Engineered immune effector cells expressing exogenously introduced cytokines

Abstract

The present application provides immune effector cells that express an exogenously introduced p40 subunit of IL-12; an exogenously introduced ligand for CCR7 (e.g., CCL-19 and CCL-21); and a functional exogenous receptor (e.g., a chimeric antigen receptor), the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2021 / 078227, filed February 26, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application incorporates by reference the Sequence Listing entitled "14651-039-228_SEQ_LISTING.txt" created on February 18, 2022, which was submitted herewith in text form and is 145,395 bytes in size.

[0003] 3. Field This disclosure relates to polypeptides comprising functional exogenous receptors (e.g., chimeric antigen receptors), engineered immune effector cells, and methods of use thereof. This disclosure further relates to the activation and expansion of cells for therapeutic uses, particularly chimeric antigen receptor-based T cell immunotherapy. [Background technology]

[0004] 4. Background Adoptive transfer of engineered immune effector cells represents a new and innovative therapeutic strategy. For example, chimeric antigen receptor (CAR)-engineered T cells have been shown to induce effective clinical responses and have a favorable prognosis compared with conventional treatments. However, low in vivo survival efficiency, insufficient activation of endogenous immune cells, immunosuppressive microenvironment, T cell depletion, and difficulty in infiltrating diseased tissues are common reasons why CAR-T cells are challenging for some diseases. Therefore, there is still a need in the art for improved constructs or engineered immune effector cells, such as CAR-T cells, to treat diseases or disorders. Summary of the Invention

[0005] 5. Overview In one aspect, the description provides an immune effector cell that expresses an exogenously introduced p40 subunit of IL-12; an exogenously introduced ligand for CCR7 (e.g., CCL-19 and CCL-21); and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0006] In some embodiments, the description provides immune effector cells that express an exogenously introduced p40 subunit of IL-12; an exogenously introduced CCL-19; and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0007] In some embodiments, the description provides immune effector cells that express an exogenously introduced p40 subunit of IL-12; an exogenously introduced CCL-21; and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0008] In some embodiments, p40 is human p40 or a fragment or variant thereof. In some embodiments, p40 comprises the amino acid sequence of SEQ ID NO:5. In other embodiments, p40 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, a p40 polypeptide in accordance with the present disclosure is a secreted polypeptide. In some embodiments, a p40 polypeptide in accordance with the present disclosure is a membrane-bound polypeptide (e.g., MB12).

[0009] In some embodiments, the CCL-19 is human CCL-19 or a fragment or variant thereof. In some embodiments, the CCL-19 comprises the amino acid sequence of SEQ ID NO:6. In other embodiments, the CCL-19 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:6.

[0010] In some embodiments, the CCL-21 is human CCL-21 or a fragment or variant thereof. In some embodiments, the CCL-21 comprises the amino acid sequence of SEQ ID NO:22. In other embodiments, the CCL-21 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:22.

[0011] In some embodiments, the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen binder (TAC)-like chimeric receptor. In some embodiments, the functional exogenous receptor is a CAR.

[0012] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is from CD8α or CD28.

[0013] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is from CD3ζ.

[0014] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

[0015] In some embodiments, the functional exogenous receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, hi some embodiments, the hinge domain is from CD8α.

[0016] In some embodiments, the functional exogenous receptor comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is from CD8α.

[0017] In some embodiments, the immune effector cells are T cells.

[0018] In another aspect, the present specification provides polypeptides, the polypeptides comprising an exogenously introduced p40 subunit of IL-12; an exogenously introduced CCR7 ligand (e.g., CCL-19 and CCL-21); and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0019] In some embodiments, the description provides a polypeptide comprising an exogenously introduced p40 subunit of IL-12; an exogenously introduced CCL-19; and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0020] In another embodiment, the description provides a polypeptide comprising an exogenously introduced p40 subunit of IL-12; an exogenously introduced CCL-21; and a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0021] In some embodiments, p40 is human p40 or a fragment or variant thereof. In some embodiments, p40 comprises the amino acid sequence of SEQ ID NO:5. In other embodiments, p40 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, a p40 polypeptide in accordance with the present disclosure is a secreted polypeptide. In some embodiments, a p40 polypeptide in accordance with the present disclosure is a membrane-bound polypeptide (e.g., MB12).

[0022] In some embodiments, the CCL-19 is human CCL-19 or a fragment or variant thereof. In some embodiments, the CCL-19 comprises the amino acid sequence of SEQ ID NO:6. In other embodiments, the CCL-19 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:6.

[0023] In some embodiments, the CCL-21 is human CCL-21 or a fragment or variant thereof. In some embodiments, the CCL-21 comprises the amino acid sequence of SEQ ID NO:22. In other embodiments, the CCL-21 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:22.

[0024] In some embodiments, the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen binder (TAC)-like chimeric receptor. In some embodiments, the functional exogenous receptor is a CAR.

[0025] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is from CD8α or CD28.

[0026] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is from CD3ζ.

[0027] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

[0028] In some embodiments, the functional exogenous receptor comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, hi some embodiments, the hinge domain is from CD8α.

[0029] In some embodiments, the functional exogenous receptor further comprises a signal peptide located at the N-terminus of the polypeptide, hi some embodiments, the signal peptide is from CD8α.

[0030] In some embodiments, p40, CCL-19, and the functional exogenous receptor are linked to each other via a peptide linker. In some embodiments, p40, CCL-21, and the functional exogenous receptor are linked to each other via a peptide linker. In some embodiments, the peptide linker is a 2A self-cleaving peptide selected from the group consisting of F2A, E2A, P2A, T2A, and variants thereof. In some specific embodiments, the 2A self-cleaving peptide is a P2A fragment comprising the amino acid sequence of SEQ ID NO: 13. In some specific embodiments, the 2A self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO: 14.

[0031] In some embodiments, p40 and CCL-19 are present within a single domain comprising the amino acid sequence of SEQ ID NO: 4. In other embodiments, p40 and CCL-19 are present within a single domain comprising an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 4.

[0032] In some embodiments, p40 and CCL-21 are present within a single domain comprising the amino acid sequence of SEQ ID NO: 20. In other embodiments, p40 and CCL-21 are present within a single domain comprising an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20.

[0033] In yet another aspect, the present description provides an isolated nucleic acid, the isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide according to the present description.

[0034] In yet another aspect, the present specification provides an isolated nucleic acid, the isolated nucleic acid comprising: an exogenously introduced first region encoding the p40 subunit of IL-12; an exogenously introduced second region encoding a CCR7 ligand (e.g., CCL-19 and CCL-21); and a third region encoding a functional exogenous receptor, the functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CCR7 ligand is CCL-19. In other embodiments, the CCR7 ligand is CCL-21.

[0035] In yet another aspect, the description provides a vector comprising an isolated nucleic acid according to the description.

[0036] In yet another aspect, the description provides a method for producing an immune effector cell, the method comprising introducing into an immune cell (i) a nucleic acid or vector according to the description, or (ii) a composition comprising two or more nucleic acids, each encoding one or two of the p40 subunit of IL-12, CCL-19, and a functional exogenous receptor, or a composition comprising two or more nucleic acids, each encoding one or two of the p40 subunit of IL-12, CCL-21, and a functional exogenous receptor.

[0037] In another aspect, the description provides an immune effector cell produced by a method according to the description.

[0038] In yet another aspect, the present description provides a pharmaceutical composition comprising an immune effector cell, polypeptide, nucleic acid, or vector according to the present description and a pharmaceutically acceptable carrier.

[0039] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to the present disclosure. [Brief explanation of the drawings]

[0040] 6. Brief description of the drawings [Figure 1] The structures of H93 CAR, H93M CAR, and H93P CAR are shown. SP refers to the signal peptide. TM refers to the transmembrane domain. TAA binding protein refers to the antigen-binding domain or binding protein of a tumor-associated antigen (i.e., anti-GPC3 scFv). [Figure 2] Figure 1 shows the positive rates of H93 CAR-T cells and H93M CAR-T cells expressing humanized anti-GPC3 scFv CAR. UnT refers to T cells not transduced with CAR. [Figure 3A] Figures 3A-3B show IL-23 (Figure 3A) and CCL-19 (Figure 3B) protein production by CAR-T cells cultured under normal conditions (RPMI-1640 + 300 IU / mL IL-2). [Figure 3B] See legend to Figure 3A. [Figure 4A] Figures 4A-4B show CAR-T cell expansion in a rechallenge assay. Cells were stimulated overnight with PLC / PRF / 5 cells at a 1:1 E / T ratio every two days. At the end of each round, the fold expansion (Figure 4A) and viability (Figure 4B) of T cells were recorded. [Figure 4B] See legend to Figure 4A. [Figure 5A] Figures 5A-5C show the in vitro cytotoxicity (Figure 5A), TNF-α release levels (Figure 5B), and IFN-γ release levels (Figure 5C) of CAR-T cells against GPC3-positive cell lines at an E / T ratio of 1:1 after treatment with various stimulation rounds in a re-challenge assay. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] Positive rates of CAR-T cells after treatment with different stimulation rounds in a re-challenge assay are shown. [Figure 7A] Figures 7A-7D show the expression of PD-1 (Figures 7A and 7B) and LAG3 (Figures 7C and 7D) on CAR-T cells 24 hours after the stimulation round in the re-challenge assay. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] Figures 8A-8B show a CAR-T cell migration assay performed using a 96-well transwell chamber. Various CAR-T cells were co-cultured with PLC / PRF / 5 cells at a 1:1 E / F ratio for 30 hours, and the cell culture supernatant was collected and added to the lower chamber. After 4, 6, and 8 hours, the number of T cells migrated from the upper chamber to the lower chamber (Figure 8A) and the concentration of CCL-19 in the co-culture supernatant (Figure 8B) were quantified. [Figure 8B] See legend to Figure 8A. [Figure 9A] Figures 9A-9D show the antitumor effect of CAR-T cells in an NCG mouse xenograft model. NCG mice were subcutaneously inoculated with Huh7 cells and treated with CAR-T cells at 0.2 M and 0.6 M doses (iv) (n = 4 mice per group). Tumor volume (Figures 9A and 9B) and CAR copy number in genomic DNA of peripheral blood of NCG mice (Figures 9C and 9D) were assessed. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10A] Figures 10A-10B show the weight changes of mice (n=4 mice per group) treated with CAR-T cells at doses of 0.2 M (Figure 10A) and 0.6 M (Figure 10B) in an NCG mouse xenograft model. [Figure 10B] See legend to Figure 10A. [Figure 11A] Figures 11A-11D show IL-23 protein (Figures 11A and 11B) and IFN-γ protein (Figures 11C and 11D) levels in the peripheral blood of NCG mice after treatment with CAR-T cells at a dose of 0.2 M or 0.6 M (n = 3 mice per group). [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12] Figure 1 shows T cell infiltration in tumor tissues from mice treated with 0.2 M UnT, H93 CAR-T cells, or H93M CAR-T cells at the endpoint of an animal study (n=3) in an NCG mouse xenograft model. Tumor tissues were fixed in formalin, embedded in paraffin, and used for immunohistochemistry (IHC) to detect T cell infiltration stained with 3,3'-diaminobenzidine (DAB). Images were obtained using 3D HISTECH (DRNJIER). The right column shows the magnified area of ​​the black rectangle. [Figure 13]Figure 1 shows the recruitment of mouse macrophages and mouse dendritic cells in tumor tissue after treatment with 0.2 M UnT, H93 CAR-T cells, or H93M CAR-T cells in an NCG mouse xenograft model. At the endpoint of the animal study, tumor tissue was fixed in formalin, embedded in paraffin, and used for immunohistochemistry (IHC). CD68-bearing macrophages (left column) and CD11c-bearing dendritic cells (right column) were stained with 3,3'-diaminobenzidine (DAB). Images were obtained using 3D HISTECH (DRNJIER). [Figure 14] The positive rates of H93 CAR-T cells, H93M CAR-T cells, and H93P CAR-T cells expressing humanized anti-GPC3 scFv CAR are shown. [Figure 15A] Figures 15A-15B show the results of the re-challenge assay. At the end of each round, the fold expansion of CAR-T cells (Figure 15A) was recorded. Figure 15B shows the in vitro cytotoxicity of CAR-T cells against GPC3-positive cell lines at a total T cell to target cell ratio of 1:2 after treatment with various stimulation rounds in the re-challenge assay. [Figure 15B] See legend to Figure 15A. [Figure 16] Figure 1 shows a CAR-T cell migration assay performed using a 96-well transwell chamber. Various CAR-T cells were co-cultured with PLC / PRF / 5 cells at a 1:1 E / T ratio for 30 hours, and the cell culture supernatant was collected and added to the lower chamber. After 2, 4, and 6 hours, the number of T cells in the lower chamber that had migrated from the upper chamber was quantified. [Figure 17A] Figures 17A-17B show the antitumor effect of CAR-T cells in an NCG mouse xenograft model. NCG mice were subcutaneously inoculated with GPC3+ Hep3B cells and injected with two doses of CAR-T cells: 0.3 M CAR+ T cells / mouse and 0.8 M CAR+ T cells / mouse (iv) (n = 4 mice per group). [Figure 17B] See legend to Figure 17A. [Figure 18A]Figures 18A-18C show the antitumor effect of CAR-T cells in an NCG mouse xenograft model. NCG mice were subcutaneously inoculated with Hep3B cells and treated with a 0.3 μM dose of H93 CAR-T cells, H93M CAR-T cells, H93IL12p40 CAR-T cells, or H93CCL19 CAR-T cells (iv) (n = 4 mice per group). Tumor volume (Figure 18A), the percentage of CD3-positive cells in the peripheral blood of NCG mice (Figure 18B), and body weight (Figure 18C) were assessed. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19A] Figures 19A-19D show the antitumor effect of CAR-T cells in a C57BL / 6 mouse xenograft model. C57BL / 6 mice were subcutaneously inoculated with LL / 2-hGPC3 cells and treated with musH93 CAR-T cells, musH93M CAR-T cells, musH93IL12p40 CAR-T cells, and musH93CCL19 CAR-T cells at doses of 10M, 6M, 6M, and 6M, respectively (iv) (n = 5 mice per group). Tumor volume (Figure 19A), the percentage of CAR+CD3+ cells in the peripheral blood of C57BL / 6 mice (Figure 19B), and body weight (Figure 19C) were assessed, and the survival rate (%) (Figure 19D) was calculated. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 20A] Figures 20A-20B show IL-23 (Figure 20A) and CCL-19 (Figure 20B) protein production after overnight co-culture of 261 and 261M CAR-T cells with CLDN18.2+NUGC4 cells. [Figure 20B] See legend to Figure 20A. [Figure 21A]Figures 21A-21C show the results of re-challenge assays of 261 and 261M CAR-T cells. Comparison of fold expansion of CAR-T cells in conventional conditions (10% 1640 + 300 IU IL-2) (Figure 21A) and the re-challenge assay (Figure 21B) suggested that 261M CAR-T cells exhibited antigen-dependent expansion after multiple challenges with NUGC4 cells. Figure 21C shows cell viability of CAR-T cells after treatment with multiple stimulation rounds in the re-challenge assay. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 22A] Figures 22A-22D show the antitumor effects of 261 and 261M CAR-T cells in an NCG mouse xenograft model. NCG mice were subcutaneously inoculated with NUGC4 cells and treated with CAR-T cells at a 0.5 M dose (iv) (n = 4 mice per group). Tumor volume (Figures 22A and B), the percentage of CD3-positive cells in the peripheral blood of NCG mice (Figure 22C), and body weight (Figure 22D) were assessed. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 23A] Figures 23A-23B show the antitumor effects of mus261M and mus261-719 CAR-T cells in a C57BL / 6 mouse xenograft model. C57BL / 6 mice were subcutaneously inoculated with LL / 2-hCLDN18.2 cells and treated with murine CAR-T cells at a 10 μg dose (iv) (n = 4 mice per group). Tumor volume (Figure 23A) and body weight (Figure 23B) were assessed. [Figure 23B] See legend to Figure 23A. [Figure 24A] Figures 24A and 24B show the CAR positivity rate (Figure 24A) and IL12p40 positivity rate (Figure 24B) of H93 CAR-T cells, H93M CAR-T cells, and H93M-MB12 CAR-T cells. [Figure 24B] See legend to Figure 24A. [Figure 25A] Figures 25A-25B show the production of IL12p40 (Figure 25A), IL-23 (Figure 25B), CCL-19 (Figure 25C), and IL-12 (Figure 25D) proteins by H93 CAR-T cells, H93M CAR-T cells, and H93M-MB12 CAR-T cells after overnight stimulation with anti-CD3 / CD28 beads. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 26A] Figures 26A-26B show cell migration assays of H93, H93M, and H93M-MB12 CAR-T cells performed using a 96-well transwell chamber. Various CAR-T cells were cocultured with PLC / PRF / 5 cells at a 1:1 E / F ratio for 24 hours, and the cell culture supernatant was collected and added to the lower chamber. After 2, 4, and 6 hours, the number of T cells migrated from the upper chamber to the lower chamber (Figure 26A) and the concentration of CCL-19 in the coculture supernatant (Figure 26B) were quantified. [Figure 26B] See legend to Figure 26A. [Figure 27A] Figures 27A-27C show the results of re-challenge assays of H93, H93M, and H93M-MB12 CAR-T cells. Figures 27A and 27B show the fold expansion of CAR-T cells in conventional conditions (10% 1640 + 300 IU IL-2) and in the re-challenge assay. Figure 27C shows the CAR-positive change of CAR-T cells after treatment with multiple stimulation rounds in the re-challenge assay. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 28A]Figures 28A-28B show in vitro cytokine secretion, TNF-α release levels (Figure 28A) and IFN-γ release levels (Figure 28B) of H93, H93M, and H93M-MB12 CAR-T cells after treatment with various stimulation rounds in a re-challenge assay. [Figure 28B] See legend to Figure 28A. DETAILED DESCRIPTION OF THE INVENTION

[0041] 7. Detailed Description The present disclosure is based, in part, on the surprising discovery that engineered immune cells expressing one or more functional exogenous receptors (e.g., CARs) and exogenously introduced p40 and CCR7 ligands (e.g., CCL-19 and CCL-21) have improved function and properties.

[0042] 7.1. Definition The techniques and procedures described or referenced herein generally include those that are well understood and / or commonly employed by those skilled in the art using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (eds. Ausubel et al., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (eds. An, 2009), Monoclonal Antibodies: Methods and Protocols (eds. Albitar, 2010), and Antibody Engineering, Vols. 1 and 2 (eds. Kontermann and Dubel, 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For purposes of interpretation of this specification, the following terminology applies, and where appropriate, terms used in the singular also include the plural, and vice versa. In the event that any explanation of a term provided herein contradicts any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0043] The terms "antibody," "immunoglobulin," or "Ig" may be used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, and full-length or intact monoclonal antibodies), antibody compositions with multiepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, single-domain antibodies (e.g., VHHs), and fragments thereof (e.g., domain antibodies). Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species (e.g., mouse, rabbit, llama, etc.). The term "antibody" is intended to include polypeptide products of B cells in the immunoglobulin family of polypeptides, which have the ability to bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (ed. by Borrebaeck, 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies further include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, where such functional fragments refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (edited by Myers, 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2nd ed., 1990). Antibodies according to the present specification can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. Antibodies can be agonist or antagonist antibodies. Antibodies can be neither agonist nor antagonist.

