellular ligand binding domain and an intracellular signaling domain. In some embodiments, the method further comprises introducing into the progenitor T cell a second nucleic acid encoding a functional exogenous receptor comprising an extracellular ligand binding domain and an intracellular signaling domain. In some embodiments, the first nucleic acid and the second nucleic acid are introduced into the T cell sequentially. Thus, in some embodiments, methods are provided for producing engineered cells (e.g., T cells, allogeneic T cells, or endogenous TCR-deficient T cells) comprising introducing into a progenitor cell a nucleic acid encoding a) a fusion protein (such as any of the fusion proteins described herein) and b) a functional exogenous receptor (such as any of the functional exogenous receptors described herein). In some embodiments, methods are provided for producing engineered cells (e.g., T cells, allogeneic T cells, or endogenous TCR-deficient T cells) comprising introducing into a progenitor cell a first nucleic acid encoding a fusion protein, and then introducing into the progenitor cell a second nucleic acid encoding a functional exogenous receptor comprising an extracellular ligand binding domain and an intracellular signaling domain. In some embodiments, the first nucleic acid and the second nucleic acid are introduced into the T cell simultaneously. In some embodiments, the first nucleic acid and the second nucleic acid are on separate vectors. In some embodiments, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to different promoters.In some embodiments, the first nucleic acid is upstream of the second nucleic acid. In some embodiments, the first nucleic acid is downstream of the second nucleic acid.

[0261] In some embodiments, methods are provided for producing engineered cells (e.g., engineered T cells, engineered allogeneic T cells, or engineered endogenous TCR-deficient T cells) comprising introducing into a progenitor cell a vector (e.g., a viral vector such as a lentiviral vector) that is, from upstream to downstream, a promoter (e.g., EF1-α), a first nucleic acid encoding a functional foreign receptor (e.g., an engineered TCR or CAR), and a second nucleic acid encoding a fusion protein described herein. In some embodiments, the first and second nucleic acids are separated by a third nucleic acid encoding a multicistronic element (e.g., a self-cleaving peptide of 2A selected from the group consisting of T2A, P2A, E2A, or F2A).

[0262] In some embodiments, methods are provided for producing engineered cells (e.g., engineered T cells, engineered allogeneic T cells, or engineered endogenous TCR-deficient T cells) comprising introducing into a progenitor cell a vector (e.g., a viral vector such as a lentiviral vector) that is, from upstream to downstream, a promoter (e.g., EF1-α), a first nucleic acid encoding a fusion protein (such as any of the fusion proteins described herein), and a second nucleic acid encoding a functional exogenous receptor (e.g., an engineered TCR or CAR). In some embodiments, the first and second nucleic acids are separated by a third nucleic acid encoding a multicistronic element (e.g., a self-cleaving peptide of 2A selected from the group consisting of T2A, P2A, E2A, or F2A).

[0263] In some embodiments, the engineered cells are T cells, and the endogenous TCR locus in the T cells is modified by a CRISPR-Cas system, thereby generating endogenous TCR-deficient T cells. See, e.g., WO2020 / 020359.

[0264] In some embodiments, the methods described herein further comprise depleting αβ T cells or enriching for γδ T cells.

[0265] In some embodiments, the promoter is selected from the group consisting of Rous sarcoma virus (RSV) promoter, Simian virus 40 (SV40) promoter, Cytomegalovirus immediate early gene promoter (CMV IE), Elongation factor 1 alpha promoter (EF1-alpha), Phosphoglycerate kinase-1 (PGK) promoter, Ubiquitin C (UBQ-C) promoter, Cytomegalovirus enhancer / Chicken β-actin (CAG) promoter, Polyoma enhancer / Herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, Myeloproliferative Sarcoma Virus Enhancer, Negative Regulatory Region Deleted, d1587rev Primer Binding Site Replacement (MND) promoter, NFAT promoter, TETON® promoter, and NFκB promoter. In some embodiments, the promoter is EF1-alpha.

[0266] In some embodiments, the multicistronic element is P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, IRES, (GS) n , (GSGGS) n , (GGGS) n , (GGGGS) n or combinations thereof, where n is at least an integer.

