GENETICALLY ENGINEERED T CELL CO-RECEPTORS AND METHODS OF USE THEREOF

Abstract

The present disclosure relates to modified receptors comprising a MyD88 domain, as well as cells comprising same and methods of use thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 321,553, filed March 18, 2022, which is incorporated by reference herein in its entirety.

[0002] Incorporation by reference of sequence listing The Sequence Listing XML associated with this application has been provided electronically in XML file format and is incorporated herein by reference. The name of the XML file containing the Sequence Listing XML is "UNCO-040_SeqList_ST26". The XML file is 29,452 bytes, was created on March 20, 2023, and has been submitted electronically via the USPTO Patent Center.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under R01CA207913 awarded by the National Institute of Health. The Government has certain rights in this invention.

[0004] The present disclosure relates to modified receptors and fusion proteins comprising a MyD88 domain and a CD3 subunit, as well as T cell receptors and cells comprising same, and methods of use thereof. [Background technology]

[0005] Adoptive cell therapy, including TIL and CAR T cell therapy, as well as TCR therapy, have shown promise in the treatment of hematological and solid tumors, however, CAR-T therapy is currently the only T cell therapy approved by the FDA (e.g., Yescarta, Kymriah, Tecartus, Abecma, and Breyanzi).

[0006] T cell therapy has several limitations that have hindered clinical development. For example, cancer patients undergoing treatment with cell therapy such as CAR T cells, Natural Killer (NK) cells, tumor-infiltrating lymphocytes (TILs), or T-cell receptor (TCR) engineered cells must undergo a preconditioning step using chemotherapy to promote immune cell engraftment. Patients receiving TILs also receive high doses of IL-2. Unfortunately, the preconditioning step results in a transient depletion of endogenous T cells as well as other important immune cells, platelets, and red blood cells. High doses of IL-2 are highly toxic and pose a high risk of developing toxicity, necessitating inpatient services in intensive care units.

[0007] In addition to toxic effects on patients, lymphodepletion and IL-2 are expensive and differentiated cells lose potency rapidly in vivo, limiting their therapeutic efficacy.

[0008] Thus, there remains an unmet need for cell therapies that do not require lymphodepletion and / or IL-2 therapy for T cell engraftment and expansion. Summary of the Invention

[0009] In one aspect, provided herein is a cell comprising: (a) a modified T cell receptor (TCR) comprising an α chain, a β chain, CD3ζ, an intracellular domain, and a MyD88 polypeptide comprising a death domain (DD) of MyD88 and an intermediate domain (ID) of MyD88, wherein the MyD88 domain is fused to a CD3ζ subunit; or (b) a fusion protein comprising a CD3ζ, intracellular domain, and a MyD88 DD. In some embodiments, the MyD88 polypeptide comprises an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9.

[0010] In another aspect, provided herein is a cell comprising: (a) a modified T cell receptor (TCR) comprising an alpha chain, a beta chain, a CD3 zeta intracellular domain, and a MyD88 polypeptide comprising an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2, wherein the MyD88 domain is fused to the CD3 zeta subunit; or (b) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9.

[0011] In another aspect, provided herein is a modified T cell receptor (TCR) comprising an α chain, a β chain, and CD3 subunits CD3γ, CD3ζ, CD3ε, and CD3δ, wherein a MyD88 domain comprising the death domain (DD) of MyD88 and the intermediate domain (ID) of MyD88 is fused to the CD3ζ subunit. In some embodiments, the T cell receptor targets a cancer antigen. In some embodiments, the MyD88 domain comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0012] In another aspect, provided herein is a cell comprising a modified TCR as described herein.

[0013] In another aspect, provided herein is a pharmaceutical composition comprising a cell described herein and a pharma- ceutically acceptable carrier.

[0014] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a cell or pharmaceutical composition described herein. In some embodiments, the patient does not undergo lymphodepletion prior to administration of the T cells. In some embodiments, the patient does not receive cytokines while being administered the cells. In some embodiments, the cancer is breast cancer, sarcoma, melanoma, or lung cancer.

[0015] In another aspect, provided herein is a method of improving a therapeutic cell, the method comprising transfecting the therapeutic cell with a modified TCR as described herein.

[0016] In another aspect, provided herein is a method of improving a therapeutic cell, comprising modifying a receptor of the therapeutic cell to fuse a MyD88 domain to a CD3ζ domain present in the receptor of the therapeutic cell. In some embodiments, the MyD88 domain comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0017] In another aspect, provided herein is a method for improving a therapeutic cell, comprising introducing a fusion protein comprising a CD3 ζ intracellular domain and MyD88 DD into the cell. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9. In some embodiments, the cell is an immune cell. In some embodiments, the cell is derived from a patient.