[0044] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In some embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0045] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In some embodiments, an intact antibody has one or more effector functions.

[0046] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0047] As used herein, "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of binding to an antigen. Single domain antibodies comprise a VHH domain as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains, such as antibodies from Camelidae species (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and bovine. For example, as described herein, single domain antibodies may be derived from antibodies produced from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside of Camelidae can produce heavy chain antibodies naturally devoid of light chains, and VHHs derived from such other species are within the scope of the present disclosure. In some embodiments, a single domain antibody (e.g., VHH) according to the present invention has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0048] The term "binds" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex may further include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between a single antigen-binding site on an antibody and a single epitope on a target molecule (e.g., an antigen) is the affinity of the antibody or functional fragment for that epitope. The ratio of the dissociation rate (koff) to the association rate (kon) of a binding molecule (e.g., an antibody) with a monovalent antigen (koff / kon) is the dissociation constant, KD, which is inversely proportional to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for each antibody-antigen complex and depends on kon and koff. The dissociation constant KD of an antibody according to the present invention can be determined using any method according to the present invention or any other method known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants (e.g., a multivalent antigen) contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site increases the likelihood of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0049] In the context of the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen (e.g., a polypeptide). Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques well known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigen, as measured using laboratory techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise and may be more than 10 times the background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul, ed., 2nd ed. 1989). In some embodiments, the extent to which a binding molecule or antigen-binding domain binds to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as measured, for example, by FACS analysis or RIA. A binding molecule or antigen-binding domain that binds to an antigen includes, for example, a binding molecule or antigen-binding domain that can bind to an antigen with sufficient affinity so that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen.In some embodiments, the binding molecule or antigen-binding domain that binds to an antigen has a concentration of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or less than 0.1 nM, or 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the binding molecule or antigen-binding domain binds to an antigenic epitope that is conserved in antigens from different species.

[0050] In some embodiments, a binding molecule or antigen-binding domain may comprise a "chimeric" sequence, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may also include humanized sequences.

[0051] In some embodiments, binding molecules or antigen-binding domains may comprise portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies, which comprise sequences from a human immunoglobulin (e.g., a recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., a donor antibody) such as camelid, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Note that humanized antibodies may also comprise residues that are not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. A humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In some embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0052] In some embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "fully human antibody" or "human antibody," where these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. A binding molecule may comprise a single-domain antibody sequence. In specific embodiments, the term refers to an antibody comprising variable and constant regions of human origin. In some embodiments, a "fully human" antibody may further encompass antibodies that bind to a polypeptide and are encoded by nucleic acid sequences, which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences, e.g., as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)). In addition, methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) can also be used to prepare human monoclonal antibodies.Human antibodies can be prepared by administering antigen to transgenic animals (e.g., mice) that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). For further information on human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0053] In some embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomic for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and closely related to human germline VH and VL sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0054] In some embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, each antibody of which is identical except for, for example, possible naturally occurring mutations or well-known post-translational modifications (e.g., amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation) that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced in bacterial or eukaryotic animal or plant cells using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and monoclonal antibodies expressed by clonal cell lines are well known in the art. See, for example, Short Protocols in Molecular Biology (ed. Ausubel et al., 5th ed., 2002).

[0055] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is usually approximately 150,000 daltons. Each L chain is linked to an H chain via one covalent disulfide bond, while the two H chains are linked to each other via one or more disulfide bonds, depending on the H chain isotype. Each H and L chain further has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of a VH and a VL together forms a single antigen-binding site. For the structural characteristics of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (ed. Stites et al., 8th ed., 1994) and Immunobiology (ed. Janeway et al., 5th ed., 2001).

[0056] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. A conventional IgG typically contains two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, similar to the Fab region of a conventional IgG, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on a single polypeptide. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0057] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody light or heavy chain, typically located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, and responsible for the binding and specificity of each particular antibody to its specific antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that some segments of the variable region in antibodies exhibit significant sequence variability. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed within the 110 amino acid span of the variable region. Instead, V regions are composed of less variable (e.g., relatively invariant) segments of about 15-30 amino acids called framework regions (FRs), separated by shorter regions of greater (e.g., extreme) variability called "hypervariable regions," each about 9-12 amino acids in length. The heavy and light chain variable regions each contain four FRs that primarily employ a β-sheet structure, connected by three hypervariable regions that form loops and, in some cases, part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region varies widely in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0058] The terms "Kabat variable region residue numbering" or "Kabat et al. amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody sequences in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues in a given antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology. When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system (e.g., Kabat et al., supra) is typically used. When referring to residues in an immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" (e.g., the EU index reported in Kabat et al., supra) is usually used. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0059] The term "heavy chain," when used in reference to antibodies, refers to a polypeptide chain of approximately 50 to 70 kDa, in which the amino-terminal portion contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion contains a constant region. The amino acid sequence of the heavy chain constant region can be one of five different types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Individual heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains each produce one of the five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0060] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types of amino acid sequences based on the constant domain, called kappa (κ) or lambda (λ).

[0061] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. CDR1, CDR2, and CDR3 in a VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in a VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0062] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., supra; Nick Deschacht et al., J Immunol 2010;184:5696-5704). Chothia instead refers to the position of the structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering, Vol. 2 (eds. Kontermann and Dubel, 2nd ed., 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another commonly used numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system specialized in immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. As used herein, CDRs refer to amino acid sequences and positions within a light or heavy chain. Because the "positions" of CDRs within the structure of immunoglobulin variable domains are conserved across species and reside in structures called loops, CDR and framework residues can be easily identified by using a numbering system that aligns variable domain sequences according to structural features.This information can be used to graft and replace CDR residues from one species' immunoglobulin onto an acceptor framework, usually from a human antibody. Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001), developed an additional numbering system (AHon). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT proprietary numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Each residue from these hypervariable regions or CDRs is illustrated in Table 1 below.

[0063] Table 1: Exemplary CDRs with various numbering systems TIFF2024507958000001.tif86163

[0064] The boundaries of a given CDR may vary depending on the recognition regimen used. Therefore, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof (e.g., a variable region), as well as each CDR of an antibody or region thereof (e.g., CDR-H1, CDR-H2), should be understood to encompass the complementarity-determining regions defined by any of the above known regimens. In some cases, the regimen for recognizing one or more specific CDRs is specified, e.g., CDRs defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of a CDR is given. It should be noted that a CDR region may be defined by a combination of various numbering systems, e.g., a combination of the Kabat and Chothia numbering systems, or a combination of the Kabat and IMGT numbering systems. Thus, a term such as "a CDR as described in a particular VH or VHH" includes, but is not limited to, any CDR1 defined by the above exemplary CDR numbering systems. Given a variable region (eg, VHH, VH, or VL), one skilled in the art will understand that the CDRs within that region may be defined by various numbering systems or combinations thereof.

[0065] The hypervariable regions may also include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0066] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in antigen binding by an antibody but exhibit various effector functions, such as interacting with Fc receptors. The term refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than other portions of the immunoglobulin, i.e., the variable region, and which contains the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0067] The term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, and human antibodies, domain antibodies (e.g., single-domain antibodies), diabodies, linear, and bispecific antibodies. FR residues are hypervariable region or variable domain residues other than CDR residues.

[0068] The term "Fc region" herein is intended to define the C-terminal region of an immunoglobulin heavy chain and includes, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226 or Pro230 toward its carboxy terminus. For example, the C-terminal lysine (residue 447 in the EU numbering system) of the Fc region can be removed during antibody production or purification, or by recombinantly manipulating the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies containing and excluding the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, cell surface receptor (e.g., B cell receptor) down-regulation, and the like. Such effector functions typically require combining the Fc region with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays well known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion). In some embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc regions herein have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or have at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0069] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single-chain antibody sequence) can specifically bind. An epitope may be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope may be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope may comprise amino acids from two or more non-contiguous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will generally understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0070] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percent sequence identity, and after any conservative substitutions are not considered as part of the sequence identity. Alignment and percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art 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 needed to achieve maximal alignment over the full length of the sequences under comparison.

[0071] As used herein, the term "functional exogenous receptor" refers to an exogenous receptor (e.g., a recombinant or engineered TCR, cTCR, TAC-like chimeric receptor, or TCR such as a CAR) that retains its biological activity after introduction of an immune effector cell (e.g., a T cell). Biological activity includes, but is not limited to, specific binding to a molecule by the exogenous receptor, appropriate transduction of downstream signals, e.g., the ability to induce cell proliferation, cytokine production, and / or execution of regulatory or cytolytic effector functions.

[0072] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transfer one or more antigens into immune effector cells, such as T cells. Some CARs are also referred to 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 (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR.

[0073] As used herein, the term "recombinant or engineered TCR" refers to a peptide contained as a functional exogenous receptor and expressed in an immune cell according to the present invention. The function of a recombinant or engineered TCR may include, for example, redirecting the immune activity of an immune cell against a desired type of cell, such as cancer cells and infected cells bearing a specific marker on their surface. They can replace or be coexpressed with an endogenous TCR. In some embodiments, such recombinant TCRs are single-chain TCRs containing an open reading frame in which the variable Vα and Vβ domains are paired with a protein linker. This involves molecular cloning of TCR genes known to be specific for a selected antigen, and then introducing these chains into T cells, typically via a retroviral vector. Expression of the cloned TCRα and TCRβ genes thus confers functional specificity to the transduced T cell determined by the pairing of these new genes. Components of a recombinant or engineered TCR can be any functional subunit of a TCR, such as recombinant TCRα and TCRβ, encoded by an exogenous polynucleotide sequence introduced into a cell.

[0074] In some embodiments, the functional exogenous receptor according to the present invention is a chimeric TCR (cTCR) that simultaneously possesses antigen-binding and T cell activation functions. For example, the cTCR may comprise: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3, claudin 18.2), (b) an optional linker, (c) an optional extracellular domain or portion thereof of a first TCR subunit (e.g., CD3ε), (d) a transmembrane domain comprising a transmembrane domain of a second TCR subunit (e.g., CD3ε), and (e) an intracellular signaling domain comprising an intracellular signaling domain of a third TCR subunit (e.g., CD3ε), wherein the first, second, and third TCR subunits are all selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the first, second, and third TCR subunits are the same (e.g., all are CD3ε). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the cTCR 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 cTCR further comprises a signal peptide located at the N-terminus of the cTCR, for example, a signal peptide derived from CD8α.

[0075] In some embodiments, the functional exogenous receptor is a T cell antigen binder (TAC), e.g., comprising: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3, claudin 18.2); (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3ε); (d) an optional second linker; (e) an optional extracellular domain or portion thereof of a first TCR co-receptor (e.g., CD4); (f) a transmembrane domain comprising the transmembrane domain of a second TCR co-receptor (e.g., CD4); and (g) an optional intracellular signaling domain comprising the intracellular signaling domain of a third TCR co-receptor (e.g., CD4). wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ, and wherein the first, second, and third TCR co-receptors are all selected from the group consisting of CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC 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 TAC further comprises a signal peptide located at the N-terminus of the TAC, for example, a signal peptide derived from CD8α. In some embodiments, the extracellular ligand-binding domain is located at the N-terminus of the extracellular TCR binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR binding domain.

[0076] In some embodiments, the functional exogenous receptor is a TAC-like chimeric receptor, for example, comprising: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3, claudin 18.2); (b) an optional first linker; (c) an extracellular TCR-binding domain that specifically recognizes the extracellular domain of a first TCR subunit (e.g., TCRα); (d) an optional second linker; and (e) a second TCR binding domain that specifically recognizes the extracellular domain of a second TCR subunit (e.g., TCRα). (f) a transmembrane domain comprising the transmembrane domain of a third TCR subunit (e.g., CD3ε); and (g) an optional intracellular signaling domain comprising the intracellular signaling domain of a fourth TCR subunit (e.g., CD3ε), wherein the first, second, third, and fourth TCR subunits are all selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the second, third, and fourth TCR subunits are the same. In some embodiments, the first, second, third, and fourth TCR subunits are different. In some embodiments, the second, third, and fourth TCR subunits are the same but different from the first TCR subunit. In some embodiments, the extracellular ligand-binding domain is located N-terminal to the extracellular TCR binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR-binding domain. In some embodiments, the TAC-like chimeric receptor 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 TAC-like chimeric receptor further comprises a signal peptide, for example, a signal peptide derived from CD8α, located at the N-terminus of the TAC-like chimeric receptor.

[0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms further encompass amino acid polymers that have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition further includes, for example, polypeptides containing one or more amino acid analogs (including, but not limited to, unnatural amino acids) and other modifications known in the art. It should be understood that, because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a "polypeptide" can exist as a single chain or two or more associated chains.

[0078] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or a synthetic reaction. A polynucleotide may also include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded, synthetic polynucleotide, usually (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is referred to as the transcription direction. The sequence region 5' of the 5' end of the RNA transcript that has the same sequence on the DNA strand as the RNA transcript is referred to as the "upstream sequence," and the sequence region 3' of the 3' end of the RNA transcript that has the same sequence on the DNA strand as the RNA transcript is referred to as the "downstream sequence."

[0079] An "isolated nucleic acid" is a nucleic acid (e.g., RNA, DNA, or mixture of nucleic acids) that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Note that an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the single-chain antibodies or antibodies described herein are isolated or purified. The terms encompass nucleic acid sequences that have been removed from their naturally occurring environment and include recombinant or cloned DNA isolates and chemically synthesized analogs or heterologous biologically synthesized analogs. A substantially pure molecule may include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding a CAR, as described herein, is a nucleic acid molecule that is identified and isolated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0080] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0081] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused), when used to refer to nucleic acids or amino acids, refers to the operable linkage of nucleic acid or amino acid sequences, respectively, which are placed in a functional relationship with each other. For example, operably linking a promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequence results in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements cause transcription of the open reading frame, ultimately resulting in the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one whose functional domains are positioned appropriately from each other to confer their intended function.

[0082] The term "vector" refers to a substance for carrying or containing a nucleic acid sequence, such as a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, for introduction into a host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain a selection sequence or a marker capable of stable integration into a host cell chromosome. The vector may also contain one or more selection marker genes and appropriate expression control sequences. The included selection marker genes may, for example, provide resistance to antibiotics or toxins, complement nutrient deficiencies, or supply important nutrients missing from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, all of which are well known in the art. When two or more nucleic acid molecules are to be coexpressed (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules may be inserted, for example, into a single expression vector or separate expression vectors. For expression in a single vector, the encoding nucleic acids may be operably linked to a common expression control sequence or may be linked to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as RNA blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblot used to detect gene product expression, or other suitable analytical methods to test for expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0083] As used herein, the term "host" refers to an animal, for example, a mammal (e.g., a human).

[0084] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0085] As used herein, the term "autologous" refers to any material derived from the same individual, where the material is later reintroduced into the individual.

[0086] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0087] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0088] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, or for use in animals, and more specifically, in humans.

[0089] "Excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0090] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed., 1990).

[0091] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, consistent with a reasonable benefit-to-risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed., edited by Rowe et al.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed., edited by Ash and Ash; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed., edited by Gibson; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0092] In some embodiments, excipients are sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline, aqueous dextrose, and glycerin solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients may further include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, ethylene glycol, water, ethanol, or the like. The composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral compositions may include formulations and standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0093] A composition containing a pharmaceutical compound may include, for example, a binding molecule (eg, an antibody) in isolated or purified form and a suitable amount of an excipient.

[0094] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of engineered immune effector cells, or a therapeutic molecule, or pharmaceutical composition comprising an agent and engineered immune effector cells according to the present invention, sufficient to bring about a desired result.

[0095] The terms "subject" and "patient" may be used interchangeably. As used herein, in some embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0096] "Administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0097] As used herein, the terms "treat," "treatment," and "treating" refer to reducing or ameliorating the progression, severity, and / or duration of a disease or condition through the administration of one or more therapies. Treatment may be determined by assessing whether one or more symptoms associated with the underlying condition are reduced, ameliorated, and / or alleviated, such that the patient is observed to improve, even though the patient may still have the underlying condition. The term "treatment" includes the management and amelioration of disease. The terms "manage," "managing," and "management" refer to the beneficial effect a subject derives from a therapy that does not necessarily result in a cure of the disease.

[0098] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of a disease, disorder, condition, or associated symptom (e.g., cancer) occurring (or recurring).

[0099] As used herein, "delaying" the onset of cancer refers to delaying, inhibiting, slowing, extending, stabilizing, and / or slowing the onset of the disease. This delay may have different lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" the onset of cancer is one that reduces the probability of disease development within a given time range and / or reduces the extent of disease within a given time range, compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. Cancer onset can be detected 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 may also refer to the progression of cancer, which may be initially undetectable, and includes occurrence, recurrence, and attack.

[0100] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0101] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0102] It is to be understood that wherever this specification describes an embodiment with the term "comprising," it also provides similar embodiments where the phrase "consisting essentially of" and / or "consisting essentially of" is used. It is further to be understood that wherever this specification describes an embodiment with the phrase "consisting essentially of," it also provides similar embodiments where the phrase "consisting of" is used.

[0103] The term "between" used in the phrase "between A and B" or "between A and B" refers to a range that includes A and B.

[0104] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0105] 7.2. Engineered immune effector cells As described in Section 8 below, introducing specific combinations of the p40 subunit of IL-12 and a CCR7 ligand (e.g., CCL-19 and CCL-21) into immune effector cells expressing one or more functional exogenous receptors can significantly improve the properties / function of the immune effector cells. Thus, in one aspect, the present specification provides host cells (e.g., immune effector cells) comprising exogenously introduced p40 subunit of IL-12 and a CCR7 ligand (e.g., CCL-19 and CCL-21), wherein the host cell expresses one or more functional exogenous receptors.

[0106] CCR7 (CC chemokine receptor type 7) is a protein encoded by the CCR7 gene in humans. CCR7 is also referred to as BLR2, CC-CKR-7, CCR-7, CD197, CDw197, CMKBR7, EBI1, and CC motif chemokine receptor 7. In some embodiments, CCR7 according to the present specification is identified as OMIM:600242, MGI:103011, HomoloGene:1387, and / or GeneCards:CCR7. Two natural ligands for the receptor, CCL-19 and CCL-21, have been identified. A ligand of CCR7 according to the present specification may be a natural ligand of CCR7 of any species. In other embodiments, a ligand of CCR7 according to the present specification may be an artificial or synthetic ligand of CCR7 of any species.