[0267] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, vaccinia vectors, lentiviral vectors, herpes simplex viral vectors, and derivatives thereof. In some embodiments, the vector is a non-viral vector, such as an episomal expression vector, an enhanced episomal vector (EEV), a PiggyBac transposase vector, or a Sleeping Beauty (SB) transposon system. In some embodiments, the functional foreign receptor is an engineered TCR. In some embodiments, the functional foreign receptor is a non-TCR receptor, such as a CAR (e.g., an anti-antigen CAR, a ligand / receptor CAR, an ACTR).

[0268] In some embodiments, the method further comprises formulating the engineered cells expressing the fusion protein with at least one pharma- ceutically acceptable carrier. In some embodiments, the method further comprises administering to an individual a pharmaceutical formulation comprising an effective amount of the engineered cells expressing the fusion protein, or an effective amount of the engineered cells expressing the fusion protein (e.g., T cells) and at least one pharma- ceutically acceptable carrier. In some embodiments, the individual has cancer. In some embodiments, the individual is a human.

[0269] Cell sources, cell preparation, and culture Prior to expansion and genetic modification of cells (e.g., T cells), a source of cells is obtained from an individual. For example, immune cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of cell lines having the desired cells (e.g., T cells) available in the art can be used. In some embodiments, cells (e.g., T cells) can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many, but not all, divalent cations. More surprisingly, an initial activation step in the absence of calcium leads to increased activation. As one of skill in the art will readily recognize, the wash step may be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be immersed in a variety of biocompatible buffers (e.g., Ca 2+ Free Mg 2+ The cells may be resuspended in culture medium (such as free PBS, PlasmaLyte A, or other saline solutions with or without buffers) or in apheresis medium (such as PBS, PlasmaLyte A, or other saline solutions with or without buffers). Alternatively, undesired components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0270] In some embodiments, the cells (T cells) are provided from a cord blood bank, a peripheral blood bank, or derived from induced pluripotent stem cells (iPSCs), multipotent and pluripotent stem cells, or human embryonic stem cells. In some embodiments, the cells (e.g., T cells) are derived from a cell line. The cells (e.g., T cells) are obtained in some embodiments from a heterologous source (e.g., from mouse, rat, non-human primate, and pig). In some embodiments, the cells (e.g., T cells) are human cells. In some aspects, the cells (e.g., T cells) are primary cells (e.g., isolated directly from a subject and / or isolated and frozen from a subject). In some embodiments, the cells include one or more subsets of T cells, such as the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof (e.g., defined by function, activation state, maturation, and potential for differentiation, proliferation, recirculation, localization, and / or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation). The cells can be allogeneic and / or autologous with respect to the subject being treated. In some cases, the cells (e.g., T cells) are allogeneic with respect to one or more intended recipients.

[0271] Among the subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, (stem cell memory T (TSC M ) cells, central memory T (TC M ) cells, effector memory T(T EM) cells, or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells).

[0272] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells (such as CD3+ T cells, CD28+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, and CD45RO+ T cells) can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads (such as DYNABEADS® M-450 CD3 / CD28 T) for a period of time sufficient for positive selection of the desired T cells. In some embodiments, the period is about 30 minutes. In further embodiments, the period of time ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10-24 hours. In some embodiments, the incubation period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where there are few T cells compared to other cell types (such as isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals). Furthermore, the use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or extending the time for binding T cells to anti-CD3 / anti-CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio (as described further herein), subpopulations of T cells can be preferentially selected for at the beginning of the culture or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for at the initiation of culture or at other desired time points. One of skill in the art will recognize that multiple rounds of selection can also be used.In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. The "unselected" cells may also be subjected to additional rounds of selection.