[0018] In some embodiments, the method results in an increase in therapeutic cell expansion in vivo of at least 50% compared to the therapeutic cell prior to the modification. In some embodiments, the method results in an increase in cytokine expression in therapeutic cells in vivo of at least 50% compared to the therapeutic cell prior to the modification. In some embodiments, the method results in a decrease in exhaustion of therapeutic cells in vivo of at least 20% compared to the therapeutic cell prior to the modification. In some embodiments, the therapeutic cells retain comparable antigen specificity following transfection. [Brief description of the drawings]

[0019] [Figure 1A] Four T cell receptor (TCR) constructs are shown, including the α and β TCRs of the TCR known as "DMF5" that are reactive to MART-1 (amino acids 27-35). CD3z:MyD88 was added to the α and / or β TCR chains. Only the intracellular (ICS) domain of CD3z was used. MyD88 lacked the toll / interleukin-1 (IL-1) receptor (TIR). Figure 1A shows a schematic diagram of DNA constructs that can be cloned into a vector (e.g., pMSGV retroviral vector). Figure 1B shows a schematic diagram of TCR constructs that can be expressed on the T cell surface. LS, human CD8 alpha leader sequence; α chain / β chain, human α / β chain of the DMF5 TCR; G4S, GGGGS linker sequence; CD3z_ICS, intracellular domain of CD3z; MyD88=death domain and intermediate domain of MyD88; EGFRt, transmembrane endothelial growth factor receptor. EGFRt is used as a reporter to determine transduction efficiency. It can also be used as a means to select transduced cells using a reagent (i.e., anti-EGFR antibody). Alternatively, EGFRt can also be used to target cells for in vivo tracking using labeled anti-EGFR antibody. EGFRt can also be used as a target to destroy transduced T cells using anti-EGFR antibody drug conjugate. [Figure 1B]Four T cell receptor (TCR) constructs are shown, including the α and β TCRs of the TCR known as "DMF5" that are reactive to MART-1 (amino acids 27-35). CD3z:MyD88 was added to the α and / or β TCR chains. Only the intracellular (ICS) domain of CD3z was used. MyD88 lacked the toll / interleukin-1 (IL-1) receptor (TIR). Figure 1A shows a schematic diagram of DNA constructs that can be cloned into a vector (e.g., pMSGV retroviral vector). Figure 1B shows a schematic diagram of TCR constructs that can be expressed on the T cell surface. LS, human CD8 alpha leader sequence; α chain / β chain, human α / β chain of the DMF5 TCR; G4S, GGGGS linker sequence; CD3z_ICS, intracellular domain of CD3z; MyD88=death domain and intermediate domain of MyD88; EGFRt, transmembrane endothelial growth factor receptor. EGFRt is used as a reporter to determine transduction efficiency. It can also be used as a means to select transduced cells using a reagent (i.e., anti-EGFR antibody). Alternatively, EGFRt can also be used to target cells for in vivo tracking using labeled anti-EGFR antibody. EGFRt can also be used as a target to destroy transduced T cells using anti-EGFR antibody drug conjugate. [Figure 1C] Figure 1 shows the various proteins that make up the T cell receptor complex, including the T cell receptor alpha and beta chains, and the CD3 subunits, CD3γ, CD3ζ, CD3ε, and CD3δ. CD3ζ forms dimers with CD3γ and CD3δ, while CD3ζ forms homodimers. We developed novel synthetic CD3 signaling molecules by engineering CD3ε ("ε-amdTCR") and CD3ζ (ζ-ampTCR) to express MyD88 death and intermediate domains. [Figure 1D] The CD3ζ:MyD88 fusion protein is shown without the TCR complex. This representation provides a better view of the dimerized CD3ζ:MyD88 fusion protein in the absence of other TCR signaling molecules. [Diagram 2] FIG. 1 shows expression of TCR:CD3z:MyD88 constructs in human T cells as determined by flow cytometry. [Figure 3A] Figure 3A shows effector cytokine expression in T cells engineered with DMF5 CD3z:MyD88 appended to the TCR beta or TCR alpha chain. Figure 3A shows cytokine expression in Jurkat cells engineered to express the DMF5 TCR:CD3z:MyD88 construct. Each point represents the mean of three technical replicates, error bars = 1 standard deviation. * indicates significant differences (p<0.05) between the DMF5α:CD3z:MyD88 and DMF5β:CD3z:MyD88 groups with wild-type DMF5 but not with each other, ** indicates significant differences (p<0.05) between the α:CD3z:MyD88, β:CD3z:MyD88, and wild-type groups with each other by one-way ANOVA. Figure 3B shows cytokine expression in 624 melanoma cells (naturally expressing HLA-A2+MART1+). Each point represents one technical replicate, each bar represents the mean of the replicates, error bars = 1 standard deviation, p values ​​are as indicated on the graphs and were determined by one-way ANOVA. [Figure 3B]Figure 3A shows effector cytokine expression in T cells engineered with DMF5 CD3z:MyD88 appended to the TCR beta or TCR alpha chain. Figure 3A shows cytokine expression in Jurkat cells engineered to express the DMF5 TCR:CD3z:MyD88 construct. Each point represents the mean of three technical replicates, error bars = 1 standard deviation. * indicates significant differences (p<0.05) between the DMF5α:CD3z:MyD88 and DMF5β:CD3z:MyD88 groups with wild-type DMF5 but not with each other, ** indicates significant differences (p<0.05) between the α:CD3z:MyD88, β:CD3z:MyD88, and wild-type groups with each other by one-way ANOVA. Figure 3B shows cytokine expression in 624 melanoma cells (naturally expressing HLA-A2+MART1+). Each point represents one technical replicate, each bar represents the mean of the replicates, error bars = 1 standard deviation, p values ​​are as indicated on the graphs and were determined by one-way ANOVA. [Figure 4A] Figure 4 shows the response and polyfunctionality of T cells engineered with TCRB:CD3z:MyD88, wild-type TCR, or control TCR:CD3z. Figure 4A shows the expression of TNFα and TNFβ. Figure 4B shows the expression of IL-2 and GM-CSF. [Figure 