[0107] The functional exogenous receptor may be, for example, a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), and a chimeric receptor like a T cell antigen binder (TAC). In some embodiments, the functional exogenous receptor is a CAR. In some embodiments, the functional exogenous receptor is a TCR. In some embodiments, the functional exogenous receptor is a cTCR. In other embodiments, the functional exogenous receptor is a TAC. Any immune effector cell capable of performing an immune effector function can be used in the present invention, including, but not limited to, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0108] In some embodiments, the present specification provides a host cell (e.g., an immune effector cell) comprising any one of the polypeptides, polynucleotides, or vectors described herein (e.g., as provided in the sections below).

[0109] In certain embodiments, the description provides CAR-T cells, which express exogenously introduced p40 and CCL-19. In certain embodiments, the description provides TCR-T cells, which express exogenously introduced p40 and CCL-19. In certain embodiments, the description provides TAC-T cells, which express exogenously introduced p40 and CCL-19.

[0110] In certain embodiments, the description provides CAR-T cells, which express exogenously introduced p40 and CCL-21. In certain embodiments, the description provides TCR-T cells, which express exogenously introduced p40 and CCL-21. In certain embodiments, the description provides TAC-T cells, which express exogenously introduced p40 and CCL-21.

[0111] 7.2.1. P40, CCL-19 and CCL-21 In some embodiments, the engineered immune effector cells of the invention comprise a specific combination of exogenously introduced p40 subunit of IL-12 or a variant thereof and CCL-19 or a variant thereof, hi other embodiments, the engineered immune effector cells of the invention comprise a specific combination of exogenously introduced p40 subunit of IL-12 or a variant thereof and CCL-21 or a variant thereof.

[0112] As used herein, p40 is the subunit β of interleukin-12, also known as IL-12B, natural killer cell-stimulating factor 2, cytotoxic lymphocyte maturation factor p40, or interleukin-12 subunit p40, and is a protein encoded by the IL12B gene in humans. P40 is a common subunit of interleukin-12 and interleukin-23. In some embodiments, p40 is identified as OMIM:161561, MGI:96540, HomoloGene:1648, and / or GeneCards:IL12B. As used herein, p40 includes p40 from any species. As used herein, p40 further includes any p40 variant that substantially retains at least one biological activity thereof (e.g., at least 80% of its biological activity). In some embodiments, p40 according to the present invention is human p40 or a fragment or variant thereof. In a specific embodiment, p40 according to the present invention comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, a p40 provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5. For example, in some embodiments, a p40 provided herein comprises an amino acid sequence having about 80% sequence identity to SEQ ID NO:5 and retains at least 80% of the biological activity of a p40 comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, a p40 polypeptide provided herein is a secreted polypeptide. In some embodiments, a p40 polypeptide provided herein is a membrane-bound polypeptide (e.g., MB12).

[0113] The p40 polypeptides herein are membrane-bound polypeptides formed by any technique known in the art. In some embodiments, the p40 polypeptides are membrane-bound via a membrane anchor domain. In some embodiments, the p40 polypeptides are membrane-bound via a transmembrane domain. Exemplary transmembrane domains include, but are not limited to, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, B7, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLR1), CD160, CD19, IL-2R β, IL-2R γ, CD8α, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the p40 polypeptide is membrane-associated and further comprises a second polypeptide fragment. In some embodiments, the second polypeptide fragment is selected from the group consisting of CD28, CD8α, OX40, and B7. In some embodiments, the p40 fragment and the second polypeptide fragment are linked to each other via a peptide linker.In some embodiments, the linker peptide is a flexible linker. Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GSGGS). n (SEQ ID NO: 46), (GGGS) n (SEQ ID NO: 47), and (GGGGS) n (SEQ ID NO:48), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, a membrane-bound p40 polypeptide in accordance with the present disclosure comprises, from N- to C-terminal, a signal peptide, p40, a hinge and transmembrane domain derived from CD8α, and any intracellular signaling domain from CD8α. In a specific embodiment, a membrane-bound p40 polypeptide in accordance with the present disclosure comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the membrane-bound p40 polypeptide further comprises a third polypeptide fragment. In some embodiments, the third polypeptide fragment is a dominant-negative receptor (DNR). In some embodiments, the third polypeptide fragment is a TGFβRII DNR. In some embodiments, a membrane-associated p40 polypeptide according to the present disclosure comprises, from N- to C-terminal, a signal peptide, p40, a TGFβRII DNR, a hinge and transmembrane domain derived from CD8α, and any intracellular signaling domain from CD8α.

[0114] Chemokine (C-C motif) ligand 19 (CCL-19) is a protein encoded by the CCL19 gene in humans and is also referred to as CKb11, ELC, MIP-3b, MIP3B, SCYA19, and C-C motif chemokine ligand 19. In some embodiments, CCL-19 is identified as OMIM:602227, MGI:5434459, HomoloGene:4569, and / or GeneCards:CCL19. As used herein, CCL-19 includes CCL-19 from any species. As used herein, CCL-19 further includes any CCL-19 variant that substantially retains at least one biological activity thereof (e.g., at least 80% of its biological activity). In some embodiments, a CCL-19 according to the present disclosure is human CCL-19 or a fragment or variant thereof. In a specific embodiment, a CCL-19 according to the present disclosure comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, a CCL-19 according to the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6. For example, in some embodiments, a CCL-19 according to the present disclosure comprises an amino acid sequence having about 80% sequence identity to SEQ ID NO: 6 and retains at least 80% of the biological activity of a CCL-19 comprising the amino acid sequence of SEQ ID NO: 6.

[0115] Chemokine (C-C motif) ligand 21 (CCL-21) is a cytokine belonging to the CC chemokine family. CCL-21 is also referred to as 6Ckine, CKb9, ECL, SCYA21, SLC, TCA4, and CC motif chemokine ligand 21. In some embodiments, CCL-21 is identified as OMIM:602737, MGI:1349183, HomoloGene:2247, and / or GeneCards:CCL21. As used herein, CCL-21 includes CCL-21 from any species. As used herein, CCL-21 further includes any CCL-21 variant that substantially retains at least one biological activity thereof (e.g., at least 80% of its biological activity). In some embodiments, CCL-21 according to the present specification is human CCL-21 or a fragment or variant thereof. In a specific embodiment, CCL-21 according to the present specification comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, a CCL-21 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22. For example, in some embodiments, a CCL-21 according to the present specification comprises an amino acid sequence having about 80% sequence identity to SEQ ID NO: 22 and retains at least 80% of the biological activity of a CCL-21 comprising the amino acid sequence of SEQ ID NO: 22.

[0116] In some embodiments, a p40 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, and a CCL-19 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6. In a specific embodiment, a p40 according to the present invention comprises the amino acid sequence of SEQ ID NO:5, and a CCL-19 according to the present invention comprises the amino acid sequence of SEQ ID NO:6.

[0117] In some embodiments, a p40 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, and a CCL-21 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:22. In a specific embodiment, p40 according to the present invention comprises the amino acid sequence of SEQ ID NO:5, and CCL-21 according to the present invention comprises the amino acid sequence of SEQ ID NO:22.

[0118] The percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be determined using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), as modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100 and wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches may be performed, for example, with the XBLAST program parameters set to score 50 and word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST, described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997), can be used. Alternatively, PSI BLAST may be used in an iterated search (Id.), which detects distant relationships between molecules. When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., for XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When aligning amino acid sequences using the ALIGN program, a PAM120 weight residue table can be used, with a gap length penalty of 12 and a gap penalty of 4.

[0119] In some embodiments, one or more amino acid sequence modifications or alterations of p40, CCL-19, and CCL-21 described herein are contemplated. The alteration may be a substitution, deletion, or insertion of one or more codons encoding the polypeptide, resulting in a change in the amino acid sequence relative to the original polypeptide.

[0120] Amino acid substitutions may be the result of substituting an amino acid with another amino acid having similar structural and / or chemical properties, such as substituting serine for leucine, and may be, for example, conservative amino acid substitutions. Standard techniques well known to those of skill in the art may be used to introduce mutations into nucleotide sequences encoding the molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Tolerable changes can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity of the parent polypeptide.

[0121] The present disclosure includes polypeptides produced by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As described above, the art has defined families of amino acid residues with side chains with similar charges. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly along all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be measured. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) may be made to preserve or not significantly alter properties.

[0122] Amino acids can be grouped based on similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2nd ed., 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, to improve the oxidative stability of the molecule and prevent aberrant crosslinking, any cysteine ​​residue not involved in maintaining the correct conformation of the peptide may be substituted with another amino acid, such as alanine or serine. Non-conservative substitutions involve exchanging one member of these classes for another.

[0123] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. The alterations may be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To generate polypeptide variant DNA, cloned DNA may be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques.

[0124] 7.2.2. Chimeric Antigen Receptors The engineered immune effector cells of the present invention express one or more functional exogenous receptors. Any functional exogenous receptor is encompassed by the present disclosure. Chimeric antigen receptors (CARs) are described in more detail below as exemplary functional exogenous receptors according to the present disclosure, but are not intended to limit the scope of the present disclosure.

[0125] In some embodiments, the CAR in the immune effector cells of the invention comprises a polypeptide, which comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, each and every region of which is described in more detail below.

[0126] Extracellular antigen-binding domain The extracellular antigen-binding domain of a CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain of a CAR described herein is monospecific. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is multispecific. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is monovalent. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is multivalent. In some embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains, which are fused to each other via a direct peptide bond or a peptide linker.

[0127] In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. For example, the binding domain may be derived from a monoclonal antibody (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), an antibody with multiepitopic or monoepitopic specificity, a polyclonal or monovalent antibody, a multivalent antibody, a multispecific antibody formed from at least two intact antibodies (e.g., a bispecific antibody, so long as it exhibits the desired biological activity), a single-chain antibody, a single-domain antibody, and fragments thereof (e.g., domain antibodies). The antibody may be a human antibody, a humanized antibody, a chimeric antibody, and / or an affinity-matured antibody, as well as an antibody from another species (e.g., mouse, rabbit, llama, etc.). In some embodiments, an antibody is intended to include a polypeptide product of B cells in the immunoglobulin family of polypeptides, which has the ability to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (ed. Borrebaeck, 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These functional fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies according to the present specification include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2nd ed., 1990). An antibody according to the present invention can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist nor an antagonist.

[0128] In a specific embodiment, the extracellular antigen-binding domain of the CAR of the invention comprises a single-chain Fv (sFv or scFv). An scFv is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. See Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0129] In another specific embodiment, the extracellular antigen-binding domain of the CAR of the invention comprises one or more single domain antibodies (sdAbs). The sdAbs may be of the same or different sources and of the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains from antibodies having only heavy chains (e.g., VHH or VNAR), binding molecules naturally lacking light chains, single domains (e.g., VH or VL) derived from traditional four-chain antibodies, humanized antibodies having only heavy chains, human single domain antibodies produced from transgenic mice or rats expressing human heavy chain segments, and single domain scaffolds other than those derived from engineered domains and antibodies. Any sdAb known in the art or developed in accordance with the present disclosure, including the single domain antibodies described above in this disclosure, can be used to construct the CARs described herein. sdAbs may be derived from any species, including but not limited to mouse, rat, human, camel, llama, river lamprey, fish, shark, goat, rabbit and cow. Single domain antibodies contemplated herein further include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0130] In some embodiments, naturally occurring single-domain antigen-binding molecules derived from sdAbs are referred to as heavy-chain antibodies lacking light chains (also referred to herein as "antibodies having only heavy chains"). For example, such single-domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, variable domains derived from heavy-chain molecules naturally lacking light chains are referred to herein as VHHs to distinguish them from conventional VH regions of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced by Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. Other species outside of Camelidae can produce heavy-chain molecules naturally lacking light chains, and such VHHs are within the scope of the present disclosure. Note that humanized versions and other modifications and variants of VHHs are also contemplated and within the scope of the present disclosure. In some embodiments, sdAbs are derived from the variable region of immunoglobulins found in cartilaginous fish. For example, sdAbs may be derived from an immunoglobulin isotype called a novel antigen receptor (NAR), which is found in shark serum. Methods for producing single domain molecules derived from the variable region of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0131] In some embodiments, naturally occurring VHH domains against a particular antigen or target can be obtained from a library (naive or immune) of camelid VHH sequences. Such methods may or may not involve screening such libraries for said antigen or target, or at least a portion, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries can be used, e.g., VHH libraries obtained from (naive or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO 00 / 43507.

[0132] In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized and / or produced in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions can be altered by "camelization" of specific amino acid residues in the framework regions. Camelization refers to the replacement or substitution of one or more amino acid residues in an amino acid sequence from a (naturally occurring) VH domain of a conventional four-chain antibody with one or more amino acid residues present in one or more corresponding positions in a VHH domain of a heavy-chain antibody. This may be performed in a manner known in the art and will be apparent to one skilled in the art. Such "camelizing" substitutions are preferably inserted at amino acid positions forming and / or present at the VH-VL interface and / or at so-called camelid tag residue positions, 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)).

[0133] In some embodiments, the sdAb is a human single domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US 20090307787, U.S. Patent No. 8,754,287, US 20150289489, US 20100122358 and WO 2004049794.

[0134] In some embodiments, single domain antibodies are produced using conventional four-chain antibodies. See, e.g., EP 0 368 684, Ward et al., Nature, 341(6242):544-6 (1989), Holt et al., Trends Biotechnol., 21(11):484-490 (2003), WO 06 / 030220, and WO 06 / 003388.

[0135] In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or fragment thereof. A humanized antibody may comprise human framework regions and human constant region sequences.

[0136] Humanized antibodies can be produced using a variety of techniques known in the art, including CDR-grafting (European Patent No. EP 239,400, International Publication No. WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, but not limited to, the techniques disclosed in U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55(23 Suppl):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent No. US 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0137] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are commonly referred to as "import" residues, and these residues are typically taken from an "import" variable domain. For example, humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods described by Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36.

[0138] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. revealed that only about one-third of the residues in the CDRs actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In SDR grafting technology, only the SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:25-34).

[0139] To reduce antigenicity, the selection of human (light and heavy chain) variable domains for generating humanized antibodies can be important. For example, the so-called "best-fit" method involves screening the variable domain sequence of a non-human (e.g., rodent) antibody against the entire library of known human variable domain sequences. The human sequence closest to that of a rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308, and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89, and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequence of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). Alternatively, human germline genes are used as the source of the framework regions.

[0140] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FRs is irrelevant. This method involves comparing non-human sequences with functional human germline gene repertoires. Then, genes encoding the same or closely related canonical structures as the mouse sequences are selected. Subsequently, among genes that share canonical structures with non-human antibodies, the genes with the highest homology within CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0141] Furthermore, it is usually desirable to humanize antibodies to retain their affinity for the antigen and other favorable biological properties. To achieve this goal, one method involves preparing humanized antibodies by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). ​​Inspection of these displays permits analysis of the possible effect of residues on the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, by selecting and combining FR residues from the receptor and import sequences, the desired antibody characteristic, such as increased affinity for one or more target antigens, can be obtained. Typically, the hypervariable region residues are directly and most significantly involved in influencing antigen binding.

[0142] Another antibody humanization method is based on a metric of antibody humanization called human string content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing the HSC, rather than using a global identity measure, to generate multiple distinct humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0143] In addition to the above methods, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries and by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0144] In the FR library approach, a set of residue mutations is introduced at specific positions within the FR, and the library is screened to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the "vernier" residues identified as likely to contribute to CDR structure (e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or a more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0145] In FR shuffling, entire FRs are combined with non-human CDRs without creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library can be screened for binding in a two-step process (first VL humanization, then VH). Alternatively, a one-step FR shuffling process can be used. Such methods prove more effective than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0146] The "humaneering" method is based on experimental identification of essential minimum specificity determinants (MSDs), followed by sequential replacement of non-human fragments with a human FR library and evaluation of binding. It begins with the CDR3 regions of the non-human VH and VL chains and gradually replaces other regions of the non-human antibody, including CDR1 and CDR2 of VH and VL, with human FRs. This method typically results in epitope retention and identification of antibodies from multiple subclasses with different human V-segment CDRs. Humaneering allows the isolation of antibodies with 91% to 96% homology to human germline antibodies (see, e.g., Alfreito, Cambridge Healthtech Institute's 3rd Annual PEGS, The Protein Engineering Summit, 2007).

[0147] "Human engineering" methods involve altering non-human antibodies or antibody fragments (e.g., murine or chimeric antibodies or antibody fragments) with specific modifications to their amino acid sequences to produce modified antibodies with reduced immunogenicity in humans, while still retaining the required binding characteristics of the original non-human antibody. Typically, the techniques involve classifying amino acid residues in non-human (e.g., murine) antibodies into "low risk," "intermediate risk," or "high risk" residues. Classification is performed using an overall risk / reward calculation that assesses the predicted benefit of a particular substitution (e.g., with respect to human immunogenicity) and the risk that the substitution will affect the folding of the resulting antibody. By aligning amino acid sequences from non-human antibody variable regions with the corresponding regions of specific or consensus human antibody sequences, specific human amino acid residues to be substituted at given positions (e.g., low risk or intermediate risk) in the non-human (e.g., murine) antibody sequence can be selected. Amino acid residues at low or intermediate risk positions in the non-human sequence can be substituted for the corresponding residue in the human antibody sequence by alignment. Techniques for producing human engineered proteins are described in further detail in Studnicka et al., 1994, Protein Engineering 7:805-14, U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Publication WO 93 / 11794.

[0148] Composite human antibodies can be produced, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce composite human antibodies, the variable region sequences are designed from fragments of multiple human antibody variable region sequences to avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody. Such antibodies may also contain human constant region sequences, such as human light and / or heavy chain constant regions.

[0149] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009;525:405-23, xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006). A deimmunized antibody comprises a variable region from which T cell epitopes have been deleted and a human constant region. Briefly, the VH and VL of an antibody are cloned, followed by identification of T cell epitopes by testing overlapping peptides derived from the antibody VH and VL in a T cell proliferation assay. T cell epitopes are identified using computational methods to identify peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to eliminate binding to human MHC class II. The mutated VH and VL are then used to produce a deimmunized antibody.

[0150] In some embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof, for example, the extracellular antigen-binding domain comprises multiple binding domains connected in series (e.g., multiple scFvs). In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. The term "specificity" refers to the selective recognition of a specific epitope of an antigen by the antigen-binding protein. As used herein, the term "multispecific" indicates that the antigen-binding protein has two or more antigen-binding sites, where at least two bind to different antigens. As used herein, the term "valent" indicates that an antigen-binding protein has a specified number of binding sites. A full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that the antigen-binding protein has two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites, respectively.