[0273] Enrichment of T cell populations by negative selection can be achieved by a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0274] For the isolation of a desired population of cells by positive or negative selection, the cell concentration and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In a further embodiment, more than 100 million cells / mL is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a cell concentration from 75, 80, 85, 90, 95, or 100 million cells / mL is used. In further embodiments, a concentration of 125 or 150 million cells / mL may be used. The use of a high concentration may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of a high cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest (such as CD28 negative T cells) or cells from samples where many tumor cells are present (i.e. leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of a high cell concentration allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

[0275] In some embodiments, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of cells (e.g., T cells) and surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In some embodiments, the concentration of cells used is 5×10 6 In some embodiments, the concentration used is about 1×105 / mL~1×10 6 / mL, and any integer value therebetween.

[0276] In some embodiments, cells can be incubated on a rotator at varying speeds for varying times at either 2-10° C. or at room temperature.

[0277] Stimulatory T cells may also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more homogenous product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or culture medium containing 31.25% PlasmaLyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then the cells are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen, can be used.

[0278] In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to sit at room temperature for 1 hour prior to activation.

[0279] Collection of blood samples or apheresis products from a subject at a time period before the expanded cells described herein may be needed is also contemplated in the present application. Thus, a source of cells to be expanded can be collected at any time point required, and the desired cells (such as T cells) can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy (such as those described herein). In one embodiment, blood samples or apheresis are taken from generally healthy subjects. In certain embodiments, blood samples or apheresis are taken from generally healthy subjects who are at risk of developing a disease, but who have not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and later used. In certain embodiments, samples are collected from patients shortly after diagnosis of a particular disease as described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant forms of treatment, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation). These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993).In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, concomitantly with, or after) T cell ablative therapy using either bone marrow or stem cell transplant, chemotherapeutic agents (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (such as OKT3 or CAMPATH). In another embodiment, the cells may be isolated prior to B cell ablative therapy (such as agents reactive with CD20, e.g., Rituxan) and frozen following said therapy for later use for treatment.

[0280] In some embodiments, T cells are obtained directly from a patient following treatment. In this regard, it has been observed that following certain cancer treatments (especially those using drugs that damage the immune system), the quality of the obtained T cells may be optimal or improved in terms of their ability to expand ex vivo during the period immediately following treatment, when the patient would normally recover from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells (including T cells, dendritic cells, or other cells of the hematopoietic lineage) during this recovery phase. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to generate conditions in the subject that facilitate repopulation, recirculation, regeneration, and / or expansion of certain cell types, particularly during a defined time window following the treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0281] T cell activation and proliferation In some embodiments, the cells are incubated and / or cultured prior to or in conjunction with the genetic manipulation. The incubation step may include culturing, culturing, stimulating, activating, and / or growing. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimulants. Such conditions include those designed to induce proliferation, growth, activation, and / or survival of the cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic manipulation (such as for the introduction of a genetically engineered antigen receptor). The conditions may include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors (such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other agents designed to activate the cells).

[0282] Whether before or after genetic modification of the T cells with the fusion proteins or foreign receptors (e.g., engineered TCRs, CARs) described herein, the T cells can generally be prepared using methods described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905, The cells can be activated and expanded using the methods described in U.S. Patent Application Publication No. 20060121005, and in U.S. Patent Application No. 681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0283] Generally, T cells can be expanded by contacting the surface to which they are attached with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, such as by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions for stimulation of proliferation of T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which may be used as well as others generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0284] In some embodiments, T cells are expanded by adding feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMCs)) to the culture initiation composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma radiation in the range of about 3000-3600 rads to prevent cell division. In some embodiments, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0285] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or can be coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then crosslinked to a surface (such as a cell expressing an Fc receptor or antibody, or other binding agent that binds to the agent). In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and expansion of T cells in the present invention.