4B] Figure 4 shows the response and polyfunctionality of T cells engineered with TCRB:CD3z:MyD88, wild-type TCR, or control TCR:CD3z. Figure 4A shows the expression of TNFα and TNFβ. Figure 4B shows the expression of IL-2 and GM-CSF. [Figure 5A]Cytokine production in T cells engineered with TCRB:CD3z:MyD88 compared to cells engineered with wild type TCR, TCRA:CD3z:MyD88, or TCR:CD3z. Figure 5A shows a comparison between DMF5 WT, TCRB:CD3z, and TCRB:CD3z:MyD88. Figure 5B shows a comparison between DMF5 WT, TCRB:CD3z, TCRA:CD3z:MyD88, and TCRB:CD3z:MyD88. Each dot represents the average of three technical replicates, error bars = 1 standard deviation. Figure 5A includes IL-2 production. Figure 5B shows data using a TCR with MyD88 added to both TCR alpha and TCR beta. Without wishing to be bound by theory, it is believed that adding MyD88 to both TCR chains prevents the TCR from being expressed, resulting in a non-functional receptor. [Figure 5B] Cytokine production in T cells engineered with TCRB:CD3z:MyD88 compared to cells engineered with wild type TCR, TCRA:CD3z:MyD88, or TCR:CD3z. Figure 5A shows a comparison between DMF5 WT, TCRB:CD3z, and TCRB:CD3z:MyD88. Figure 5B shows a comparison between DMF5 WT, TCRB:CD3z, TCRA:CD3z:MyD88, and TCRB:CD3z:MyD88. Each dot represents the average of three technical replicates, error bars = 1 standard deviation. Figure 5A includes IL-2 production. Figure 5B shows data using a TCR with MyD88 added to both TCR alpha and TCR beta. Without wishing to be bound by theory, it is believed that adding MyD88 to both TCR chains prevents the TCR from being expressed, resulting in a non-functional receptor. [Figure 6A] Figure 6 shows the effect of TCRB:CD3z:MyD88 on T cell expansion. Figure 6A shows a schematic of the experimental setup. Figure 6B shows cell counts of T cells from three donors engineered with DMF5 TCR or TCRB:CD3Z:MyD88. [Figure 6B-1]Figure 6 shows the effect of TCRB:CD3z:MyD88 on T cell expansion. Figure 6A shows a schematic of the experimental setup. Figure 6B shows cell counts of T cells from three donors engineered with DMF5 TCR or TCRB:CD3Z:MyD88. [Figure 6B-2] Figure 6 shows the effect of TCRB:CD3z:MyD88 on T cell expansion. Figure 6A shows a schematic of the experimental setup. Figure 6B shows cell counts of T cells from three donors engineered with DMF5 TCR or TCRB:CD3Z:MyD88. [Figure 7] The effect of TCRB:CD3z:MyD88 on T cell antitumor activity and effector molecule production is shown. [Figure 8A] The effects of CD3z and CD3z-MyD88 on intracellular levels of cytokines as determined by flow cytometry (FIG. 8A) and on the levels of polyfunctional TILs (those producing two or more cytokines, FIGS. 8B and 8C) are shown. [Figure 8B] The effects of CD3z and CD3z-MyD88 on intracellular levels of cytokines as determined by flow cytometry (FIG. 8A) and on the levels of polyfunctional TILs (those producing two or more cytokines, FIGS. 8B and 8C) are shown. [Figure 8C] The effects of CD3z and CD3z-MyD88 on intracellular levels of cytokines as determined by flow cytometry (FIG. 8A) and on the levels of polyfunctional TILs (those producing two or more cytokines, FIGS. 8B and 8C) are shown. [Figure 9A] Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 9B] Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 9C]Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 9D] Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 9E] Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 9F] Proliferation of unmodified TILs and TILs transfected in vivo with control CD3z or CD3z:MyD88 is shown in breast cancer cells (Figures 9A and 9C), sarcoma cells (Figures 9B and 9D), and melanoma cells (Figures 9E and 9F). [Figure 10] Figure 1 shows TCR diversity and repertoires in unmodified TILs and TILs transfected with control CD3z or CD3z:MyD88, with each population representing one TCR Vβ family member. [Figure 11A] Figure 11 shows the polyfunctionality of unmodified TILs and TILs transfected with control CD3z or CD3z:MyD88 over time. Figure 11A shows the intracellular levels of cytokines on T cells stimulated with anti-CD3 antibodies. Figure 11B shows the percentage of T cells capable of co-producing various effector molecules at different time points. [Figure 11B] Figure 11 shows the polyfunctionality of unmodified TILs and TILs transfected with control CD3z or CD3z:MyD88 over time. Figure 11A shows the intracellular levels of cytokines on T cells stimulated with anti-CD3 antibodies. Figure 11B shows the percentage of T cells capable of co-producing various effector molecules at different time points. [Figure 12A]Figure 12 shows the effect of CD3z:MyD88 on T cell activity (based on IFNγ production). Data are shown for breast cancer (Figure 12A), sarcoma (Figure 12B), and melanoma (Figures 12C and 12D) samples. [Figure 12B] Figure 12 shows the effect of CD3z:MyD88 on T cell activity (based on IFNγ production). Data are shown for breast cancer (Figure 12A), sarcoma (Figure 12B), and melanoma (Figures 12C and 12D) samples. [Figure 12C] Figure 12 shows the effect of CD3z:MyD88 on T cell activity (based on IFNγ production). Data are shown for breast cancer (Figure 12A), sarcoma (Figure 12B), and melanoma (Figures 12C and 12D) samples. [Figure 12D] Figure 12 shows the effect of CD3z:MyD88 on T cell activity (based on IFNγ production). Data are shown for breast cancer (Figure 12A), sarcoma (Figure 12B), and melanoma (Figures 12C and 12D) samples. [Figure 13A] Figure 13 shows the proliferation advantage of T cells transfected with CD3ζ:MyD88. Figure 13A shows the proliferation of CD4+ and CD8+ T cells with and without stimulation with anti-CD3 antibody. Figure 13B shows the mean fluorescence intensity of the proliferation dye in CD8+ T cells. [Figure 13B] Figure 13 shows the proliferation advantage of T cells transfected with CD3ζ:MyD88. Figure 13A shows the proliferation of CD4+ and CD8+ T cells with and without stimulation with