[0151] Multispecific antibodies, such as bispecific antibodies, are antibodies that have binding specificities for at least two different antigens. Methods for producing multispecific antibodies, such as co-expressing two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities, are known in the art (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (ed. by Kontermann, 2011).

[0152] The antibody may be a multivalent antibody (e.g., a tetravalent antibody) having two or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. In some embodiments, the multivalent antibody comprises (or is composed of) three to about eight antigen-binding sites. In such embodiments, the multivalent antibody comprises (or is composed of) four antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable domains. For example, the one or more polypeptide chains may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, one or more polypeptide chains may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain, or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides. For example, the multivalent antibody herein may comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally comprise a CL domain.

[0153] When multiple binding domains are present in the extracellular antigen-binding domain of the CAR of the present invention, the various domains can be fused to each other via peptide linkers. In some embodiments, the domains are fused to each other directly without any peptide linkers. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence, depending on the structural and / or functional characteristics of the various domains. Each peptide linker can be independently selected and optimized. The length, flexibility, and / or other properties of the peptide linker(s) used in the CAR may have some effect on the properties, including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. In some embodiments, the peptide linker contains flexible residues (e.g., glycine and serine), allowing adjacent domains to move freely relative to each other. For example, a glycine-serine bipartite may be a suitable peptide linker.

[0154] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, the sequence of a hinge region from an antibody having only a heavy chain may be used as a linker. See, e.g., WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO:46), (GGGS)n (SEQ ID NO:47), and (GGGGS)n (SEQ ID NO:48), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art may be included in a CAR according to the present specification, for example, WO 2016014789, WO 2015158671, WO 2016102965, US 20150299317, WO 2018067992, US 7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference.

[0155] In some embodiments, the extracellular antigen-binding domain provided in the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. Tumors express proteins that can act as target antigens for immune responses (particularly T cell-mediated immune responses). The antigen targeted by the CAR may be an antigen in a single diseased cell or an antigen expressed in different cells that each affect the disease. The antigen targeted by the CAR may be directly or indirectly involved in the disease.

[0156] In some embodiments, the antigen of the target cell is an antigen on the surface of a cancer cell, hi some embodiments, the antigen is a tumor-specific antigen, a tumor-associated antigen, or a neoantigen.

[0157] In some embodiments, the target cell is a cancer cell, for example, a cell of the following cancers: adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational choriocarcinoma, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some embodiments, the cancer is adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational choriocarcinoma, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some embodiments, the cancer is adrenal gland cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is appendix cancer. In some embodiments, the cancer is bile duct cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is gestational choriocarcinoma. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is Hodgkin's lymphoma. In some embodiments, the cancer is intestinal cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is leukemia.In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is oral cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is sinonasal cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is soft tissue sarcoma or spinal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is pharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is uterine cancer or endometrial cancer. In some embodiments, the cancer is vaginal cancer. In some embodiments, the cancer is vulvar cancer.

[0158] In some embodiments, the adrenal cancer is adrenocortical carcinoma (ACC), adrenocortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is squamous cell carcinoma, cloacal carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments, the appendix cancer is neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal adenocarcinoma, or signet ring cell adenocarcinoma. In some embodiments, the cholangiocarcinoma is extrahepatic bile duct carcinoma, adenocarcinoma, perihilar cholangiocarcinoma, hilar region cholangiocarcinoma, distal bile duct carcinoma, or intrahepatic cholangiocarcinoma. In some embodiments, the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, squamous carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma. In some embodiments, the bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary adenocarcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, meloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, breast cancer in situ, breast sarcoma, metaplastic carcinoma, adenoid cystic carcinoma, follicular tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, the gestational trophoblastic disease (GTD) is hydatidiform mole, gestational trophoblastic neoplasia (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cancer, oropharyngeal cancer, or tonsillar cancer.In some embodiments, the Hodgkin's lymphoma is classical Hodgkin's lymphoma, nodular sclerosing, mixed cytology, lymphocyte-rich, lymphocyte-reduced, or nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL). In some embodiments, the intestinal cancer is small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvic carcinoma, or renal sarcoma. In some embodiments, the leukemia is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS). In a specific embodiment, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, large cell undifferentiated carcinoma, lung nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, pulmonary sarcomatoid carcinoma, or malignant granular cell lung tumor. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna, amelanotic melanoma, connective tissue proliferative melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is symptomatic or smoldering myeloma. In some embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a pulmonary neuroendocrine tumor.In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, follicular lymphoma, cutaneous T-cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small cell lymphocytic lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T-lymphoblastic leukemia / lymphoma, acute lymphocytic leukemia (ALL), adult T-cell lymphoma / leukemia (ATLL), hairy cell leukemia, B-cell lymphoma, and leukemia. lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, intranodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, Alibert-Bazin syndrome, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathy-associated T-cell lymphoma (EATL), or hepatosplenic gamma / delta T-cell lymphoma. In some embodiments, the oral cancer is squamous cell carcinoma, verrucous carcinoma, minor salivary gland cancer, lymphoma, benign oral tumor, eosinophilic granulomatosis, fibroma, granular cell tumor, keratoacanthoma, leiomyoma, osteochondromatosis, lipoma, schwannoma, neurofibroma, papillary cytoma, condyloma acuminata, verrucous xanthomas, pyogenic granuloma, rhabdomyoma, odontogenic tumor, vitiligo, erythema, squamous cell lip cancer, basal cell lip cancer, mouth cancer, gum cancer, or tongue cancer.In some embodiments, the ovarian cancer is epithelial ovarian cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, epithelial ovarian clear cell carcinoma, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumor, primary peritoneal carcinoma, fallopian tube cancer, germinoma, teratoma, dysgerminoma, ovarian germ cell carcinoma, endodermal sinus tumor, sex cord-stromal tumor, sex cord-gonadal stromal tumor, ovarian stromal tumor, granular cell tumor, granulosa-thecal cell tumor, Sertoli-Leydig tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg tumor, or ovarian cyst. In some embodiments, the pancreatic cancer is pancreatic exocrine adenocarcinoma, pancreatic endocrine adenocarcinoma, pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is prostate adenocarcinoma, prostatic sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In some embodiments, the paranasal sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, undifferentiated carcinoma of the nasal and paranasal sinuses, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoechinoderm. In some embodiments, the soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), lipomatous liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, renal sarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is a metastatic spinal tumor. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL-cell carcinoid, type II ECL-cell carcinoid, or type III ECL-cell carcinoid. In some embodiments, the testicular cancer is seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratocele, gonadal stromal tumor, testicular Leydig cell tumor, or testicular Sertoli cell tumor.In some embodiments, the pharyngeal cancer is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal cancer, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous carcinoma, undifferentiated carcinoma, or lymph node carcinoma. In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, medullary thyroid carcinoma, or undifferentiated carcinoma. In some embodiments, the uterine cancer is endometrial carcinoma, endometrial adenocarcinoma, endometrioid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

[0159] Tumor antigens are proteins produced by tumor cells that are involved in immune responses, particularly T cell-mediated immune responses. Exemplary tumor antigens include, but are not limited to, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroid globulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0160] In some embodiments, the cancer antigen is CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EpCAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family antigens, CAGE family antigens, GAGE ​​family antigens, MAGE family antigens, SAGE family antigens, XAGE family antigens, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, P polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, or MUC1. In some embodiments, the cancer antigen is CEA. In some embodiments, the cancer antigen is immature laminin receptor. In some embodiments, the cancer antigen is TAG-72. In some embodiments, the cancer antigen is HPV E6. In some embodiments, the cancer antigen is HPV E7. In some embodiments, the cancer antigen is BING-4. In some embodiments, the cancer antigen is calcium-activated chloride channel 2. In some embodiments, the cancer antigen is cyclin-B1. In some embodiments, the cancer antigen is 9D7. In some embodiments, the cancer antigen is EpCAM. In some embodiments, the cancer antigen is EphA3. In some embodiments, the cancer antigen is Her2 / neu. In some embodiments, the cancer antigen is telomerase. In some embodiments, the cancer antigen is mesothelin. In some embodiments, the cancer antigen is SAP-1. In some embodiments, the cancer antigen is survivin. In some embodiments, the cancer antigen is a BAGE family antigen. In some embodiments, the cancer antigen is a CAGE family antigen. In some embodiments, the cancer antigen is a GAGE ​​family antigen.In some embodiments, the cancer antigen is a MAGE family antigen. In some embodiments, the cancer antigen is a SAGE family antigen. In some embodiments, the cancer antigen is a XAGE family antigen. In some embodiments, the cancer antigen is NY-ESO-1 / LAGE-1. In some embodiments, the cancer antigen is PRAME. In some embodiments, the cancer antigen is SSX-2. In some embodiments, the cancer antigen is Melan-A. In some embodiments, the cancer antigen is MART-1. In some embodiments, the cancer antigen is Gp100. In some embodiments, the cancer antigen is pmel17. In some embodiments, the cancer antigen is tyrosinase. In some embodiments, the cancer antigen is TRP-1. In some embodiments, the cancer antigen is TRP-2. In some embodiments, the cancer antigen is P. polypeptide. In some embodiments, the cancer antigen is MC1R. In some embodiments, the cancer antigen is prostate-specific antigen. In some embodiments, the cancer antigen is β-catenin. In some embodiments, the cancer antigen is BRCA1. In some embodiments, the cancer antigen is BRCA2. In some embodiments, the cancer antigen is CDK4. In some embodiments, the cancer antigen is CML66. In some embodiments, the cancer antigen is fibronectin. In some embodiments, the cancer antigen is MART-2. In some embodiments, the cancer antigen is p53. In some embodiments, the cancer antigen is Ras. In some embodiments, the cancer antigen is TGF-βRII. In some embodiments, the cancer antigen is MUC1.

[0161] In some embodiments, the tumor antigen is one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used as target antigens for immune challenges. 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 molecules associated with transformation, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0162] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present in other cells in the body. TAA-associated antigens are not unique to tumor cells and can instead be expressed on normal cells under conditions that do not allow for immune tolerance to the antigen to be induced. Expression of an antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0163] 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, tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15, overexpressed embryonic antigens such as CEA, overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu, unique tumor antigens produced by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0164] Other large 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, 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, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0165] Other non-limiting exemplary targets of CARs according to the present disclosure include GPC2, CD276, delta-like protein ligand 3 (DLL3), NY-ESO-1, melanoma-associated antigen 4, survivin, synovial sarcoma X breakpoint 2 protein, CD3, epidermal growth factor receptor (EGFR), erbb2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, ntrkr1 tyrosine kinase receptor, GPC3, claudin 18.2, mesothelin, glutamate carboxypeptidase II, PMSA, PD-L1, folate receptor alpha, PSCA, mucin 1, HLA antigens (e.g., HLA class I antigen A-2 alpha, HLA class I antibody A-11 alpha, and HLA class II antigens), c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS), IL-15 receptor, Kit tyrosine kinase, PDGF receptor beta, RET tyrosine kinase receptor, Raf 1 protein kinase, Raf B protein kinase, thymidylate synthase, topoisomerase II, Brachyury proteins, Flt3 tyrosine kinase, VEGF, VEGF receptors (VEGF-1 receptor, VEGF-2 receptor, and VEGF-3 receptor), estrogen receptor, neoantigen, human papillomavirus E6, and heat shock proteins.

[0166] In some specific embodiments, at least one target antigen of the CAR of the present invention is CD19. In other specific embodiments, at least one target antigen of the CAR of the present invention is CD20. In other specific embodiments, at least one target antigen of the CAR of the present invention is CD22. In other specific embodiments, at least one target antigen of the CAR of the present invention is BCMA. In other specific embodiments, at least one target antigen of the CAR of the present invention is VEGFR2. In other specific embodiments, at least one target antigen of the CAR of the present invention is FAP. In other specific embodiments, at least one target antigen of the CAR of the present invention is EpCam. In other specific embodiments, at least one target antigen of the CAR of the present invention is GPC3. In other specific embodiments, at least one target antigen of the CAR of the present invention is claudin 18.2. In other specific embodiments, at least one target antigen of the CAR of the present invention is CD133. In other specific embodiments, at least one target antigen of the CAR of the present invention is IL13Ra. In other specific embodiments, at least one target antigen of the CAR of the present invention is EGFRIII. In other specific embodiments, at least one target antigen of the CAR of the present invention is EphA2. In another specific embodiment, at least one target antigen of the CAR of the present invention is Mucl. In another specific embodiment, at least one target antigen of the CAR of the present invention is CD70. In another specific embodiment, at least one target antigen of the CAR of the present invention is CD123. In another specific embodiment, at least one target antigen of the CAR of the present invention is ROR1. In another specific embodiment, at least one target antigen of the CAR of the present invention is PSMA. In another specific embodiment, at least one target antigen of the CAR of the present invention is CD5. In another specific embodiment, at least one target antigen of the CAR of the present invention is GD2. In another specific embodiment, at least one target antigen of the CAR of the present invention is GAP. In another specific embodiment, at least one target antigen of the CAR of the present invention is CD33.In another specific embodiment, at least one target antigen of the CAR of the present invention is CEA. In another specific embodiment, at least one target antigen of the CAR of the present invention is PSCA. In another specific embodiment, at least one target antigen of the CAR of the present invention is Her2. In another specific embodiment, at least one target antigen of the CAR of the present invention is mesothelin.