[0286] In some embodiments, T cells are combined with agent-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated, but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force (such as a magnetic force), resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0287] As an example, cell surface proteins can be ligated by contacting the T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 ~10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Again, one of skill in the art can readily recognize that any cell concentration can be used. For example, the target cells may be very rare in the sample and may constitute only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Thus, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and the particles. For example, in one embodiment, a concentration of about 2 billion cells / mL is used. In another embodiment, more than 100 million cells / mL is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL are used. In yet another embodiment, cell concentrations from 75, 80, 85, 90, 95, or 100 million cells / mL are used. In further embodiments, concentrations of 125 or 150 million cells / mL may be used. The use of high concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest (such as CD28-negative T cells). Such cell populations may have therapeutic value and may be desirable to obtain in certain embodiments. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

[0288] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the present invention, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may be desired, as the culture time of the T cells may be 60 days or more. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or other additives for the proliferation of cells known to those of skill in the art. Other additives for the growth of cells include, but are not limited to, surfactants, plasmanate, and reducing agents (such as N-acetyl-cysteine ​​and 2-mercaptoethanol). Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokine(s) for the growth and expansion of T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures, not in cultures of cells to be infused into a subject. Target cells are cultured in the conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO ). 2) under a constant 10-20% CO2 concentration. T cells exposed to various stimulation times may exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product has a higher helper T cell population (TH, CD4+) than a cytotoxic or suppressive T cell population (TC, CD8). Ex vivo expansion of T cells by stimulation of the CD3 and CD28 receptors produces a population of T cells consisting predominantly of TH cells before about 8-9 days, while after about 8-9 days the population of T cells increasingly comprises a greater population of TC cells. Thus, depending on the goal of the treatment, it may be advantageous to infuse a subject with a T cell population that contains predominantly TH cells. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand this subset to a greater extent.

[0289] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers vary significantly but are largely reproducible during the course of the cell expansion process, thus allowing the ability to tailor activated T cell products for specific purposes.

[0290] In some embodiments, the method involves assessing the expression of one or more markers on the surface of the modified or to-be-engineered cells. In one embodiment, the method involves assessing surface expression of TCR or CD3ε, for example by affinity-based detection methods (such as by flow cytometry). In some embodiments, if the method reveals surface expression of the antigen or other marker, the gene encoding the antigen or other marker is disrupted or otherwise silenced, for example using the methods described herein.

[0291] VI. Pharmaceutical Compositions Further provided by the present application is a pharmaceutical composition comprising any one of the engineered cells (e.g., engineered T cells) expressing the fusion proteins described herein and / or a functional foreign receptor (e.g., engineered TCR, CAR, such as an anti-BCMA CAR) described herein, and a pharmaceutical acceptable carrier. The pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution by mixing a plurality of engineered cells with an optional pharmaceutical acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0292] Acceptable carriers, excipients, or stabilizing substances are nontoxic to recipients at the dosages and concentrations employed and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonicity agents, stabilizing agents, metal complexes (e.g., Zn-protein complexes); chelating agents (such as EDTA and / or nonionic surfactants).

[0293] Buffers are used to control the pH within a range that optimizes therapeutic efficacy, especially if the stability is pH dependent. The buffer is preferably present at a concentration ranging from about 50 mM to about 250 mM. Buffering agents suitable for use according to the invention include both organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may include histidine and trimethylamine salts (such as Tris).

[0294] Preservatives are added to retard microbial growth and are typically present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens (such as methyl paraben or propyl paraben); catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0295] Tonicity agents (sometimes known as "stabilizers") are present to adjust or maintain the isotonicity of the liquid in the composition. When used with large charged biomolecules (such as proteins and antibodies), they are often referred to as "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount from 0.1% to 25% by weight (preferably 1-5%), taking into account the relative amounts of other ingredients. Preferred tonicity agents include polyhydric phenolic sugar alcohols, preferably trihydric or higher sugar alcohols (such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol).

[0296] Additional excipients include agents that may act as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizing agents, and (4) agents that prevent denaturation or sticking to container walls. Such excipients include polyhydric sugar alcohols (as listed above); amino acids (alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.); organic sugars or sugar alcohols (sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol, polyethylene glycol, etc.); sulfur-containing reducing agents (such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate); low molecular weight proteins (such as human serum albumin, bovine serum albumin or gelatin or other immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (raffinose); and polysaccharides (dextrin or dextran).

[0297] The non-ionic surfactant or detergent (also known as a "wetting agent") is present to help solubilize the therapeutic agent and to protect the therapeutic protein against agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant ranges from about 0.05 mg / mL to about 1.0 mg / mL, preferably from about 0.07 mg / mL to about 0.2 mg / mL.