anti-CD3 antibody. Figure 13B shows the mean fluorescence intensity of the proliferation dye in CD8+ T cells. [Figure 14A] Figure 14 shows cytokine production in CD3z:MyD88 cells. Figure 14A shows IFNγ production assessed by ELISA in T cells stimulated with various concentrations of gp100 antigen or B16 tumor cells. Figure 14B shows TNFα and IFNγ production determined by intracellular FACS. Figure 14C summarizes the number of cytokines expressed by each cell population. [Figure 14B]Figure 14 shows cytokine production in CD3z:MyD88 cells. Figure 14A shows IFNγ production assessed by ELISA in T cells stimulated with various concentrations of gp100 antigen or B16 tumor cells. Figure 14B shows TNFα and IFNγ production determined by intracellular FACS. Figure 14C summarizes the number of cytokines expressed by each cell population. [Figure 14C] Figure 14 shows cytokine production in CD3z:MyD88 cells. Figure 14A shows IFNγ production assessed by ELISA in T cells stimulated with various concentrations of gp100 antigen or B16 tumor cells. Figure 14B shows TNFα and IFNγ production determined by intracellular FACS. Figure 14C summarizes the number of cytokines expressed by each cell population. [Figure 15A] Antitumor effect of TILs transfected with control CD3z or CD3z:MyD88. One representative experiment out of three is shown (n=4-5 mice / group). Figures 15A and 15B show tumor size of B16 melanoma in mice after preconditioning with radiation followed by treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs, Figure 15A shows data for each animal in the group for one representative experiment out of three shown (n=4-5 mice per group) and Figure 15B shows the average for each group. Figure 15C shows survival of mice bearing B16 melanoma after radiation and treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs. Statistical significance was determined by a mixed-effects ANOVA model with Geisser Greenhouse correction and Tukey's multiple comparison test. ***p<0.001. Error bars represent standard mean deviation from the mean of 5 mice per group. [Figure 15B]Antitumor effect of TILs transfected with control CD3z or CD3z:MyD88. One representative experiment out of three is shown (n=4-5 mice / group). Figures 15A and 15B show tumor size of B16 melanoma in mice after preconditioning with radiation followed by treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs, Figure 15A shows data for each animal in the group for one representative experiment out of three shown (n=4-5 mice per group) and Figure 15B shows the average for each group. Figure 15C shows survival of mice bearing B16 melanoma after radiation and treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs. Statistical significance was determined by a mixed-effects ANOVA model with Geisser Greenhouse correction and Tukey's multiple comparison test. ***p<0.001. Error bars represent standard mean deviation from the mean of 5 mice per group. [Figure 15C] Antitumor effect of TILs transfected with control CD3z or CD3z:MyD88. One representative experiment out of three is shown (n=4-5 mice / group). Figures 15A and 15B show tumor size of B16 melanoma in mice after preconditioning with radiation followed by treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs, Figure 15A shows data for each animal in the group for one representative experiment out of three shown (n=4-5 mice per group) and Figure 15B shows the average for each group. Figure 15C shows survival of mice bearing B16 melanoma after radiation and treatment with PBS or T cells expressing control CD3z constructs or T cells expressing CD3z:MyD88 constructs. Statistical significance was determined by a mixed-effects ANOVA model with Geisser Greenhouse correction and Tukey's multiple comparison test. ***p<0.001. Error bars represent standard mean deviation from the mean of 5 mice per group. [Figure 16A]The body weights of animals treated with PBS, or with T cells expressing a control CD3z construct, or with T cells expressing CD3z:MyD88 are shown. Each Figure 16A-C shows data for one of three experiments. The x-axis in Figures 16A-C shows days. [Figure 16B] The body weights of animals treated with PBS, or with T cells expressing a control CD3z construct, or with T cells expressing CD3z:MyD88 are shown. Each Figure 16A-C shows data for one of three experiments. The x-axis in Figures 16A-C shows days. [Figure 16C] The body weights of animals treated with PBS, or with T cells expressing a control CD3z construct, or with T cells expressing CD3z:MyD88 are shown. Each Figure 16A-C shows data for one of three experiments. The x-axis in Figures 16A-C shows days. [Figure 17A] Figure 17 shows the effect of CD3z:MyD88 on neoantigen-reactive TILs. Figure 17A shows a schematic of the experimental setup. Figure 17B shows INFγ levels in TILs engineered to express CD3z:MyD88 (black bars) compared to CD3z (grey bars). Figure 17C shows the number of INFγ-producing cells and TCRvB family gene usage as determined by flow cytometry. [Figure 17B] Figure 17 shows the effect of CD3z:MyD88 on neoantigen-reactive TILs. Figure 17A shows a schematic of the experimental setup. Figure 17B shows INFγ levels in TILs engineered to express CD3z:MyD88 (black bars) compared to CD3z (grey bars). Figure 17C shows the number of INFγ-producing cells and TCRvB family gene usage as determined by flow cytometry. [Figure 17C]Figure 17 shows the effect of CD3z:MyD88 on neoantigen-reactive TILs. Figure 17A shows a schematic of the experimental setup. Figure 17B shows INFγ levels in TILs engineered to express CD3z:MyD88 (black bars) compared to CD3z (grey bars). Figure 17C shows the number of INFγ-producing cells and TCRvB family gene usage as determined by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Provided herein are synthetic T cell receptors (TCRs) that contain a MyD88 domain. Also provided herein are cells that contain the TCRs described herein and methods of use thereof.