[0167] In some embodiments, a CAR according to the present disclosure binds to a B cell antigen, in some embodiments, the B cell antigen is CD1a, CD1b, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11b, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32b, CD35, CD37, CD38 , CD39, CD40, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, CD49d, CD50, CD52 , CD53, CD54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD75, CD75S, CD77, CD 79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I, CD85J, CD86, CD92, CD95, CD97, CD98, CD99, CD 100, CD102, CD108, CD119, CD120a, CD120b, CD121b, CD122, CD124, CD125, CD126, CD130, CD132, CD137, C D138, CD139, CD147, CD148, CD150, CD152, CD162, CD164, CD166, CD167a, CD170, CD171, CD175, CD175s, C D180, CD184, CD185, CD192, CD196, CD197, CD200, CD205, CD201a, CDw210b, CD212, CD213a1, CD213a2, CD 215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD224, CD225, CD226, CD227, CD229, CD230, CD232, CD252, CD252, CD254, CD255, CD256, CD257, CD258, CD259, CD2 60, CD261, CD262, CD263, CD264, CD267, CD268, CD269, CD270, CD272, CD274, CD275, CD277, CD279, CD283, CD289, CD290, CD295, CD298, CD300, CD300c, CD305, CD306,In some embodiments, the B cell antigen is a CD307a, CD307b, CD307c, CD307d, CD307e, CD314, CD215, CD316, CD317, CD319, CD321, CD327, CD328, CD329, CD338, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD360, CD361, CD362, or CD363 antigen. In some embodiments, the B cell antigen is a CD1a antigen. In some embodiments, the B cell antigen is a CD1b antigen. In some embodiments, the B cell antigen is a CD1c antigen. In some embodiments, the B cell antigen is a CD1d antigen. In some embodiments, the B cell antigen is a CD2 antigen. In some embodiments, the B cell antigen is a CD5 antigen. In some embodiments, the B cell antigen is a CD6 antigen. In some embodiments, the B cell antigen is a CD9 antigen. In some embodiments, the B cell antigen is a CD11a antigen. In some embodiments, the B cell antigen is a CD11b antigen. In some embodiments, the B cell antigen is a CD11c antigen. In some embodiments, the B cell antigen is a CD17 antigen. In some embodiments, the B cell antigen is a CD18 antigen. In some embodiments, the B cell antigen is a CD19 antigen. In some embodiments, the B cell antigen is a CD20 antigen. In some embodiments, the B cell antigen is a CD21 antigen. In some embodiments, the B cell antigen is a CD22 antigen. In some embodiments, the B cell antigen is a CD23 antigen. In some embodiments, the B cell antigen is a CD24 antigen. In some embodiments, the B cell antigen is a CD25 antigen. In some embodiments, the B cell antigen is a CD26 antigen. In some embodiments, the B cell antigen is a CD27 antigen. In some embodiments, the B cell antigen is a CD29 antigen. In some embodiments, the B cell antigen is a CD30 antigen. In some embodiments, the B cell antigen is CD31 antigen. In some embodiments, the B cell antigen is CD32a antigen. In some embodiments, the B cell antigen isIn some embodiments, the B cell antigen is a CD32b antigen. In some embodiments, the B cell antigen is a CD35 antigen. In some embodiments, the B cell antigen is a CD37 antigen. In some embodiments, the B cell antigen is a CD38 antigen. In some embodiments, the B cell antigen is a CD39 antigen. In some embodiments, the B cell antigen is a CD40 antigen. In some embodiments, the B cell antigen is a CD45 antigen. In some embodiments, the B cell antigen is a CD45RA antigen. In some embodiments, the B cell antigen is a CD45RB antigen. In some embodiments, the B cell antigen is a CD45RC antigen. In some embodiments, the B cell antigen is a CD45RO antigen. In some embodiments, the B cell antigen is a CD46 antigen. In some embodiments, the B cell antigen is a CD47 antigen. In some embodiments, the B cell antigen is a CD48 antigen. In some embodiments, the B cell antigen is a CD49b antigen. In some embodiments, the B cell antigen is a CD49c antigen. In some embodiments, the B cell antigen is a CD49d antigen. In some embodiments, the B cell antigen is a CD50 antigen. In some embodiments, the B cell antigen is a CD52 antigen. In some embodiments, the B cell antigen is a CD53 antigen. In some embodiments, the B cell antigen is a CD54 antigen. In some embodiments, the B cell antigen is a CD55 antigen. In some embodiments, the B cell antigen is a CD58 antigen. In some embodiments, the B cell antigen is a CD60a antigen. In some embodiments, the B cell antigen is a CD62L antigen. In some embodiments, the B cell antigen is a CD63 antigen. In some embodiments, the B cell antigen is a CD68 antigen. In some embodiments, the B cell antigen is a CD69 antigen. In some embodiments, the B cell antigen is a CD70 antigen. In some embodiments, the B cell antigen is a CD72 antigen. In some embodiments, the B cell antigen is a CD73 antigen. In some embodiments, the B cell antigen is a CD74 antigen. In some embodiments,The B cell antigen is a CD75 antigen. In some embodiments, the B cell antigen is a CD75S antigen. In some embodiments, the B cell antigen is a CD77 antigen. In some embodiments, the B cell antigen is a CD79a antigen. In some embodiments, the B cell antigen is a CD79b antigen. In some embodiments, the B cell antigen is a CD80 antigen. In some embodiments, the B cell antigen is a CD81 antigen. In some embodiments, the B cell antigen is a CD82 antigen. In some embodiments, the B cell antigen is a CD83 antigen. In some embodiments, the B cell antigen is a CD84 antigen. In some embodiments, the B cell antigen is a CD85E antigen. In some embodiments, the B cell antigen is a CD85I antigen. In some embodiments, the B cell antigen is a CD85J antigen. In some embodiments, the B cell antigen is a CD86 antigen. In some embodiments, the B cell antigen is a CD92 antigen. In some embodiments, the B cell antigen is a CD95 antigen. In some embodiments, the B cell antigen is a CD97 antigen. In some embodiments, the B cell antigen is a CD98 antigen. In some embodiments, the B cell antigen is a CD99 antigen. In some embodiments, the B cell antigen is a CD100 antigen. In some embodiments, the B cell antigen is a CD102 antigen. In some embodiments, the B cell antigen is a CD108 antigen. In some embodiments, the B cell antigen is a CD119 antigen. In some embodiments, the B cell antigen is a CD120a antigen. In some embodiments, the B cell antigen is a CD120b antigen. In some embodiments, the B cell antigen is a CD121b antigen. In some embodiments, the B cell antigen is a CD122 antigen. In some embodiments, the B cell antigen is a CD124 antigen. In some embodiments, the B cell antigen is a CD125 antigen. In some embodiments, the B cell antigen is a CD126 antigen. In some embodiments, the B cell antigen is a CD130 antigen. In some embodiments, the B cell antigen isThe B cell antigen is a CD132 antigen. In some embodiments, the B cell antigen is a CD137 antigen. In some embodiments, the B cell antigen is a CD138 antigen. In some embodiments, the B cell antigen is a CD139 antigen. In some embodiments, the B cell antigen is a CD147 antigen. In some embodiments, the B cell antigen is a CD148 antigen. In some embodiments, the B cell antigen is a CD150 antigen. In some embodiments, the B cell antigen is a CD152 antigen. In some embodiments, the B cell antigen is a CD162 antigen. In some embodiments, the B cell antigen is a CD164 antigen. In some embodiments, the B cell antigen is a CD166 antigen. In some embodiments, the B cell antigen is a CD167a antigen. In some embodiments, the B cell antigen is a CD170 antigen. In some embodiments, the B cell antigen is a CD171 antigen. In some embodiments, the B cell antigen is a CD175 antigen. In some embodiments, the B cell antigen is a CD175s antigen. In some embodiments, the B cell antigen is a CD180 antigen. In some embodiments, the B cell antigen is a CD184 antigen. In some embodiments, the B cell antigen is a CD185 antigen. In some embodiments, the B cell antigen is a CD192 antigen. In some embodiments, the B cell antigen is a CD196 antigen. In some embodiments, the B cell antigen is a CD197 antigen. In some embodiments, the B cell antigen is a CD200 antigen. In some embodiments, the B cell antigen is a CD205 antigen. In some embodiments, the B cell antigen is a CD201a antigen. In some embodiments, the B cell antigen is a CDw210b antigen. In some embodiments, the B cell antigen is a CD212 antigen. In some embodiments, the B cell antigen is a CD213a1 antigen. In some embodiments, the B cell antigen is a CD213a2 antigen. In some embodiments, the B cell antigen is a CD215 antigen. In some embodiments, the B cell antigen is a CD217 antigen. In some embodiments,In some embodiments, the B cell antigen is a CD218a antigen. In some embodiments, the B cell antigen is a CD218b antigen. In some embodiments, the B cell antigen is a CD220 antigen. In some embodiments, the B cell antigen is a CD221 antigen. In some embodiments, the B cell antigen is CD22, 2 antigen. In some embodiments, the B cell antigen is a CD224 antigen. In some embodiments, the B cell antigen is a CD225 antigen. In some embodiments, the B cell antigen is a CD226 antigen. In some embodiments, the B cell antigen is a CD227 antigen. In some embodiments, the B cell antigen is a CD229 antigen. In some embodiments, the B cell antigen is a CD230 antigen. In some embodiments, the B cell antigen is a CD232 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD254 antigen. In some embodiments, the B cell antigen is a CD255 antigen. In some embodiments, the B cell antigen is a CD256 antigen. In some embodiments, the B cell antigen is a CD257 or CD258 antigen. In some embodiments, the B cell antigen is a CD259 antigen. In some embodiments, the B cell antigen is a CD260 antigen. In some embodiments, the B cell antigen is a CD261 antigen. In some embodiments, the B cell antigen is a CD262 antigen. In some embodiments, the B cell antigen is a CD263 antigen. In some embodiments, the B cell antigen is a CD264 antigen. In some embodiments, the B cell antigen is a CD267 antigen. In some embodiments, the B cell antigen is a CD268 antigen. In some embodiments, the B cell antigen is a CD269 antigen. In some embodiments, the B cell antigen is a CD270 antigen. In some embodiments, the B cell antigen is a CD272 antigen. In some embodiments, the B cell antigen is a CD274 antigen. In some embodiments, the B cell antigen is a CD275 antigen. In some embodiments, the B cell antigen is a CD277 antigen. In some embodiments, the B cell antigen is a CD279 antigen. In some embodiments, the B cell antigen is a CD283 antigen. In some embodiments, the B cell antigen is a CD289 antigen.In some embodiments, the B cell antigen is a CD290 antigen. In some embodiments, the B cell antigen is a CD295 antigen. In some embodiments, the B cell antigen is a CD298 antigen. In some embodiments, the B cell antigen is a CD300 antigen. In some embodiments, the B cell antigen is a CD300c antigen. In some embodiments, the B cell antigen is a CD305 antigen. In some embodiments, the B cell antigen is a CD306 antigen. In some embodiments, the B cell antigen is a CD307a antigen. In some embodiments, the B cell antigen is a CD307b antigen. In some embodiments, the B cell antigen is a CD307c antigen. In some embodiments, the B cell antigen is a CD307d antigen. In some embodiments, the B cell antigen is a CD307e antigen. In some embodiments, the B cell antigen is a CD314 antigen. In some embodiments, the B cell antigen is a CD215 antigen. In some embodiments, the B cell antigen is a CD316 antigen. In some embodiments, the B cell antigen is a CD317 antigen. In some embodiments, the B cell antigen is the CD319 antigen. In some embodiments, the B cell antigen is the CD321 antigen. In some embodiments, the B cell antigen is the CD327 antigen. In some embodiments, the B cell antigen is the CD328 antigen. In some embodiments, the B cell antigen is the CD329 antigen. In some embodiments, the B cell antigen is the CD338 antigen. In some embodiments, the B cell antigen is the CD351 antigen. In some embodiments, the B cell antigen is the CD352 antigen. In some embodiments, the B cell antigen is the CD353 antigen. In some embodiments, the B cell antigen is the CD354 antigen. In some embodiments, the B cell antigen is the CD355 antigen. In some embodiments, the B cell antigen is the CD356 antigen. In some embodiments, the B cell antigen is the CD357 antigen. In some embodiments, the B cell antigen is the CD358 antigen. In some embodiments, the B cell antigen is the CD360 antigen.In some embodiments, the B cell antigen is the CD361 antigen. In some embodiments, the B cell antigen is the CD362 antigen. In some embodiments, the B cell antigen is the CD363 antigen.

[0168] In one embodiment, the target of the CAR of the invention is a pathogen. In some embodiments, the target cell is a cell containing a pathogen.

[0169] In some embodiments, the pathogen causes an infectious disease selected from the group consisting of acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, campylobacteriosis, carbapenem-resistant infections, chancroid, chikungunya virus infection, chlamydia disease, fish botulism, difficile infection, clostridium perfringens disease, coccidioides fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jacob disease, Transmissible Spongiform Encephalopathy (TSE), Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue Fever Types 1, 2, 3, or 4, Diphtheria, Shiga Toxin-producing E. coli (STEC) infection, Eastern Equine Encephalitis, Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arbovirus or Parainfections, Non-Enterovirus, Enterovirus D68 Disease (EV-D68), Giardiasis, Glanders, Gonococcal Infection, Granulomatous Inguinalis, Haemophilus Influenza Type B Infection (Hib or H-Influenza), Hemophagocytic Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Shingles (Herpes Zoster), Histoplasmosis infection, Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Legionella infection (Legionnaires' disease), Leprosy (Hansen's disease), Leptospirosis, Listeriosis (Listeria), Lyme disease, Lymphogranuloma venereum (LGV) infection, Malaria, Measles, Melioidosis, Meningitis (viral), Meningococcal meningitis (meningitis (bacterial)), Middle East Respiratory Syndrome Coronavirus Disease (MERS-CoV), Parotitis,Norwalk virus disease, pediculosis, pelvic inflammatory disease (PID), whooping cough (spastic cough), plague (bubonic plague, septicemic plague, pneumonic plague), pneumococcal infection (pneumonia), acute polio (Polio), Powassan encephalitis, psittacosis, pubic lice infestation (Pthiriasis), exanthematous pustulosis (smallpox, monkeypox, cowpox), Q fever, rabies, rickettsial infections (Rocky Mountain spotted fever), rubella (German measles), Measles), Salmonella gastroenteritis (Salmonella), Scabies, Scombroid food poisoning (Scombroid), Pyemia, Severe Acute Respiratory Syndrome (SARS), Shigella gastroenteritis (Shigella), Smallpox, Methicillin-resistant Staphylococcus aureus (MRSA) infection, Staphylococcal food poisoning Enterotoxin B poisoning (Staphylococcal food poisoning), Vancomycin-intermediate-resistant Staphylococcus aureus infection (VISA), Vancomycin-resistant Staphylococcus aureus infection (VRSA), Group A Streptococcus infection (invasive) (Streptococcus aureus) In some embodiments, the infectious disease is acute flaccid myelitis (AFM), group B streptococcal infection (Strep-B), streptococcal toxic shock syndrome (STSS), syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomoniasis, trichinosis infection, tuberculosis (TB), latent tuberculosis infection (LTBI), tularemia, group D typhoid, vaginosis, Varicella (chickenpox), Vibrio cholerae (cholera), vibriosis (vibrio), Ebola hemorrhagic fever, Lassa virus hemorrhagic fever, Marburg hemorrhagic fever, West Nile virus, yellow fever, yersiniosis, and Zika virus infection. In some embodiments, the infectious disease is acute flaccid myelitis (AFM). In some embodiments, the infectious disease is anaplasmosis. In some embodiments, the infectious disease is anthrax. In some embodiments, the infectious disease is babesiosis. In some embodiments, the infectious disease is botulism. In some embodiments, the infectious disease is brucellosis. In some embodiments, the infectious disease is a campylobacter infection. In some embodiments, the infectious disease is a carbapenem-resistant infection. In some embodiments, the infectious disease isIn some embodiments, the infectious disease is chancroid. In some embodiments, the infectious disease is chikungunya virus infection. In some embodiments, the infectious disease is chlamydia disease. In some embodiments, the infectious disease is fish botulism. In some embodiments, the infectious disease is a B. difficile infection. In some embodiments, the infectious disease is Clostridium perfringens disease. In some embodiments, the infectious disease is a Coccidioides fungal infection. In some embodiments, the infectious disease is a coronavirus disease. In some embodiments, the infectious disease is Covid-19 (SARS-CoV-2). In some embodiments, the infectious disease is Creutzfeldt-Jakob disease / transmissible spongiform encephalopathy. In some embodiments, the infectious disease is cryptosporidiosis. In some embodiments, the infectious disease is cyclosporiasis. In some embodiments, the infectious disease is dengue fever type 1, 2, 3, or 4. In some embodiments, the infectious disease is diphtheria. In some embodiments, the infectious disease is a Shiga toxin-producing Escherichia coli (STEC) infection. In some embodiments, the infectious disease is Eastern equine encephalitis. In some embodiments, the infectious disease is hemorrhagic fever (Ebola). In some embodiments, the infectious disease is ehrlichiosis. In some embodiments, the infectious disease is encephalitis. In some embodiments, the infectious disease is an arbovirus or parainfectious disease. In some embodiments, the infectious disease is a non-enterovirus. In some embodiments, the infectious disease is enterovirus D68 disease (EV-D68). In some embodiments, the infectious disease is giardiasis. In some embodiments, the infectious disease is glanders. In some embodiments, the infectious disease is gonorrhea. In some embodiments, the infectious disease is granuloma groin. In some embodiments, the infectious disease is Haemophilus influenzae type B infection (Hib or H-influenza). In some embodiments, the infectious disease isIn some embodiments, the infectious disease is hemophagocytic syndrome (HPS). In some embodiments, the infectious disease is hemolytic uremic syndrome (HUS). In some embodiments, the infectious disease is hepatitis A (Hep A). ​​In some embodiments, the infectious disease is hepatitis B (Hep B). In some embodiments, the infectious disease is hepatitis C (Hep C). In some embodiments, the infectious disease is hepatitis D (Hep D). In some embodiments, the infectious disease is hepatitis E (Hep E). In some embodiments, the infectious disease is herpes. In some embodiments, the infectious disease is shingles (herpes zoster). In some embodiments, the infectious disease is histoplasmosis infection. In some embodiments, the infectious disease is human immunodeficiency virus / AIDS (HIV / AIDS). In some embodiments, the infectious disease is human papillomavirus (HPV). In some embodiments, the infectious disease is influenza (Flu). In some embodiments, the infectious disease is Legionella infection (Legionnaires' disease). In some embodiments, the infectious disease is leprosy (Hansen's disease). In some embodiments, the infectious disease is leptospirosis. In some embodiments, the infectious disease is listeriosis (Listeria). In some embodiments, the infectious disease is Lyme disease. In some embodiments, the infectious disease is lymphogranuloma venereum (LGV) infection. In some embodiments, the infectious disease is malaria. In some embodiments, the infectious disease is measles. In some embodiments, the infectious disease is melioidosis. In some embodiments, the infectious disease is meningitis (viral). In some embodiments, the infectious disease is meningococcal meningitis (meningitis (bacterial)). In some embodiments, the infectious disease is Middle East Respiratory Syndrome Coronavirus disease (MERS-CoV). In some embodiments, the infectious disease is parotitis. In some embodiments, the infectious disease is Norwalk virus disease. In some embodiments, the infectious disease isIn some embodiments, the infectious disease is pediculosis. In some embodiments, the infectious disease is pelvic inflammatory disease (PID). In some embodiments, the infectious disease is whooping cough (spastic cough). In some embodiments, the infectious disease is plague (bubonic plague, in some embodiments, the infectious disease is septicemic plague, in some embodiments, the infectious disease is pneumonic plague). In some embodiments, the infectious disease is pneumococcal infection (pneumonia). In some embodiments, the infectious disease is polio (polio). In some embodiments, the infectious disease is Powassan encephalitis. In some embodiments, the infectious disease is psittacosis. In some embodiments, the infectious disease is pubic lice infestation. In some embodiments, the infectious disease is exanthematous pustulosis (smallpox, in some embodiments, the infectious disease is monkeypox, in some embodiments, the infectious disease is cowpox). In some embodiments, the infectious disease is Q fever. In some embodiments, the infectious disease is rabies. In some embodiments, the infectious disease is a rickettsial infection (Rocky Mountain spotted fever). In some embodiments, the infectious disease is rubella (three-day measles). In some embodiments, the infectious disease is Salmonella gastroenteritis (Salmonella). In some embodiments, the infectious disease is scabies. In some embodiments, the infectious disease is scombroid food poisoning. In some embodiments, the infectious disease is pyemia. In some embodiments, the infectious disease is severe acute respiratory syndrome (SARS). In some embodiments, the infectious disease is Shigella gastroenteritis (Shigella). In some embodiments, the infectious disease is smallpox. In some embodiments, the infectious disease is methicillin-resistant Staphylococcus aureus (MRSA) infection. In some embodiments, the infectious disease is staphylococcal food poisoning enterotoxin B poisoning (staphylococcal food poisoning). In some embodiments, the infectious disease is vancomycin-intermediate Staphylococcus aureus infection (VISA).In some embodiments, the infectious disease is a vancomycin-resistant Staphylococcus aureus infection (VRSA). In some embodiments, the infectious disease is a Group A Streptococcus infection (invasive) (Strep A (invasive)). In some embodiments, the infectious disease is a streptococcal disease. In some embodiments, the infectious disease is a Group B disease (Strep-B). In some embodiments, the infectious disease is streptococcal toxic shock syndrome (STSS). In some embodiments, the infectious disease is syphilis (primary; in some embodiments, the infectious disease is secondary; in some embodiments, the infectious disease is early latent; in some embodiments, the infectious disease is late latent; in some embodiments, the infectious disease is congenital). In some embodiments, the infectious disease is tetanus infection. In some embodiments, the infectious disease is trichomoniasis. In some embodiments, the infectious disease is trichinellosis infection. In some embodiments, the infectious disease is tuberculosis (TB). In some embodiments, the infectious disease is latent tuberculosis infection (LTBI). In some embodiments, the infectious disease is tularemia. In some embodiments, the infectious disease is group D typhoid. In some embodiments, the infectious disease is vaginosis. In some embodiments, the infectious disease is chickenpox (varicella), Vibrio cholerae infection (cholera), or Vibrio (Vibrio). In some embodiments, the infectious disease is Ebola hemorrhagic fever. In some embodiments, the infectious disease is Lassa virus hemorrhagic fever. In some embodiments, the infectious disease is Marburg hemorrhagic fever. In some embodiments, the infectious disease is West Nile virus. In some embodiments, the infectious disease is yellow fever. In some embodiments, the infectious disease is yersiniosis. In some embodiments, the infectious disease is Zika virus infection.

[0170] In some embodiments, the pathogen is a bacterium, hi some embodiments, the bacterium is a bacillus, bartonella, bordetella, borrelia, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, francisella, haemophilus, helicobacter, or the like. In some embodiments, the bacterium is a bacterium of the genus Bacillus, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia. In some embodiments, the bacterium is a bacterium of the genus Bacillus. In some embodiments, the bacterium is a bacterium of the genus Bartonella. In some embodiments, the bacterium is a bacterium of the genus Bordetella. In some embodiments, the bacterium is a bacterium of the genus Borrelia. In some embodiments, the bacterium is a bacterium of the genus Brucella. In some embodiments, the bacterium is a bacterium of the genus Campylobacter. In some embodiments, the bacterium is a bacterium of the genus Chlamydia. In some embodiments, the bacterium is a bacterium of the genus Chlamydiae. In some embodiments, the bacterium is a bacterium of the genus Clostridium. In some embodiments, the bacterium is a bacterium of the genus Corynebacterium.In some embodiments, the bacterium is a bacterium of the genus Enterococcus. In some embodiments, the bacterium is a bacterium of the genus Escherichia. In some embodiments, the bacterium is a bacterium of the genus Francisella. In some embodiments, the bacterium is a bacterium of the genus Haemophilus. In some embodiments, the bacterium is a bacterium of the genus Helicobacter. In some embodiments, the bacterium is a bacterium of the genus Legionella. In some embodiments, the bacterium is a bacterium of the genus Leptospira. In some embodiments, the bacterium is a bacterium of the genus Listeria. In some embodiments, the bacterium is a bacterium of the genus Mycobacterium. In some embodiments, the bacterium is a bacterium of the genus Mycoplasma. In some embodiments, the bacterium is a bacterium of the genus Neisseria. In some embodiments, the bacterium is a bacterium of the genus Pseudomonas. In some embodiments, the bacterium is a bacterium of the genus Rickettsia. In some embodiments, the bacterium is a bacterium of the genus Salmonella. In some embodiments, the bacterium is a bacterium of the genus Shigella. In some embodiments, the bacterium is a bacterium of the genus Staphylococcus. In some embodiments, the bacterium is a bacterium of the genus Streptococcus. In some embodiments, the bacterium is a bacterium of the genus Treponema. In some embodiments, the bacterium is a bacterium of the genus Ureaplasma. In some embodiments, the bacterium is a bacterium of the genus Vibrio. In some embodiments, the bacterium is a bacterium of the genus Yersinia.