[0298] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methyl cellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0299] For pharmaceutical compositions to be used for in vivo administration, they must be sterilized. Pharmaceutical compositions can be sterilized by filtration through a sterile filtration membrane. The pharmaceutical compositions herein are generally placed into a container with a sterile access port (for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle).

[0300] Routes of administration are in accordance with known and accepted methods, such as by single or multiple boluses or infusions over an extended period of time in a suitable manner (e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular routes), by topical administration, inhalation, or by sustained or extended release means.

[0301] Sustained release preparations can be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antagonists, said matrices being in the form of shaped articles (e.g., films or microcapsules).Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid.

[0302] The pharmaceutical compositions described herein may contain two or more active compounds or agents as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are preferably present in combination in an amount effective for the intended purpose.

[0303] The active ingredient may also be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, for example in microcapsules prepared by coascervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0304] VII. Treatment Methods The application further provides a method of treating a disease or condition in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an engineered cell described herein. In some embodiments, the disease or condition involves immunosuppression. In some embodiments, the diseased tissue has a higher expression level of TGFβ (e.g., higher expression of TGFβ mRNA or TGFβ protein) than a corresponding tissue in an individual without the disease or condition. In some embodiments, the diseased tissue has a higher expression level of TGFβR (e.g., higher expression of TGFβR1 / TGFβR2 mRNA or TGFβR1 / TGFβR2 protein) than a corresponding tissue in an individual without the disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the disease or condition is an infectious disease or a condition associated with an infectious disease.

[0305] The application also provides a method of reducing immunosuppressive signals in a diseased tissue in an individual, comprising administering to the individual a pharmaceutical composition comprising an effective amount of the engineered cells described herein. In some embodiments, reducing immunosuppressive signals comprises reducing signaling through TGFβR. In some embodiments, the diseased tissue has a higher expression level of TGFβ (e.g., higher expression of TGFβ mRNA or TGFβ protein) than a corresponding tissue in an individual without the disease or condition. In some embodiments, the diseased tissue has a higher expression level of TGFβR (e.g., higher expression of TGFβR1 / TGFβR2 mRNA or TGFβR1 / TGFβR2 protein) than a corresponding tissue in an individual without the disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the disease or condition is an infectious disease or a condition associated with an infectious disease.

[0306] In some embodiments, the method further comprises assessing the level of TGFβ or a TGFβ receptor (e.g., TGFβR1 and / or TGFβR2) in the diseased tissue prior to treatment. In some embodiments, the method further comprises selecting the individual for treatment based on the level of TGFβ or a TGFβ receptor (e.g., TGFβR1 and / or TGFβR2) in the diseased tissue. In some embodiments, the individual is selected for treatment if the diseased tissue has a TGFβ level (e.g., a mean TGFβ level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than that in a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, an individual is selected for treatment if the diseased tissue has a TGFβR level (e.g., average TGFβR1 level and / or average TGFβR2 level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than in a reference tissue (e.g., a corresponding tissue in a healthy individual).

[0307] In some embodiments, a method is provided for treating a disease or condition (e.g., cancer, solid tumor, hematological cancer, or infectious disease) in an individual, comprising administering a composition (e.g., pharmaceutical composition) comprising an effective amount of engineered cells (e.g., immune cells, e.g., T cells) comprising (or expressing) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the extracellular domain of TGFβR1; ii) a first transmembrane domain comprising the transmembrane domain of TGFβR1; iii) a first intracellular domain comprising the intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising the extracellular domain of TGFβR2; ii) a second transmembrane domain comprising the transmembrane domain of TGFβR2; and iii) a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, the engineered cells are allogeneic cells (e.g., allogeneic T cells). In some embodiments, the engineered cells are autologous cells (e.g., autologous T cells). In some embodiments, the engineered cells are γδ T cells. In some embodiments, the engineered cells further comprise a functional exogenous receptor (e.g., a CAR, e.g., an engineered TCR) or a nucleic acid sequence encoding same. In some embodiments, the diseased tissue has a TGFβ level (e.g., an average TGFβ level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than in a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the diseased tissue has a TGFβR level (e.g., an average TGFβR1 level and / or an average TGFβR2 level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than in a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the engineered cells are administered parenterally (e.g., intravenously) into the individual. In some embodiments, the individual is a human.