[0021] Modified Receptors Engineered T cell receptors can be used in adoptive T cell therapy to harness the body's immune system to target, for example, cancer cells. In the context of chimeric antigen receptors (CARs), immune cells are transfected to express an artificial receptor that binds to a given antigen. TCR therapy relies on the ability of TcRs to recognize antigens presented by major histocompatibility complex (MHC) molecules. See, for example, Zhao and Cao, 2019; Front. Immunol. 10:2250, which is incorporated herein by reference in its entirety.

[0022] The T cell receptor complex consists of various proteins including the T cell receptor alpha and beta chains and the CD3 subunits, CD3γ, CD3ζ, CD3ε, and CD3δ. CD3ε forms dimers with CD3γ and CD3δ, while CD3ζ forms homodimers. The α and β chains each contain a constant region and a variable region. The variable regions of the α and β chains confer antigen specificity to the receptor. Thus, there are three complementarity-determining regions (CDRs) in each of the α and β chains, with CDR1 and CDR2 typically contacting the MHC molecule, while CDR3 usually contacts the antigen. See Wonget et al., 2019, Front Immuno. 10:2454, incorporated herein by reference in its entirety. The TCR is activated upon binding to an antigen presented by the MHC.

[0023] CARs generally include an extracellular domain that includes an antigen-binding region (e.g., an antibody or a portion of an antibody such as a single chain variable fragment (scFv)), a transmembrane domain, and an intracellular domain. The intracellular domain often includes a CD3ζ signaling domain and one or two costimulatory domains, which may be derived from, for example, CD8, CD28, OX40, or 4-1BB, or a combination thereof. See Jayaraman et al., EBioMedicine 58(2020)102931, which is incorporated herein by reference in its entirety.