[0171] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In some embodiments, the protozoan is an Entamoeba, Giardia, Leishmania, Balantidium, Plasmodium, or Cryptosporidium. In some embodiments, the helminth is a trematode, a tapeworm, an accipitridae, or an ascarid. In some embodiments, the ectoparasite is an arthropod.

[0172] In some embodiments, the pathogen is a virus. In some embodiments, the virus is a virus selected from the family Adenoviridae, Arenaviridae, Astroviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Orthomyxoviridae, or any of the family Orthomyxoviridae. In some embodiments, the virus is a virus of the Adenoviridae family. In some embodiments, the virus is a virus of the Arenaviridae family. In some embodiments, the virus is a virus of the Astroviridae family. In some embodiments, the virus is a virus of the Bunyaviridae family. In some embodiments, the virus is a virus of the Caliciviridae family. In some embodiments, the virus is a virus of the Coronaviridae family. In some embodiments, the virus is a virus of the Filoviridae family. In some embodiments, the virus is a Flaviviridae virus. In some embodiments, the virus is a Hepadnaviridae virus. In some embodiments, the virus is a Hepeviridae virus. In some embodiments, the virus is an Orthomyxoviridae virus.In some embodiments, the virus is a Papillomaviridae virus. In some embodiments, the virus is a Paramyxoviridae virus. In some embodiments, the virus is a Parvoviridae virus. In some embodiments, the virus is a Picornaviridae virus. In some embodiments, the virus is a Polyomaviridae virus. In some embodiments, the virus is a Poxviridae virus. In some embodiments, the virus is a Reoviridae virus. In some embodiments, the virus is a Retroviridae virus. In some embodiments, the virus is a Rhabdoviridae virus. In some embodiments, the virus is a Togaviridae virus.

[0173] In some embodiments, the virus is an adenovirus, a coronavirus, a coxsackievirus, an Epstein-Barr virus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus type 2, a cytomegalovirus, a human herpesvirus type 8, a human immunodeficiency virus, an influenza virus, a measles virus, a parotid virus, a human papillomavirus, a parainfluenza virus, a poliovirus, a rabies virus, a respiratory syncytial virus, a rubella virus, or a varicella-zoster virus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is Covid-19 (SARS-CoV-2). In some embodiments, the virus is a coxsackievirus. In some embodiments, the virus is an Epstein-Barr virus. In some embodiments, the virus is a hepatitis A virus. In some embodiments, the virus is a hepatitis B virus. In some embodiments, the virus is a hepatitis C virus. In some embodiments, the virus is herpes simplex virus type 2. In some embodiments, the virus is cytomegalovirus. In some embodiments, the virus is human herpesvirus type 8. In some embodiments, the virus is human immunodeficiency virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is measles virus. In some embodiments, the virus is parotid virus. In some embodiments, the virus is human papillomavirus. In some embodiments, the virus is parainfluenza virus. In some embodiments, the virus is poliovirus. In some embodiments, the virus is rabies virus. In some embodiments, the virus is respiratory syncytial virus. In some embodiments, the virus is rubella virus. In some embodiments, the virus is varicella-zoster virus.

[0174] Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain may be derived from a natural or synthetic source. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane (preferably a eukaryotic cell membrane). A transmembrane domain suitable for the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it may be a synthetic, non-natural protein segment, such as a hydrophobin segment, that is thermodynamically stable in a cell membrane.

[0175] Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain can form an α-helix, a complex of two or more α-helices, a β-barrel, or any other stable structure that can span a cellular phospholipid bilayer. Transmembrane domains can also or alternatively be classified based on their topological structure, including the number of times the transmembrane domain spans the membrane and the orientation of the protein. For example, single-pass membrane proteins span the cellular membrane once, while multi-pass membrane proteins span the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their ends relative to the inside and outside of the cell and the topological structure of one or more transmembrane segments. Type I membrane proteins have a single transmembrane region and are oriented so that the N-terminus of the protein is located on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is located on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented so 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 classified based on the number of transmembrane segments and the location of the N- and C-termini.

[0176] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may be used to adapt a CAR described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helical or beta sheet structures. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer, while the C-terminus of the protein is on the extracellular side.

[0177] The transmembrane domain used in the CARs described herein may further 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 about 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, e.g., U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0178] A transmembrane domain according to the present disclosure may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, favors orientation of 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 comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0179] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR according to the present specification comprises a hydrophobic artificial sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminal position of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily 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 polyleucine-alanine sequence. The hydrophilic or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0180] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0181] In some specific embodiments, the transmembrane domain is derived from CD8α, hi other specific embodiments, the transmembrane domain is derived from CD28α.

[0182] Intracellular signaling domains The intracellular signaling domain in a CAR according to the present specification is responsible for activating at least one normal effector function of an immune effector cell expressing the CAR. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or accessory activity (including cytokine secretion). Thus, the term "cytoplasmic signaling domain" refers to the portion of a protein that transduces an effector function signal and instructs the cell to perform a specific function. Typically, the entire cytoplasmic signaling domain can be used, but in many cases it is not necessary to use the entire chain. Regarding the use of truncated portions of the cytoplasmic signaling domain, such truncated portions may be used instead of the intact chain, so long as they transduce the effector function signal. Thus, the term cytoplasmic signaling domain is intended to refer to any truncated portion that includes a sufficient cytoplasmic signaling domain to transduce an effector function signal.

[0183] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, a 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 stimulates and induces immune effectors to function. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" is a conserved protein motif typically found in the tails of signaling molecules expressed in 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 mediated, at least in part, by phosphorylation of tyrosine residues in ITAMs following activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G), FcR beta (Fc ε Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0184] Costimulatory signaling domain In some embodiments, the CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response (e.g., effector function). In addition to stimulating antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival and to activate cellular effector functions.

[0185] The costimulatory signaling domain of the chimeric receptors described herein can be a cytoplasmic signaling domain from a costimulatory protein, which transduce signals and regulate responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "costimulatory signaling domain" can also be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding partner in an immune cell (e.g., a T cell) that mediates a costimulatory response of the immune cell, including, but not limited to, proliferation and survival, by specifically binding to a costimulatory ligand.

[0186] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any one 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 domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins (e.g., any two or more costimulatory signaling domains described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) are fused to each other via any peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0187] 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 is applicable to the CARs described herein. The type or types of costimulatory signaling domains are selected based on factors such as, for example, the type of immune effector cell (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) on which the effector molecule will be expressed and the desired immune effector function (e.g., ADCC effector). Examples of costimulatory signaling domains used in CARs can be the cytoplasmic signaling domains of costimulatory proteins, including 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) and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF, BLyS ... 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 TNFRII / TNFRSF1B), SLAM family members (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 any other costimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of ligands that specifically bind to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0188] In some embodiments, the costimulatory signaling domain is a variant of any costimulatory signaling domain described herein, such that the costimulatory signaling domain can modulate an 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 variants compared to a wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid variants may be referred to as variants. Compared to a costimulatory signaling domain that does not contain the mutation, mutations in amino acid residues in the costimulatory signaling domain can result in increased signal transduction and enhanced stimulation of the immune response. Compared to a costimulatory signaling domain that does not contain the mutation, mutations in amino acid residues in the costimulatory signaling domain can result in decreased signal transduction and decreased stimulation of the immune response.

[0189] signal peptide In some embodiments, a CAR according to the present disclosure may comprise a signal peptide (also referred to as a signal sequence) at the N-terminal position of the polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the cell's secretory pathway and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides applicable to the CARs described herein, including signal sequences of naturally occurring proteins or synthetic, non-natural signal sequences, will be apparent to those skilled 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.

[0190] Hinge Area In some embodiments, a CAR according to the present disclosure may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is typically expressed between two domains of a protein and can allow one or two flexible domains of a protein to move 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 can be used.

[0191] The hinge domain of an antibody (e.g., an IgG, IgA, IgM, IgE, or IgD antibody) is also applicable to the pH-dependent chimeric receptor system described herein. In some embodiments, the hinge domain is the hinge domain connecting the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is the hinge domain of an antibody and comprises the hinge domain of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0192] Non-naturally occurring peptides may be used as the hinge domain of 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 are independently integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0193] The hinge domain may comprise any one of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the length of the hinge domain may be at least any one of 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.

[0194] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Any hinge domain of a protein known in the art that contains a hinge domain is applicable to the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and provides flexibility to the chimeric receptor.

[0195] In some specific embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., comprises a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In other embodiments, the hinge domain is derived from CD28α. In some embodiments, the hinge domain is a portion of the hinge domain of CD28α, e.g., comprises a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28α.

[0196] Exemplary CAR Notable drugs include Kymria(drug)(tisagenlecel), Yescart(drug)(axicabtagene ciloleucel), ALEXIS AIDT-2 EOC(Kiromic Biopharma Inc.) CIK-CAR.PSMA(Formula Pharmaceuticals Inc.) ADI-002(Adicet Bio Inc.) TSC-200(TScan Therapeutics Inc.) TSC-100(TScan Therapeutics Inc.) RB-H21(Refuge Biotechnologies). Inc., ADP-A2AFP (Adapimmune Therapeunics plc), CT-0729 (Charisma Therapeutics), CT-1119 (Charisma Therapeutics), CCT-301-59 (EXUMA Biotech Corp.), BOXR-889 (Unum Therapeutics). Inc., meso-CAR-T+PD-78 (MirImmune LLC), MAGE-A10C796T (Adaptimune Therapeunics plc), NKG2D-DARIC T antibody (Bluebird Bio Inc.), AGENT-NY-ESO-1 X (AgenTus Therapeutics Inc.), MB-105 (City of Hope Medical Center, P-MUC1C-101 (Poseida Therapeutics Inc.), TT-16 (Baylor College of Medicine), ACTR-087 (Unum Therapeutics Inc.), EGFR-806 (Seattle Children's Hospital), TAC01-ROR1 (Triumvira Immunologics). Inc., CCT-301-38 (EXUMA Biotech Corp.), MB-103 (Mustang Bio Inc.), TAB-28z (OncoTab Inc.), ET-1402 (Eureka Therapeutics Inc.), ITI-1000 (Duke University), CIDeCAR (Bellicum Pharmaceuticals).Inc), MT-201 (Myeloid Therapeunics Inc), CT-0508 (Carisma Therapeustics), ADP-A2M4 (Adapeimune Therapeutics plc), AMG-119 (Amgen Inc), iCasp9M28z T cells (Memorial Sloan-Kettering Cancer Center), P-PSMA-101 (Poseida Therapeutics Inc), ACE-1702 (Acepodia Inc), AIC-100 (Cornell University), PRGN-3005 (Precigen Inc), UniCAR-T-PSMA (GEMoaB Monoclonals GmbH), IMA-204 (MD Anderson Cancer Center), DSG3-CAART (University of Pennsylvania), LXF-821 (University of Pennsylvania), MOv19-BBz CAR T cells (University of Pennsylvania), Tn MUC-1 CAR-T (University of Pennsylvania), huMesoCART (University of Pennsylvania), IMA-203 (Immatics NV), IMA-202 (Immatics NV), XYP-317 (Xyphos Inc), CYAD-101 (Celyad Oncology), huMNC2-CAR44 T cells (Minerva Biotechnologies Corp), HER2Bi-armed ATC (Roger Williams Medical Center) and CYAD-200 series (Celdara Medical LLC), Lisocabtagene Maralequel (liso-cel), FT596 (Fate Therapeutics), KTE-C19 (Kite Pharma Inc), KTE-X19 (Kite Pharma Inc), KITE-718 (Kite Pharma Inc), KITE-439 (Kite Pharma Inc), JCAR-024 (Fred Hutchinson Cancer ResearchCenter), JCAR-023 (Juno Therapeutics Inc), BPX-201 (Bellicum Pharmaceuticals Inc), BPX-601 (Bellicus Pharmacueticals Inc), BPX-603 (Bellicom Pharmaceuticals Inc), MCY-M11 (MaxCyte Inc), KUR-503 (Baylor College of Medicine), ICS-200 (University of Alabama at Birmingham), ADP-A2M4CD8 (Adapeimune Therapeutics plc), and GLYCAR (Baylor College of Medicine).

[0197] In some embodiments, a CAR according to the present specification comprises an amino acid sequence having a certain percent identity to any one of the exemplary CARs described above and infra in Section 8. In some embodiments, the present specification provides a CAR comprising or consisting of an extracellular domain, which has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a CAR described above and infra in Section 8.

[0198] In some embodiments, modifications of one or more amino acid sequences of the CARs described herein are contemplated. For example, optimization of the binding affinity and / or other biological properties of the extracellular domain may be required, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to the extracellular domains described herein, it is also contemplated to prepare variants of the domains described herein. For example, scFv variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. It will be understood by those skilled in the art that amino acid changes may alter post-translational processing of the antibody.

[0199] The alterations may be a substitution, deletion, or insertion of one or more codons that encode the polypeptide, resulting in a change in the amino acid sequence relative to the original antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0200] Amino acid substitutions may be the result of substituting an amino acid with another amino acid having similar structural and / or chemical properties, such as substituting serine for leucine, and may be, for example, conservative amino acid substitutions. Standard techniques well known to those of skill in the art may be used to introduce mutations into nucleotide sequences encoding the molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Tolerable changes can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity of the parent polypeptide.

[0201] The present disclosure includes polypeptides produced by conservative amino acid substitutions. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) may be made to retain or not significantly alter properties.

[0202] One type of substitutional variant involves substituting one or more hypervariable region residues or fragments thereof of a parent antibody (e.g., a humanized or human antibody) in the extracellular antigen-binding domain of a CAR of the invention. Typically, the resulting mutations selected for further study have an alteration (e.g., improvement) in some biological property (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or substantially retain some biological property of the parent antibody. Exemplary substitutional variants are affinity-matured antibodies, which can be readily produced using, for example, phage-display-based affinity maturation techniques, as described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0203] Alterations (e.g., substitutions) can be made in CDRs to, for example, improve antibody affinity. Such alterations may be made in CDR "hot spots," i.e., residues encoded by codons frequently mutated during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), where the resulting mutant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (ed. by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis), and a secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR orientation, in which a number of CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

[0204] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions according to the present disclosure) may be made in a CDR that do not significantly reduce binding affinity. In some embodiments, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0205] As described in Cunningham and Wells, Science, 244:1081-1085 (1989), a useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis." Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or removed as substitution candidates. The mutants can be screened to determine whether they contain the desired properties.

[0206] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antigen-binding domain antibody.

[0207] Changes may be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To produce antibody variant DNA, cloned DNA may be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques.

[0208] The present disclosure further includes immune effector cells that comprise two or more functional exogenous receptors, such as dual CARs that bind to two different targets.

[0209] 7.2.3. Immune effector cells "Immune effector cell" refers to an immune cell capable of exerting immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and exerts ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0210] In some embodiments, the immune effector cells are T cells. In some embodiments, these T cells are CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cells produce IL-2, TFN, and / or TNF after expressing a CAR and binding to a target cell. In some embodiments, the CD8+ T cells lyse antigen-specific target cells after expressing a CAR and binding to a target cell.

[0211] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells may be an established cell line, such as NK-92 cells.

[0212] In some embodiments, immune effector cells are differentiated from stem cells (eg, hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells).

[0213] The engineered immune effector cells are prepared by introducing a polypeptide according to the present disclosure into immune effector cells, such as T cells. In some embodiments, the polypeptide is introduced into the immune effector cells by transfecting them with any one of the isolated nucleic acids or any one of the vectors described above.

[0214] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0215] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, vectors are introduced into cells by electroporation.

[0216] Biological methods for introducing vectors into immune effector 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.

[0217] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0218] In some embodiments, RNA molecules encoding any of the polypeptides described herein can be prepared by conventional methods (e.g., in vitro transcription) and introduced into immune effector cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0219] In some embodiments, the transduced or transfected immune effector cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured and expanded for at least about 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 immune effector cells are further evaluated or screened to select engineered mammalian cells.

[0220] Reporter genes can be used to identify transfectable cells and evaluate the function of regulatory sequences. Typically, reporter genes are genes that are absent or not expressed in the recipient organism or tissue, and encode a polypeptide whose expression is indicated by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is detected at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (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.

[0221] Other methods for confirming the presence of nucleic acids encoding polypeptides in engineered immune effector cells include molecular biological assays well known to those skilled in the art, such as DNA and RNA blotting, RT-PCR and PCR, and biochemical assays that detect the presence or absence of specific peptides, for example, by immunological methods (e.g., ELISA and Western blot).

[0222] 7.2.4. Source of T cells In some embodiments, a source of T cells is obtained from a subject prior to expansion and genetic modification. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, multiple T cell lines available in the art can be used. In some embodiments, T cells can be obtained from blood collected from a subject using multiple techniques well known to those skilled in the art (e.g., Ficoll™ isolation). In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis are washed to remove the plasma portion, and the cells may be used for subsequent processing steps in an appropriate buffer or medium. 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 (if not all) divalent cations. An initial activation step in the absence of calcium can lead to expanded activation. As one skilled in the art will readily appreciate, the wash step can be completed by methods known in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, for example, a centrifuge containing Ca may be used. 2+ None, Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as PBS without buffer, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0223] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, 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., 3x28) conjugated beads (e.g., 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 approximately 30 minutes. In other embodiments, the period is 30 minutes to 36 hours or more, and all integer values ​​therebetween. In other embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For the isolation of T cells from leukemia patients, a longer incubation time (e.g., 24 hours) can increase cell yield. Longer incubation times can be used to isolate T cells in any case where T cells are scarce compared to other cell types, such as tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Longer incubation times can also improve the efficiency of CD8+ T cell capture. Thus, in some embodiments, subpopulations of T cells can be preferentially selected for or deselected at the initiation of culture or at other time points during culture by simply shortening or lengthening the time allowed for binding of T cells to CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio. Additionally, subpopulations of T cells can be preferentially selected for or deselected at the initiation of culture or at other desired time points by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface. Those skilled 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 for the activation and expansion process. The "unselected" cells can also be subjected to further rounds of selection.