[0308] In some embodiments, a method is provided for treating a disease or condition (e.g., cancer, solid tumor, hematological cancer, or infectious disease) in an individual, comprising administering a composition (e.g., pharmaceutical composition) comprising an effective amount of an engineered cell (e.g., immune cell, e.g., T cell) comprising (or expressing) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising the extracellular domain of TGFβR2; ii) a first transmembrane domain comprising the transmembrane domain of TGFβR2; iii) a first intracellular domain comprising the intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising the extracellular domain of TGFβR1; ii) a second transmembrane domain comprising the transmembrane domain of TGFβR1; and iii) a second intracellular domain comprising the intracellular domain of IL-12Rβ1. In some embodiments, the engineered cell is an allogeneic cell (e.g., an allogeneic T cell). In some embodiments, the engineered cell is an autologous cell (e.g., an autologous T cell). In some embodiments, the engineered cells are γδ T cells. In some embodiments, the engineered cells further comprise a functional exogenous receptor (e.g., a CAR, e.g., an engineered TCR) or a nucleic acid sequence encoding same. In some embodiments, the diseased tissue has a TGFβ level (e.g., an average TGFβ level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than in a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the diseased tissue has a TGFβR level (e.g., an average TGFβR1 level and / or an average TGFβR2 level) that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than in a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the engineered cells are administered parenterally (e.g., intravenously) into the individual. In some embodiments, the individual is a human.

[0309] In some embodiments, a method is provided for treating a disease or condition (e.g., cancer, solid tumor, hematological cancer, or infectious disease) in an individual, comprising administering a composition (e.g., pharmaceutical composition) comprising an effective amount of engineered cells (e.g., immune cells, e.g., T cells) comprising (or expressing) a fusion protein comprising: a) a first polypeptide comprising: i) a first extracellular domain comprising an extracellular domain of TGFβR1; ii) a first transmembrane domain comprising a transmembrane domain of IL-23R; and iii) a first intracellular domain comprising an intracellular domain of IL-23R; and b) a second polypeptide comprising: i) a second extracellular domain comprising an extracellular domain of TGFβR2; ii) a second transmembrane domain comprising a transmembrane domain of IL-12Rβ1; and iii) a second intracellular domain comprising an intracellular domain of IL-12Rβ1. In some embodiments, the engineered cells are allogeneic cells (e.g., allogeneic T cells). In some embodiments, the engineered cells are autologous cells (e.g., au...

Claims

1. a) a first polypeptide comprising: i) a first extracellular domain comprising the extracellular domain of one of TGFβR1 or TGFβR2, ii) a first transmembrane domain, and iii) a first intracellular domain comprising the intracellular domain of one of IL-12Rβ1 or IL-23R; b) a second polypeptide comprising: i) a second extracellular domain comprising the extracellular domain of the other of TGFβR1 or TGFβR2; ii) a second transmembrane domain; and iii) a second intracellular domain comprising the intracellular domain of the other of IL-12Rβ1 or IL-23R; A fusion protein comprising:

2. the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of TGFβR1, TGFβR2, IL-12Rβ1, and IL-23R; Optionally, the first transmembrane domain and the second transmembrane domain each comprise a transmembrane domain of one of IL-12Rβ1 or IL-23R. The fusion protein of claim 1.