[0024] In some embodiments, the CD3 ζ domain is linked or fused to the α chain of the TCR. In some embodiments, the CD3 ζ domain is linked or fused to the β chain of the CDR. In some embodiments, one CD3 ζ subunit is linked to each of the α and β chains of the TCR. Schematic diagrams of constructs illustrating these four configurations are shown in Figures 1A-1C.

[0025] Provided herein is a modified TCR comprising an α chain, a β chain, and CD3 subunits CD3γ, CD3ζ, CD3ε, and CD3δ, where the CD3ζ or CD3ε subunit is fused to a MyD88 domain. MyD88 is an adaptor for signaling pathways including Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor family. MyD88 contains three major domains: an N-terminal death domain (DD), an intermediate domain (INT), and a C-terminal Toll interleukin-1 receptor domain (TIR). MyD88 normally induces pro-inflammatory signaling, but can have anti-inflammatory effects depending on the context. The DD of MyD88 associates with IRAK family members, and overexpression of the DD leads to NFκB activation and c-Jun N-terminal kinase (JNK) activation. The TIR domain mediates interactions with other TIR domain-containing proteins. The INT domain links the TIR and DD, and variants of MyD88 lacking the INT domain act as dominant-negative forms of MyD88. See Deguine and Barton, F1000 Prime Reports 2014, 6:97, which is incorporated herein by reference in its entirety.

[0026] In some embodiments, the modified receptor provided herein is a modified TCR that comprises a MyD88 domain linked or fused to a CD3ζ or CD3ε subunit. In some embodiments, the modified receptor provided herein is a CAR that comprises a MyD88 domain linked or fused to a CD3ζ domain.

[0027] In some embodiments, the MyD88 domain comprises the DD and / or INT of MyD88.

[0028] An exemplary sequence of a MyD88 domain containing DD and INT is set forth in SEQ ID NOs: 1 and 2: MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGVDTRRAKR (SEQ ID NO: 1). MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLG (SEQ ID NO: 2).

[0029] In some embodiments, the modified receptor provided herein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the modified receptor provided herein comprises the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, the modified receptor provided herein comprises the sequence set forth in SEQ ID NO: 1.

[0030] A "conservative amino acid substitution" is an amino acid substitution that replaces one amino acid with another amino acid with similar properties, such as substituting a hydrophobic amino acid (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) for another hydrophobic amino acid, a hydrophobic amino acid with a bulky side chain (Phe, Tyr, Trp) for another hydrophobic amino acid with a bulky side chain, an amino acid with a positively charged side chain (Arg, His, Lys) for another amino acid with a positively charged side chain, an amino acid with a negatively charged side chain (Asp, Glu) for another amino acid with a negatively charged side chain, or an amino acid with a polar uncharged side chain (Ser, Thr, Asn, Gln) for another amino acid with a polar uncharged side chain.

[0031] In some embodiments, the modified receptors provided herein comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 2. In some embodiments, the modified receptors provided herein comprise the amino acid sequence set forth in SEQ ID NO: 2 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, the modified receptors provided herein comprise the sequence set forth in SEQ ID NO: 2.

[0032] An exemplary sequence of a CD3ζ domain fused to a MyD88 domain containing the DD and INT domains is set forth in SEQ ID NO:3, where the CD3ζ domain is underlined and the MyD88 domain is in bold. The T2A self-cleaving fragment that can be included to facilitate transfection and expression of the modified TCR is shown in italics.

[0033] [Table 1] In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 175-338 of SEQ ID NO: 3. In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 1-338 of SEQ ID NO: 3. In some embodiments, the modified TCRs provided herein comprise amino acids 1-338 of SEQ ID NO: 3.

[0034] Alternative sequences for the CD3 zeta domain fused to the myD88 domain are shown in SEQ ID NO: 9, where the CD3 zeta domain is underlined and the MyD88 domain is in bold, and the linker sequence is in italics.

[0035] [Table 2]

[0036] In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the modified TCRs provided herein comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0037] Another alternative sequence of a CD3 zeta domain fused to a myD88 domain is shown in SEQ ID NO: 16, with the CD3 zeta domain underlined and the MyD88 domain in bold.

[0038] [Table 3]

[0039] In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the modified TCRs provided herein comprise the amino acid sequence set forth in SEQ ID NO: 16.

[0040] It will be clear to one skilled in the art that, as an alternative to fusing the MyD88 domain to a CD3 domain (e.g., CD3ζ domain), the MyD88 domain can be linked to the CD3 domain (e.g., CD3ζ domain) via a linker. Suitable linkers are known in the art and include, for example, a G4S linker or a GGGS linker. In another aspect, the MyD88 domain can be linked to a cell surface antigen of a T cell. In some embodiments, the MyD88 domain is linked to CD8a. CD8a is a membrane glycoprotein expressed on a subset of T cells that acts as a co-receptor for MHC class I.

[0041] An exemplary sequence of a CD8a domain linked to a MyD88 domain containing the DD and INT domains is set forth in SEQ ID NO:4, where the CD8a domain is underlined, the MyD88 domain is in bold, and the G4S linker linking the MyD88 and CD8a domains is in italics.