[0224] Enrichment of a T cell population by negative selection can be achieved by binding with antibodies against surface markers unique to the negatively selected cells. One method involves cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0225] The concentration of cells and surfaces (e.g., particles (e.g., beads)) may be varied to isolate the desired cell population by positive or negative selection. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase cell expansion) to ensure maximum cell-to-bead contact. 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 another embodiment, greater than 100 million cells / mL is used. In another 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 of 75, 80, 85, 90, 95, or 100 million cells / mL is used. In other embodiments, a concentration of 1.25 or 1.50 billion cells / mL may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high concentrations of cells can allow for more effective capture of cells that may weakly express a target antigen of interest (e.g., 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 are desirable to obtain. In some embodiments, the use of high concentrations of cells allows for more effective selection of CD8+ T cells, which normally have weak CD28 expression.

[0226] In some embodiments, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of 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 for binding to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more effectively than dilute concentrations of CD8+ T cells. In some embodiments, the concentration of cells used is 5×10 6 In some embodiments, the concentration used is about 1 x 105 pieces / mL~1×10 6 The value can be any integer value between 0.01 and 0.15, and can be any integer value between 0.01 and 0.15.

[0227] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at 2°C to 10°C or at room temperature.

[0228] T cells for stimulation may be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing steps may provide a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in such cases, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 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 freezing the cells to -80°C at a rate of 1°C / min and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods and uncontrolled immediate freezing at -20°C or liquid nitrogen can be used.

[0229] In some embodiments, cryopreserved cells are thawed, washed, and allowed to stand at room temperature for one hour before activation, as described herein.

[0230] The present disclosure also contemplates collecting a blood sample or apheresis product from a subject at a time prior to the potential need for the expanded cells described herein. Thus, a cell source for the expanded subject can be collected at any desired time, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy, such as for multiple diseases or conditions that would benefit from T cell therapy, as described herein. In one embodiment, the blood sample or apheresis blood component is taken from a normal, healthy subject. In some embodiments, the blood sample or apheresis blood component is taken from a normal, healthy subject who is at risk for developing a disease but does not have the disease, and desired cells are isolated and frozen for later use. In some embodiments, the T cells can be expanded, frozen, and used at a later time. In some embodiments, the sample is taken from the patient shortly after diagnosis of a particular disease, as described herein, but prior to any treatment. In another embodiment, cells are isolated from a subject's blood sample or apheresis blood component prior to multiple associated therapeutic modalities, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228), and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (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 another embodiment, the patient's cells are isolated and frozen for later use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplant, T cell ablative therapy using chemotherapeutic agents (e.g., fludarabine), external beam radiation therapy (XRT), cytoxan, or antibodies (e.g., OKT3 or CAMPATH). In another embodiment, the cells may be isolated before B cell ablative therapy (e.g., an agent reactive with CD20, such as Rituxan) and frozen for use in subsequent treatment.

[0231] In some embodiments, T cells are obtained during treatment or directly from a patient. In this regard, it has been observed that after some cancer treatments, particularly treatment with drugs that damage the immune system, the quality of T cells obtained may be optimal or the ex vivo expansion capacity of the T cells may be improved immediately after treatment, during the period when the patient would normally be recovering from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, in the context of the present disclosure, it is contemplated to collect blood cells during this recovery period, including T cells, dendritic cells, or other hematopoietic cells. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in a subject that are favorable for the repopulation, recirculation, regeneration, and / or expansion of cell types, particularly during a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0232] 7.2.5. T Cell Activation and Expansion In some embodiments, before or after genetic modification of the T cells with a CAR described herein, the T cells are typically engineered to express a CAR-containing T cell vector, such as those described in, e.g., U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,1 44,575, 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.

[0233] Typically, T cells can be expanded by contact with a surface bearing an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, as described herein, T cell populations can be stimulated by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) bound to a calcium ion vector. A ligand that binds to a helper molecule can be used to costimulate helper molecules on the T cell surface. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell expansion. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the expansion of CD4+ T cells or CD8+ T cells. Examples of anti-CD3 antibodies include UCHT1, OKT3, and HIT3a (BioLegend, San Diego, US), and may be used as well as other methods known in the art (Graves J et al., J. Immunol. 146:2102 (1991); Li B et al., Immunology 116:487 (2005); Rivollier A et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), as well as other methods 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)).

[0234] In some embodiments, the primary and costimulatory signals for T cells can be provided by various regimens. For example, the agents providing each signal can be in solution or coupled to a surface. If coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to a separate surface (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and another agent 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 some embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which are contemplated for use in activating and expanding T cells in some embodiments of the present disclosure.

[0235] In some embodiments, T cells are combined with agent-coated beads, followed by separation of the beads and cells and culturing the cells. In another embodiment, the agent-coated beads and cells are not separated but are cultured together prior to culturing. In another embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, to increase ligation of cell surface markers, thereby inducing cell stimulation.

[0236] For example, cell surface proteins can be linked by allowing T cells to contact paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 ~4×10 8T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles with a recommended titer of 1:100) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). One of skill in the art will readily appreciate that any cell concentration can be used. For example, the target cells may be present in very small amounts in the sample, comprising as little as 0.01% of the sample, or the entire sample (i.e., 100%) may contain the desired target cells. Thus, any cell number is within the scope of the present disclosure. In some embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase cell enrichment) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In another 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 of 75, 80, 85, 90, 95, or 100 million cells / mL is used. In other embodiments, a concentration of 1.25 or 1.50 billion cells / mL may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of higher cell concentrations may allow for more effective capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are desirable in some embodiments. For example, the use of higher cell concentrations allows for more effective selection of CD8+ T cells, which normally have weak CD28 expression.

[0237] In some embodiments, the mixture can be cultured for any integer time between a few hours (about 3 hours) and about 14 days, or both. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, 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 stimulation cycles may be necessary, as T cell culture times can be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors essential for expansion and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, 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, serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, optimizer, and / or cytokines in amounts sufficient to grow and expand T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and are not included in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to different stimulation times may exhibit various characteristics. For example, a typical peripheral blood mononuclear cell product of blood or apheresis blood components has a higher helper T cell population (TH, CD4+) than cytotoxic or suppressor T cell populations (TC, CD8).Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population that is primarily composed of TH cells before about day 8-9, and after about day 8-9, the T cell population includes an increasingly abundant TC cell population. Thus, for therapeutic purposes, it may be advantageous to infuse a T cell population that is primarily composed of TH cells into a subject. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand that subset to a greater extent.

[0238] It should be noted that in addition to CD4 and CD8 markers, other phenotypic markers also vary significantly but are highly reproducible during cell expansion, allowing for the customization of activated T cell products for specific purposes.

[0239] 7.3. Polypeptides Comprising a Functional Exogenous Receptor and One or More Additional Domains In another aspect, the present specification provides a polypeptide, wherein the polypeptide comprises at least one functional exogenous receptor (e.g., CAR, TCR, and TAC), at least one of the p40 subunit of IL-12, and a ligand of CCR7 (e.g., CCL-19 and CCL-21).

[0240] In some embodiments, the present specification provides a polypeptide, wherein the polypeptide comprises at least one of a functional exogenous receptor (e.g., a CAR, a TCR, and a TAC), the p40 subunit of IL-12, and CCL-19.

[0241] In some embodiments, the description provides a polypeptide comprising CAR and p40. In some embodiments, the description provides a polypeptide comprising CAR and CCL-19. In other embodiments, the description provides a polypeptide comprising CAR, p40, and CCL-19. The CAR in the polypeptides of the present invention may be any one of those described in Section 7.2 above. The p40 and CCL-19 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0242] In some embodiments, the present specification provides a polypeptide comprising a TCR and p40. In some embodiments, the present specification provides a polypeptide comprising a TCR and CCL-19. In other embodiments, the present specification provides a polypeptide comprising a TCR, p40, and CCL-19. The p40 and CCL-19 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0243] In some embodiments, the present specification provides a polypeptide comprising TAC and p40. In some embodiments, the present specification provides a polypeptide comprising TAC and CCL-19. In other embodiments, the present specification provides a polypeptide comprising TAC, p40, and CCL-19. The p40 and CCL-19 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0244] In some embodiments, the p40 in accordance with the present disclosure is human p40 or a fragment or variant thereof. In specific embodiments, the p40 in accordance with the present disclosure comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the p40 in accordance with the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the p40 polypeptide in accordance with the present disclosure is a secreted polypeptide. In some embodiments, the p40 polypeptide in accordance with the present disclosure is a membrane-bound polypeptide (e.g., MB12).

[0245] In some embodiments, the CCL-19 provided herein is human CCL-19 or a fragment or variant thereof. In specific embodiments, the CCL-19 provided herein comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the CCL-19 provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0246] In the polypeptides of the invention, CAR, p40, and / or CCL-19 can be arranged in any order. For example, when CAR, p40, and CCL-19 are all present in a polypeptide, in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CAR, p40, and CCL-19; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CAR, CCL-19, and p40; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CCL-19, p40, and CAR; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, p40, CCL-19, and CAR; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, p40, CAR, and CCL-19; and in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CCL-19, CAR, and p40.

[0247] In some embodiments of the various polypeptides herein, CAR, p40, and / or CCL-19 are linked to one another via a peptide linker. In some embodiments, the peptide linker is a self-cleaving peptide, such as the 2A self-cleaving peptide, such that CAR, p40, and / or CCL-19 become a single polypeptide after cleavage within the cell.

[0248] In other embodiments, the present specification provides a polypeptide, wherein the polypeptide comprises at least one of a functional exogenous receptor (e.g., a CAR, a TCR, and a TAC), the p40 subunit of IL-12, and / or CCL-21.

[0249] In some embodiments, the description provides a polypeptide comprising CAR and p40. In some embodiments, the description provides a polypeptide comprising CAR and CCL-21. In other embodiments, the description provides a polypeptide comprising CAR, p40, and CCL-21. The CAR in the polypeptides of the present invention may be any one of those described in Section 7.2 above. The p40 and CCL-21 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0250] In some embodiments, the present specification provides a polypeptide comprising a TCR and p40. In some embodiments, the present specification provides a polypeptide comprising a TCR and CCL-21. In other embodiments, the present specification provides a polypeptide comprising a TCR, p40, and CCL-21. The p40 and CCL-21 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0251] In some embodiments, the present specification provides a polypeptide comprising TAC and p40. In some embodiments, the present specification provides a polypeptide comprising TAC and CCL-21. In other embodiments, the present specification provides a polypeptide comprising TAC, p40, and CCL-21. The p40 and CCL-21 in the polypeptides of the present invention may be any one of those described in Section 7.2 above.

[0252] In some embodiments, the p40 in accordance with the present disclosure is human p40 or a fragment or variant thereof. In specific embodiments, the p40 in accordance with the present disclosure comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the p40 in accordance with the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the p40 polypeptide in accordance with the present disclosure is a secreted polypeptide. In some embodiments, the p40 polypeptide in accordance with the present disclosure is a membrane-bound polypeptide (e.g., MB12).

[0253] In some embodiments, the CCL-21 provided herein is human CCL-21 or a fragment or variant thereof. In specific embodiments, the CCL-21 provided herein comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the CCL-21 provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:22.

[0254] In the polypeptides of the invention, CAR, p40, and / or CCL-21 can be arranged in any order. For example, when CAR, p40, and CCL-21 are all present in the polypeptide, in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CAR, p40, and CCL-21; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CAR, CCL-21, and p40; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CCL-21, p40, and CAR; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, p40, CCL-21, and CAR; in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, p40, CAR, and CCL-21; and in some embodiments, a polypeptide in accordance with the present disclosure comprises, from N-terminal to C-terminal, CCL-21, CAR, and p40.

[0255] In some embodiments of the various polypeptides herein, CAR, p40, and / or CCL-21 are linked to one another via a peptide linker. In some embodiments, the peptide linker is a self-cleaving peptide, such as the 2A self-cleaving peptide, such that CAR, p40, and / or CCL-21 become a single polypeptide after being cleaved intracellularly.

[0256] Members of the 2A peptide family are named after the viruses that first described them. For example, the first described 2A peptide, F2A, is derived from the foot-and-mouth disease virus. Self-cleaving 2A peptides, 18–22 amino acids in length, mediate "ribosomal skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides enable multiple proteins to be encoded as polyproteins, which dissociate during translation into their component proteins. Self-cleaving peptides are present in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), zosea azygna virus (TaV), and porcine teschovirus-1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82:1027-101 (2001); Ryan et al., J. Gen. Virol., 72:2727-2732 (2001)), and cardioviruses such as Theiloviruses (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively, and are included in the present disclosure, as described in, for example, Donnelly et al., J. Gen. Virol., 78:13-21 (1997), Ryan and Drew, EMBO J., 13:928-933 (1994), Szymczak et al., Nature Biotech., 5:589-594 (2004), and Hasegawa et al., Stem Cells, 25(7):1707-12 (2007). In other embodiments, an intein-mediated protein splicing system is used, as described herein in Shah and Muir, Chem Sci., 5(1):446-461 (2014) and Topilina and Mills, Mobile DNA, 5(5) (2014). Other methods known in the art can also be used with the constructs of the present invention.

[0257] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 13. In a specific embodiment, the self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO: 14.

[0258] In some specific embodiments, p40 and CCL-19 are linked via a first self-cleaving peptide. In some embodiments, the first self-cleaving peptide is a T2A fragment of a 2A self-cleaving peptide comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, a domain comprising the p40 subunit of IL-12 and CCL-19 according to the present specification comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, a domain comprising the p40 subunit of IL-12 and CCL-19 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, a functional exogenous receptor (e.g., CAR) is linked to the domain via a second self-cleaving peptide. In some embodiments, the second self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO:13.

[0259] In certain other embodiments, p40 and CCL-21 are linked via a first self-cleaving peptide. In some embodiments, the first self-cleaving peptide is a T2A fragment of the 2A self-cleaving peptide comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, a domain comprising the p40 subunit of IL-12 and CCL-21 according to the present specification comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, a domain comprising the p40 subunit of IL-12 and CCL-21 according to the present specification comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, a functional exogenous receptor (e.g., CAR) is linked to the domain via a second self-cleaving peptide. In some embodiments, the second self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO:13.

[0260] In another aspect, the present specification provides nucleic acids encoding the above-described polypeptides, wherein the polypeptides comprise a functional exogenous receptor (e.g., a CAR) according to the present specification, the p40 subunit of IL-12, and / or a ligand of CCR7 (e.g., CCL-19 and CCL-21), which are described in more detail below.

[0261] Polynucleotides In another aspect, the disclosure provides polynucleotides encoding polypeptides according to the present invention, including those described in Sections 7.2 and 7.3 above.

[0262] More specifically, in some embodiments, the description provides a polynucleotide encoding a polypeptide, the polypeptide comprising a chimeric antigen receptor (CAR), p40, and CCL-19, wherein the CAR, p40, and CCL-19 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0263] In yet another aspect, the present disclosure provides a polynucleotide, the polynucleotide comprising a region encoding a CAR, a region encoding p40, and / or a region encoding CCL-19. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0264] In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding a CAR, a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-19. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding a CAR and a second polynucleotide containing a region encoding p40 and a region encoding CCL-19. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding a CAR and a region encoding CCL-19. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding CCL-19 and a second polynucleotide containing a region encoding a CAR and a region encoding p40.

[0265] In yet another aspect, the description provides a polynucleotide encoding a polypeptide, the polypeptide comprising a TCR (or a subunit thereof), p40, and CCL-19, wherein the TCR (or subunit thereof), p40, and CCL-19 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0266] In yet another aspect, the present disclosure provides a polynucleotide, the polynucleotide comprising a region encoding a TCR (or a subunit thereof), a region encoding p40, and / or a region encoding CCL-19. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0267] In yet another aspect, the description provides a composition comprising a first polynucleotide encoding a TCR (or a subunit thereof), a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-19. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding a TCR (or a subunit thereof), and a second polynucleotide containing a region encoding p40 and a region encoding CCL-19. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding a TCR (or a subunit thereof) and a region encoding CCL-19. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding CCL-19 and a second polynucleotide containing a region encoding a TCR (or a subunit thereof) and a region encoding p40.

[0268] In yet another aspect, the description provides a polynucleotide encoding a polypeptide, the polypeptide comprising TAC (or a domain thereof), p40, and CCL-19, wherein the TAC (or a domain thereof), p40, and CCL-19 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0269] In yet another aspect, the polynucleotide provided herein comprises a region encoding TAC (or a domain thereof), a region encoding p40, and / or a region encoding CCL-19. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0270] In yet another aspect, the present specification provides a composition comprising a first polynucleotide encoding TAC (or a domain thereof), a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-19. In yet another aspect, the present specification provides a composition comprising a first polynucleotide encoding TAC (or a domain thereof), and a second polynucleotide containing a region encoding p40 and a region encoding CCL-19. In yet another aspect, the present specification provides a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding TAC (or a domain thereof) and a region encoding CCL-19. In yet another aspect, the present specification provides a composition comprising a first polynucleotide encoding CCL-19 and a second polynucleotide containing a region encoding TAC (or a domain thereof) and a region encoding p40.

[0271] In other embodiments, the description provides a polynucleotide encoding a polypeptide comprising a chimeric antigen receptor (CAR), p40, and CCL-21, wherein the CAR, p40, and CCL-21 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0272] In yet another aspect, the present disclosure provides a polynucleotide, the polynucleotide comprising a region encoding a CAR, a region encoding p40, and / or a region encoding CCL-21. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0273] In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding a CAR, a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-21. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding a CAR and a second polynucleotide containing a region encoding p40 and a region encoding CCL-21. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding a CAR and a region encoding CCL-21. In yet another embodiment, provided herein is a composition comprising a first polynucleotide encoding CCL-21 and a second polynucleotide containing a region encoding a CAR and a region encoding p40.

[0274] In yet another aspect, the description provides a polynucleotide encoding a polypeptide, the polypeptide comprising a TCR (or a subunit thereof), p40, and CCL-21, wherein the TCR (or a subunit thereof), p40, and CCL-21 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0275] In yet another aspect, the present specification provides a polynucleotide, the polynucleotide comprising a region encoding a TCR (or a subunit thereof), a region encoding p40, and / or a region encoding CCL-21. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present specification uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0276] In yet another aspect, the description provides a composition comprising a first polynucleotide encoding a TCR (or a subunit thereof), a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding a TCR (or a subunit thereof), and a second polynucleotide containing a region encoding p40 and a region encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding a TCR (or a subunit thereof) and a region encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding CCL-21 and a second polynucleotide containing a region encoding a TCR (or a subunit thereof) and a region encoding p40.