3. a) the first extracellular domain comprises the extracellular domain of TGFβR1, the first transmembrane domain comprises the transmembrane domain of TGFβR1, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR2, the second transmembrane domain comprises the transmembrane domain of TGFβR2, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; b) the first extracellular domain comprises the extracellular domain of TGFβR2, the first transmembrane domain comprises the transmembrane domain of TGFβR2, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR1, the second transmembrane domain comprises the transmembrane domain of TGFβR1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; c) the first extracellular domain comprises the extracellular domain of TGFβR1, the first transmembrane domain comprises the transmembrane domain of IL-23R, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR2, the second transmembrane domain comprises the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; or d) the first extracellular domain comprises the extracellular domain of TGFβR2, the first transmembrane domain comprises the transmembrane domain of IL-23R, the first intracellular domain comprises the intracellular domain of IL-23R, the second extracellular domain comprises the extracellular domain of TGFβR1, the second transmembrane domain comprises the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprises the intracellular domain of IL-12Rβ1; The fusion protein of claim 2. (i) the first polypeptide and / or the second polypeptide further comprises a signal peptide at the N-terminus of the polypeptide; (ii) the first polypeptide and the second polypeptide are in the form of a single polypeptide, and the first polypeptide and the second polypeptide are separated by a multicistronic element; optionally, the multicistronic element comprises a 2A autocleaving peptide selected from the group consisting of T2A, P2A, E2A, or F2A; and / or (iii) the first polypeptide is N-terminal to the second polypeptide or C-terminal to the second polypeptide; The fusion protein of claim 1.

5. The single polypeptide comprises, in the N-terminal to C-terminal direction: a) the first extracellular domain comprising the extracellular domain of TGFβR1, the first transmembrane domain comprising the transmembrane domain of TGFβR1, the first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR2, the second transmembrane domain comprising the transmembrane domain of TGFβR2, and the second intracellular domain comprising the intracellular domain of IL-12Rβ1; b) the first extracellular domain comprising the extracellular domain of TGFβR2, the first transmembrane domain comprising the transmembrane domain of TGFβR2, the first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR1, the second transmembrane domain comprising the transmembrane domain of TGFβR1, and the second intracellular domain comprising the intracellular domain of IL-12Rβ1; c) the first extracellular domain comprising the extracellular domain of TGFβR1, the first transmembrane domain comprising the transmembrane domain of TGFβR1, the first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR2, the second transmembrane domain comprising the transmembrane domain of TGFβR2, and the second intracellular domain comprising the intracellular domain of IL-23R; d) the first extracellular domain comprising the extracellular domain of TGFβR1, the first transmembrane domain comprising the transmembrane domain of IL-23R, the first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR2, the second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprising the intracellular domain of IL-12Rβ1; e) the first extracellular domain comprising the extracellular domain of TGFβR2, the first transmembrane domain comprising the transmembrane domain of IL-23R, the first intracellular domain comprising the intracellular domain of IL-23R, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR1, the second transmembrane domain comprising the transmembrane domain of IL-12Rβ1, and the second intracellular domain comprising the intracellular domain of IL-12Rβ1; or f) the first extracellular domain comprising the extracellular domain of TGFβR1, the first transmembrane domain comprising the transmembrane domain of IL-12Rβ1, the first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, the second extracellular domain comprising the extracellular domain of TGFβR2, the second transmembrane domain comprising the transmembrane domain of IL-23R, and the second intracellular domain comprising the intracellular domain of IL-23R. The fusion protein of claim 4 .

6. The fusion protein of claim 1, wherein the first extracellular domain and the second extracellular domain form a binding site for TGFβ, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and binding of the fusion protein to TGFβ transduces signaling via the IL-23 receptor complex.

7. a) the transmembrane domain of IL-23R comprises the amino acid sequence of SEQ ID NO:7, or a functional variant having at least about 90% sequence identity; b) the transmembrane domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant having at least about 90% sequence identity; c) the transmembrane domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:5, or a functional variant having at least about 90% sequence identity; d) the transmembrane domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:6, or a functional variant having at least about 90% sequence identity; e) the extracellular domain of TGFβR1 comprises the amino acid sequence of SEQ ID NO:3, or a functional variant having at least about 90% sequence identity; f) the extracellular domain of TGFβR2 comprises the amino acid sequence of SEQ ID NO:4, or a functional variant having at least about 90% sequence identity; g) the intracellular domain of IL-23R comprises the amino acid sequence of SEQ ID NO: 15, or a functional variant having at least about 90% sequence identity; and / or h) the intracellular domain of IL-12Rβ1 comprises the amino acid sequence of SEQ ID NO: 16, or a functional variant having at least about 90% sequence identity; The fusion protein according to claim 3.