[0042] [Table 4]

[0043] In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the modified TCRs provided herein comprise the amino acid sequence set forth in SEQ ID NO: 4.

[0044] An exemplary amino acid sequence of a modified TCR comprising the TCR α chain, the TCR β chain, the CD3ζ intracellular domain (ICD), and the MyD88 DD is set forth in SEQ ID NO: 11, where the TCR α chain is underlined, the TCR β chain is bolded, the CD3ζ ICD is underlined and bolded, and the MyD88 DD is bolded and italicized. The linker sequence is italicized.

[0045] [Table 5]

[0046] In some embodiments, the modified TCRs provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the modified TCRs provided herein comprise the amino acid sequence set forth in SEQ ID NO: 11. The modified TCRs described herein may be screened for the ability to bind to a specific antigen before the modifications are introduced.

[0047] Without wishing to be bound by theory, it is believed that the addition of the MyD88 domain to the modified receptor results in antigen-specific activation of the receptor, improving the clinical utility of the receptor and cells containing it.

[0048] MyD88 fusion protein Fusion proteins containing MyD88 domains are also provided herein, which can be used to enhance the intrinsic cell-killing capacity of immune cells, including natural killer (NK) cells, T cells, macrophages, and tumor-infiltrating lymphocytes (TILs).

[0049] An exemplary sequence of a fusion protein comprising the CD3 ζ intracellular domain (ICD) and the MyD88 DD is set forth in SEQ ID NO: 13, with the CD3 ζ domain underlined and the MyD88 domain in bold.

[0050] [Table 6]

[0051] In some embodiments, the fusion proteins provided herein comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the fusion proteins described herein comprise the amino acid sequence set forth in SEQ ID NO:13.

[0052] Without wishing to be bound by theory, it is believed that when the CD3ζ-MyD88 fusion protein is introduced into a cell, it localizes to the cell membrane and provides a stimulus to the cell into which it is introduced, resulting in increased cell proliferation, cytokine secretion, and / or anti-tumor activity.

[0053] Polynucleotides and Vectors Also provided herein are polynucleotides and vectors encoding the modified receptors and fusion proteins described herein, or portions thereof.

[0054] Exemplary polynucleotides comprising nucleic acid sequences encoding the MyD88 domain, including the DD domain and the INT domain, are set forth in SEQ ID NOs:5 and 6.

[0055] ATGGCAGCAGGAGGACCAGGCGCCGGCAGCGCCGCCCCCGTGAGCTCTACCAGCTCTCTGCCTTTAGCCGCCCTGAACATGCGCGTGAGAAGGCGCCTGAGCCTGTTTCTGAATGTGCGGACTC AGGTCGCCGCCGACTGGACAGCCCTGGCTGAAGAAATGGATTTTGAGTACCTGGAGATCCGCCAATTAGAGACACAGGCCGATCCAACAGGCCGGCTGCTGGATGCCTGGCAGGGCAGACCCGG AGCCTCCGTGGGCAGGCTGCTGGAACTGCTGACTAAGCTGGGCAGAGATGATGTGCTGCTGGAACTGGGCCCCTCTATCGAGGAGGACTGTCAGAAATACATCCTGAAGCAACAACAGGAAGAA GCAGAGAAGCCTCTGCAGGTGGCAGCCGTGGATTCTAGCGTGCCAAGGACAGCCGAACTGGCCGGCATTACCACACTGGATGATCCTCTGGGAGTCGACACGCGTAGAGCCAAGAGA (SEQ ID NO: 5)

[0056] ATGGCAGCAGGGGGCCCTGGAGCTGGAAGTGCCGCACCAGTAAGTAGCACTTCCAGTCTTCCCCTCGCGGCACTGAATATGAGAGTTCGCCGGCGGTTGAGCCTCTTCTTGAATGTTAGAACGCAAGTGGCTGCTGACTGGACAGCCCTCGCGGAGGAAATGGATTTCGAATATTTGGAGATTAGGCAATTGGAGACTCAAGCAGATCCGACAGGTCGCCTTCTTGACGCTTGGCA GGGTCGGCCGGGCGCTAGTGTGGGACGACTGCTTGAGTTGCTGACCAAATTGGGCCGGGACGATGTTCTCTTGGAGCTTGGCCCATCCATAGAGGAGGACTGTCAAAAATATATCCTCAAACAGCAGCAGGAAGAAGCTGAAAAACCTCTGCAGGTCGCGGCTGTCGACAGCAGCGTACCTCGAACAGCTGAGTTGGCGGGAATAACAACACTGGATGATCCGCTTGGG (SEQ ID NO: 6).

[0057] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:5. In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:6 ...

[0058] An exemplary DNA sequence encoding a CD3 zeta domain fused to a myD88 domain is shown in SEQ ID NO: 10, where the CD3 zeta domain is underlined, the MyD88 domain is in bold, and the linker sequence is in italics.