[0277] In yet another aspect, the description provides a polynucleotide encoding a polypeptide comprising TAC (or a domain thereof), p40, and CCL-21, wherein the TAC (or a domain thereof), p40, and CCL-21 are fused to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0278] In yet another aspect, the present disclosure provides a polynucleotide, the polynucleotide comprising a region encoding TAC (or a domain thereof), a region encoding p40, and / or a region encoding CCL-21. In some embodiments, two or more regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by a single promoter.

[0279] In yet another aspect, the description provides a composition comprising a first polynucleotide encoding TAC (or a domain thereof), a second polynucleotide encoding p40, and a third polynucleotide encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding TAC (or a domain thereof), and a second polynucleotide containing a region encoding p40 and a region encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding p40 and a second polynucleotide containing a region encoding TAC (or a domain thereof) and a region encoding CCL-21. In yet another aspect, the description provides a composition comprising a first polynucleotide encoding CCL-21 and a second polynucleotide containing a region encoding TAC (or a domain thereof) and a region encoding p40.

[0280] The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0281] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, polypeptide fragments, analogs, and / or derivatives of the present disclosure. In some embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a polypeptide of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide has identity to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur anywhere at or between the 5' or 3' terminal positions of the reference nucleotide sequence, interspersed among single nucleotides in the reference sequence, or in one or more contiguous groups within the reference sequence.

[0282] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions, which do not result in a change in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0283] In some embodiments, polynucleotide variants have been generated to modulate or alter the expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants have been generated to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants have been generated to decrease expression of a polypeptide. In some embodiments, polynucleotide variants exhibit increased expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants exhibit decreased expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0284] Vectors Further provided are vectors comprising the polynucleotides or nucleic acid molecules described herein. In one embodiment, the nucleic acid molecules may be incorporated into a recombinant expression vector.

[0285] The present disclosure provides vectors for cloning and expressing any one of the polypeptides 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, cowpox vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0286] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, reverse-transcription viruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into reverse-transcription viral 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. Many reverse-transcription viral systems are known in the art. In some embodiments, adenoviral vectors are used. Many 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 immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using packaging regimens known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from reverse-transcription viruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term and stable integration of transgenic vectors and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0287] In some embodiments, a vector comprises any one of the nucleic acids encoding a polypeptide described herein. The nucleic acid can 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 promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulatable promoters, e.g., inducible promoters.

[0288] In some embodiments, the nucleic acid encoding the polypeptide is operably linked to a constitutive promoter. A constitutive promoter allows for constitutive expression of a heterologous gene (also called a transgenic gene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the murine stem cell virus (MSCV) promoter, the cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF1α), the ubiquitin C promoter (UbiC), the phosphoglycerokinase promoter (PGK), the simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been extensively compared in many studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to drive chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest level of transgenic expression but is also optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8):1453-1464(2009)). In some embodiments, the nucleic acid encoding the CAR is operably linked to the hEF1α promoter. In some embodiments, the nucleic acid encoding the CAR is operably linked to the MSCV promoter.

[0289] In some embodiments, the nucleic acid encoding the polypeptide is operably linked to an inducible promoter. Inducible promoters belong to the class of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune effector cells or the physiological state of the engineered immune effector cells, an inducer (i.e., an inducer agent), or a combination thereof.

[0290] In some embodiments, the inducing conditions do not induce expression of an endogenous gene in the engineered mammalian cell and / or the subject receiving the pharmaceutical composition, hi some embodiments, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and an activation state of the engineered mammalian cell.

[0291] In some embodiments, the vector contains a selectable marker gene or reporter gene to select cells expressing a polypeptide from a population of host cells transfected via a lentiviral vector. To enable expression in the host cell, both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences. For example, the vector contains transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid sequence.

[0292] In certain embodiments, vectors herein comprise an MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, (MND) promoter replaced with dl587rev primer binding site). In certain embodiments, vectors herein comprise an MSCV promoter. In other certain embodiments, vectors herein comprise EF1α.

[0293] 7.6. Preparation method In yet another aspect, the description provides a method for generating an engineered immune effector cell (e.g., a CAR-T cell or a TCR-T cell or a TAC-T cell), the method comprising introducing one or more polynucleotides or one or more vectors according to the description (e.g., as described in Sections 7.4 and 7.5 above) into an immune effector cell (e.g., a T cell).

[0294] More specifically, in some embodiments, CAR-T cells expressing exogenously introduced p40 and CCL-19 can be produced by introducing into cells one or more nucleic acids encoding the polypeptides described in Section 7.3 or one or more nucleic acids described in Section 7.4.

[0295] CAR, p40, and CCL-19 can each be introduced into T cells individually as a single polypeptide. For example, a nucleic acid encoding a CAR according to the present disclosure, a nucleic acid encoding p40, and a nucleic acid encoding CCL-19 are each introduced into T cells. Alternatively, any or all of the three can be introduced into T cells together as a single polypeptide by one nucleic acid, which is cleaved upon translation within the cell. For example, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and p40 linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding CCL-19 is separately introduced into T cells. Similarly, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and CCL-19 linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding p40 is separately introduced into T cells. In some embodiments, a nucleic acid encoding a polypeptide comprising all three of a CAR, p40, and CCL-19 linked to each other via a self-cleaving peptide linker may be introduced into T cells. Self-cleaving peptide linkers have been described in more detail above. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 13. In a specific embodiment, the self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO: 14.

[0296] Alternatively, CAR-T cells according to the present disclosure can be produced from a polynucleotide comprising multiple regions, e.g., a region encoding a CAR, a region encoding p40, and / or a region encoding CCL-19. The different regions can be controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the different regions are controlled by a single promoter.

[0297] In yet another aspect, the description provides engineered immune effector cells (e.g., CAR-T cells) produced by the methods described herein. In addition to other properties, CAR-T cells expressing exogenously introduced p40 and CCL-19 have higher levels of IL-23 compared to CAR-T cells without exogenously introduced p40 and CCL-19. In some embodiments, the level of IL-23 is at least 20% higher. In some embodiments, the level of IL-23 is at least 30% higher. In some embodiments, the level of IL-23 is at least 40% higher. In some embodiments, the level of IL-23 is at least 50% higher. In some embodiments, the level of IL-23 is at least 60% higher. In some embodiments, the level of IL-23 is at least 70% higher. In some embodiments, the level of IL-23 is at least 80% higher. In some embodiments, the level of IL-23 is at least 90% higher. In some embodiments, the level of IL-23 is at least 2-fold, 3-fold, 4-fold, 5-fold, or more.

[0298] In other embodiments, CAR-T cells expressing exogenously introduced p40 and CCL-21 can be produced by introducing into cells one or more nucleic acids encoding the polypeptides described in Section 7.3 or one or more nucleic acids described in Section 7.4.

[0299] CAR, p40, and CCL-21 can each be introduced into T cells individually as a single polypeptide. For example, a nucleic acid encoding a CAR according to the present disclosure, a nucleic acid encoding p40, and a nucleic acid encoding CCL-21 are each introduced into T cells. Alternatively, any or all of the three can be introduced into T cells together as a single polypeptide by one nucleic acid, which is cleaved upon translation within the cell. For example, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and p40 linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding CCL-21 is separately introduced into T cells. Similarly, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and CCL-21 linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding p40 is separately introduced into T cells. In some embodiments, a nucleic acid encoding a polypeptide comprising all three of a CAR, p40, and CCL-21 linked to each other via a self-cleaving peptide linker may be introduced into T cells. Self-cleaving peptide linkers have been described in more detail above. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO: 13. In a specific embodiment, the self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO: 14.

[0300] Alternatively, CAR-T cells according to the present disclosure can be produced from a polynucleotide comprising multiple regions, e.g., a region encoding a CAR, a region encoding p40, and / or a region encoding CCL-21. The different regions can be controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the different regions are controlled by a single promoter.

[0301] In yet another aspect, the description provides engineered immune effector cells (e.g., CAR-T cells) produced by the methods described herein. In addition to other properties, CAR-T cells expressing exogenously introduced p40 and CCL-21 have higher levels of IL-23 compared to CAR-T cells without exogenously introduced p40 and CCL-21. In some embodiments, the level of IL-23 is at least 20% higher. In some embodiments, the level of IL-23 is at least 30% higher. In some embodiments, the level of IL-23 is at least 40% higher. In some embodiments, the level of IL-23 is at least 50% higher. In some embodiments, the level of IL-23 is at least 60% higher. In some embodiments, the level of IL-23 is at least 70% higher. In some embodiments, the level of IL-23 is at least 80% higher. In some embodiments, the level of IL-23 is at least 90% higher. In some embodiments, the level of IL-23 is at least 2-fold, 3-fold, 4-fold, 5-fold, or more.

[0302] The methods described above in the context of CAR-T cells are also applicable to the production of other immune effector cells (e.g., TCR-T cells and TAC-T cells).

[0303] 7.7. Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising the engineered cells of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the engineered T cells of the present disclosure and a pharmaceutically acceptable excipient.

[0304] In specific embodiments, the term "excipient" may further refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier, or vehicle. Pharmaceutical excipients may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline, aqueous dextrose, and glycerin solutions can also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. The compositions, if desired, may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredients provided herein, e.g., in purified form, and a suitable amount of excipients to provide a form suitable for administration to a patient. The formulation should suit the mode of administration.

[0305] In some embodiments, the choice of excipient is determined in part by the particular cells and / or the mode of administration, and as such, multiple suitable formulations exist.

[0306] Typically, acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA, and / or non-ionic surfactants.

[0307] Buffers can be used to control the pH within a range that optimizes therapeutic efficacy, especially when stability is pH-dependent. Buffers suitable for the present disclosure include organic acids, inorganic acids, and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may include histidine and trimethylamine salts, such as Tris.

[0308] Preservatives may be added to retard microbial growth. Suitable preservatives for the present disclosure include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium halides (e.g., chlorine, bromine, iodine), benzethonium chloride, thimerosal, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0309] Tonicity agents (sometimes called "stabilizers") may be present to adjust or maintain the liquid tension in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often called "stabilizers" because they can reduce the possibility of inter- and intramolecular interactions by interacting with the charged groups on amino acid side chains. Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0310] Other exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above), amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like, sugars such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycols, and the like. These include organic sugars or sugar alcohols such as ethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; human serum albumin, bovine serum albumin, gelatin, or other immunoglobulin low molecular weight proteins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0311] A non-ionic surfactant or detergent (also called a "wetting agent") may be present to promote dissolution of the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (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, methylcellulose, and carboxymethylcellulose. Available anionic detergents include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0312] The route of administration is in accordance with known and acceptable methods, such as single or multiple bolus injections or injection or infusion in any suitable manner, e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular, topical administration, inhalation, or prolonged infusion by sustained or delayed release means.

[0313] In another embodiment, the pharmaceutical composition can be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled-release or sustained-release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials may be used to provide controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein described herein) or composition herein (see, e.g., Medical Applications of Controlled Release (eds. Langer and Wise, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (eds. Smolen and Ball, 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled-release or sustained-release system may be placed near a specific target tissue, such as the nasal passages or lungs, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to those of skill in the art may be used to produce sustained-release formulations containing one or more of the agents described herein (see, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0314] The pharmaceutical compositions described herein may further comprise two or more active compounds or agents as necessary for the particular indication being treated. Alternatively, or in addition, the compositions may comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth-inhibitory agent. Such molecules are present in any suitable combination in amounts effective for the desired purpose.

[0315] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Edition.

[0316] A variety of compositions and delivery systems are known and can be used with the therapeutic agents according to the present invention, including nucleic acids encapsulated in liposomes, microparticles, microcapsules, recombinant cells, retroviral vectors or other vectors capable of expressing the single domain antibodies or therapeutic molecules according to the present invention.

[0317] In some embodiments, pharmaceutical compositions herein contain binding molecules and / or cells in an amount effective to treat or prevent a disease or disorder, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effect is monitored by periodic evaluation of the treated subject. For repeated administration over several days or more, treatment is repeated depending on the condition until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.

[0318] 7.8. Methods and Use In another aspect, the description provides methods and uses of the engineered cells (e.g., CAR-T cells) according to the description. Such methods and uses include therapeutic methods and uses, e.g., administering the cells or compositions containing the cells to a subject having a disease or disorder. In some embodiments, the cells are administered in an effective amount to achieve treatment of the disease or disorder. Uses include the use of the cells in such methods and treatments, as well as in the preparation of medicaments for carrying out these therapeutic methods. In some embodiments, these methods are carried out by administering the cells or compositions containing these cells to a subject having or suspected of having a disease or condition. In some embodiments, these methods thereby treat the disease or disorder in the subject.

[0319] In some embodiments, treatment herein results in a complete or partial improvement or reduction of a disease or disorder or its associated symptoms, side effects, consequences, or phenotype. Ideal effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. These terms include, but do not imply, complete cure of a disease or complete elimination of any symptoms or one or more effects of all symptoms or outcomes.

[0320] As used herein, in some embodiments, treatment herein delays the onset of a disease or disorder, e.g., slows, prevents, decelerates, prevents, stabilizes, inhibits, and / or delays the onset of a disease (e.g., cance...

Claims

1. An immune effector cell, (i) an exogenously introduced p40 subunit of IL-12; (ii) an exogenously introduced ligand for CCR7, and (iii) a functional exogenous receptor, (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain; The functional exogenous receptor comprising: Expressing wherein the CCR7 ligand is CCL-19 or the CCR7 ligand is CCL-21; The immune effector cell.

2. The immune effector cell of claim 1 , wherein the p40 is human p40 or a fragment or variant thereof.

3. the p40 comprises the amino acid sequence of SEQ ID NO:5; or The p40 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:5; An immune effector cell according to claim 1 or 2.

4. The immune effector cell according to any one of claims 1 to 3, wherein the CCL-19 is human CCL-19 or a fragment or variant thereof, and / or the CCL-21 is human CCL-21 or a fragment or variant thereof.

5. 5. The immune effector cell of claim 4, wherein the CCL-19 comprises the amino acid sequence of SEQ ID NO:6 or the CCL-19 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:

6.

6. 5. The immune effector cell of claim 4, wherein the CCL-21 comprises the amino acid sequence of SEQ ID NO: 22 or the CCL-21 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:

22.

7. The p40 is membrane-bound, and optionally, the membrane-bound p40 is (i) p40, the hinge and transmembrane domains derived from CD8α, and, optionally, the intracellular signaling domain from CD8α; or (ii) p40, TGFβRII dominant negative receptor (DNR), hinge and transmembrane domains derived from CD8α, and, optionally, an intracellular signaling domain from CD8α. The immune effector cell of any one of claims 1 to 6, comprising:

8. 8. The immune effector cell of any one of claims 1 to 7, wherein the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen complex (TAC)-like chimeric receptor, optionally wherein the functional exogenous receptor is a CAR. (i) the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, optionally, the transmembrane domain is from CD8α or CD28; (ii) the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, optionally, the primary intracellular signaling domain is from CD3ζ; (iii) the intracellular signaling domain comprises a costimulatory signaling domain, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof; wherein, optionally, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137; (iv) the functional exogenous receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, optionally, the hinge domain is from CD8α; and / or (v) the functional exogenous receptor further comprises a signal peptide located at the N-terminus of the polypeptide, optionally, the signal peptide is from CD8α; An immune effector cell according to any one of claims 1 to 8.

10. The immune effector cell of any one of claims 1 to 9, which is a T cell.

11. A polypeptide comprising: (i) the p40 subunit of IL-12; (ii) a ligand of CCR7, and (iii) a functional exogenous receptor, (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain; The functional exogenous receptor comprising: Including, wherein the CCR7 ligand is CCL-19 or the CCR7 ligand is CCL-21; The polypeptide.

12. The p40 is human p40 or a fragment or variant thereof; the CCL-19 is human CCL-19 or a fragment or variant thereof; and / or The CCL-21 is human CCL-21 or a fragment or variant thereof; The polypeptide of claim 11. (i) the p40 comprises an amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:5; (ii) the CCL-19 comprises the amino acid sequence of SEQ ID NO:6, or the CCL-19 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:6; and / or (iii) the CCL-21 comprises the amino acid sequence of SEQ ID NO: 22, or the CCL-21 comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 22; A polypeptide according to claim 11 or 12.

14. The p40 is membrane-bound, and optionally, the membrane-bound p40 is (i) p40, the hinge and transmembrane domains derived from CD8α, and, optionally, the intracellular signaling domain from CD8α; or (ii) p40, TGFβRII dominant negative receptor (DNR), hinge and transmembrane domains derived from CD8α, and, optionally, an intracellular signaling domain from CD8α. The polypeptide according to any one of claims 11 to 13, comprising:

15. 15. The polypeptide of any one of claims 11 to 14, wherein the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen complex (TAC)-like chimeric receptor, optionally wherein the functional exogenous receptor is a CAR.

16. The p40, the CCL-19, and the functional exogenous receptor are linked to each other via a peptide linker, or the p40, the CCL-21, and the functional exogenous receptor are linked to each other via a peptide linker. wherein, optionally, the peptide linker is a 2A self-cleaving peptide selected from the group consisting of F2A, E2A, P2A, T2A, and variants thereof. A polypeptide according to any one of claims 11 to 15.

17. The p40 and the CCL-19 are present in one domain comprising the amino acid sequence of SEQ ID NO: 4, or the p40 and the CCL-19 are present within one domain, the domain comprising an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:4; or The p40 and the CCL-21 are present in one domain comprising the amino acid sequence of SEQ ID NO: 20, or the p40 and the CCL-21 are present within a domain, the domain comprising an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20; A polypeptide according to any one of claims 11 to 16.

18. A nucleic acid comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 11 to 17.

19. A nucleic acid comprising: (i) a first region encoding the p40 subunit of IL-12; (ii) a second region encoding a ligand for CCR7, and (iii) a third region encoding a functional exogenous receptor comprising: (a) an extracellular antigen-binding domain; (b) a transmembrane domain, and (c) Intracellular signaling domain Including, wherein the CCR7 ligand is CCL-19 or the CCR7 ligand is CCL-21; The nucleic acid.

20. A vector comprising the nucleic acid of claim 18 or 19.

21. An immune effector cell comprising a polypeptide described in any one of claims 11 to 17, a nucleic acid described in claim 18 or 19, or a vector described in claim 20.

22. A pharmaceutical composition comprising an immune effector cell according to any one of claims 1 to 10 and 21, a polypeptide according to any one of claims 11 to 17, a nucleic acid according to claim 18 or 19, or a vector according to claim 20, and a pharma- ceutical acceptable carrier.

23. 23. The pharmaceutical composition of claim 22 for use in a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition.

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