8. 1) a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:5, iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:6, and iii) a second intracellular domain comprising SEQ ID NO:16; 2) a) a first polypeptide comprising i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:6, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:5, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16; 3) a) a first polypeptide comprising: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:16; or 4) a) a first polypeptide comprising: i) a first extracellular domain comprising the amino acid sequence of SEQ ID NO:4, ii) a first transmembrane domain comprising the amino acid sequence of SEQ ID NO:7, and iii) a first intracellular domain comprising the amino acid sequence of SEQ ID NO:15; and b) a second polypeptide comprising: i) a second extracellular domain comprising the amino acid sequence of SEQ ID NO:3, ii) a second transmembrane domain comprising the amino acid sequence of SEQ ID NO:8, and iii) a second intracellular domain comprising the amino acid sequence of SEQ ID NO:

16. The fusion protein of claim 7.

9. The first polypeptide and / or the second polypeptide further comprises a linker between two domains, one of which is a transmembrane domain and the other of which is an extracellular domain or an intracellular domain; wherein optionally, the linker comprises a juxtamembrane sequence, and the juxtamembrane sequence and the transmembrane domain are derived from the same molecule; or The juxtamembrane region comprises an amino acid sequence as set forth in any of SEQ ID NOs: 9 to 12; The fusion protein of claim 8.

10. a) the first polypeptide comprises the amino acid sequence of SEQ ID NO:49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:50; b) the first polypeptide comprises the amino acid sequence of SEQ ID NO:50 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:49; c) the first polypeptide comprises the amino acid sequence of SEQ ID NO:51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:52; d) the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54; e) the first polypeptide comprises the amino acid sequence of SEQ ID NO:55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:56; f) the first polypeptide comprises the amino acid sequence of SEQ ID NO:56 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:55; or g) the fusion protein comprises an amino acid sequence as set forth in any of SEQ ID NOs: 20-25; The fusion protein of claim 9.

11. A nucleic acid comprising one or more nucleic acid sequences encoding the fusion protein of claim 1 or a portion thereof.

12. The nucleic acid of claim 11, wherein the nucleic acid further comprises a second nucleic acid sequence encoding a functional foreign receptor, the functional foreign receptor comprising an extracellular ligand binding region and optionally an intracellular signaling domain. (i) the functional exogenous receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof; and / or (ii) the functional foreign receptor comprises a chimeric antigen receptor (CAR) that specifically recognizes a tumor antigen, and optionally the CAR specifically recognizes CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, claudin 18.2, glycolipid F77, PD-L1, and / or PD-L2; The nucleic acid of claim 12.

14. 13. The nucleic acid of claim 12, wherein the nucleic acid sequence encoding the functional foreign receptor is upstream or downstream to at least one of one or more nucleic acid sequences encoding the fusion protein ("fusion protein nucleic acid sequence"), and optionally the functional foreign receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a third nucleic acid sequence encoding a second multicistronic element.

15. A vector comprising the nucleic acid of claim 11.

16. 13. An engineered cell comprising the fusion protein of claim 1.

17. and / or a functional exogenous receptor, said functional exogenous receptor comprising an extracellular ligand-binding domain and optionally an intracellular signaling domain; the functional foreign receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC), or a portion thereof; optionally, the functional foreign receptor specifically recognizes a tumor antigen.

17. The engineered cell of claim 16.

18. 13. An engineered cell comprising the nucleic acid of claim 12.

19. The engineered cell of claim 16, wherein the engineered cell is an immune cell, and optionally, the engineered cell is selected from the group consisting of a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and combinations thereof.

20. A pharmaceutical composition comprising the engineered cell of claim 16.

21. 21. A method of treating a disease or condition in an individual comprising administering to said individual a pharmaceutical composition according to claim 20, optionally wherein said disease or condition is associated with immunosuppression and / or said disease or condition is cancer.

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