[0059] [Table 7]

[0060] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10. In some embodiments, the polynucleotides provided herein comprise the nucleic acid sequence set forth in SEQ ID NO: 10.

[0061] Another exemplary DNA sequence encoding a CD3 zeta domain fused to a myD88 domain is shown in SEQ ID NO:15, where the CD3 zeta domain is underlined and the MyD88 domain is shown in bold.

[0062] [Table 8] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 15. In some embodiments, the polynucleotides provided herein comprise the nucleic acid sequence set forth in SEQ ID NO: 15.

[0063] As will be appreciated by those skilled in the art, the polynucleotides described herein may include additional portions of a TCR or CAR. Alternatively, one skilled in the art could obtain a modified TCR or CAR sequence and further modify such receptors to add the MyD88 domain provided herein.

[0064] In some embodiments, the polynucleotide further comprises a T2A self-cleaving fragment.

[0065] In another aspect, provided herein is a polynucleotide comprising a nucleic acid sequence encoding a CD3ζ domain fused to a MyD88 domain. An exemplary nucleic acid sequence encoding the CD3ζ, MyD88 domain, and T2A domain is set forth in SEQ ID NO:7, where the nucleic acid encoding the CD3ζ domain is underlined and the nucleic acid encoding the MyD88 domain is in bold. The nucleic acid encoding the T2A self-cleaving fragment that may be included to facilitate transfection and expression of the modified TCR is in italics.

[0066] [Table 9]

[0067] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the polynucleotides provided herein comprise the amino acid sequence set forth in SEQ ID NO:7.

[0068] In another aspect, provided herein is a polynucleotide comprising a nucleic acid sequence encoding a CD8a domain linked to a MyD88 domain. An exemplary nucleic acid sequence encoding the CD8a domain, the MyD88 domain, and the linker is set forth in SEQ ID NO:8, where the nucleic acid encoding the CD8a domain is underlined, the nucleic acid encoding the MyD88 domain is in bold, and the nucleic acid encoding the G4S linker connecting the MyD88 and CD8a domains is in italics.

[0069] [Table 10]

[0070] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence set forth in SEQ ID NO:8.

[0071] An exemplary nucleic acid sequence encoding a modified TCR comprising a TCR alpha chain, a TCR beta chain, a CD3zeta intracellular domain (ICD), and a MyD88 DD is set forth in SEQ ID NO: 12, in which the TCR alpha chain is underlined, the TCR beta chain is bolded, the CD3zeta ICD is underlined and bolded, and the MyD88 DD is bolded and italicized. The linker sequence is italicized.

[0072] [Table 11]

[0073] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 12. In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence set forth in SEQ ID NO: 12. An exemplary DNA sequence encoding a CD3 zeta domain fused to a MyD88 domain is shown in SEQ ID NO: 14, with the CD3 zeta domain underlined and the MyD88 domain in bold.

[0074] [Table 12]

[0075] In another aspect, provided herein is a vector comprising the polynucleotide described herein. In some embodiments, the viral vector is a non-integrating non-chromosomal vector. Exemplary non-integrating non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes virus. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno-associated vector (AAV), lentivirus, and gamma retrovirus.

[0076] Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0077] The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (for example, two) long terminal repeats (LTR), and a transgene of interest, for example, a gene encoding a CAR. The gamma retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary gamma retroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., Viruses. 2011 Jun;3(6):677-713.

[0078] Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference, in which a suitable host cell is transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat.

[0079] cell In another aspect, provided herein are therapeutic cells comprising the modified receptors and fusion proteins described herein. Such cells can be used in adoptive cell therapy to treat a variety of diseases and disorders, including cancer.

[0080] In some embodiments, the cell is a modified T cell comprising a modified TCR as described herein. In some embodiments, the cell is a modified T cell comprising a fusion protein as described herein. In some embodiments, the cell is a tumor infiltrating lymphocyte (TIL).

[0081] In one aspect, provided herein is a cell comprising (a) a modified T cell receptor (TCR) comprising an α chain, a β chain, a CD3ζ intracellular domain, and a MyD88 polypeptide comprising a death domain (DD) of MyD88 and an intermediate domain (ID) of MyD88, wherein the MyD88 domain is fused to a CD3ζ subunit, or (b) a fusion protein comprising a CD3ζ intracellular domain and a MyD88 DD. In some embodiments, the MyD88 polypeptide comprises an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9. In some embodiments, other T cell receptor subunits are co-expressed with the fusion protein. In some embodiments, a CD3γ subunit, a CD3ζ subunit, and / or a CD3ε subunit are co-expressed with the fusion protein provided herein.

[0082] In another aspect, provided herein is a cell comprising: (a) a modified T cell receptor (TCR) comprising an alpha chain, a beta chain, a CD3 zeta intracellular domain, and a MyD88 polypeptide comprising an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2, wherein the MyD88 domain is fused to the CD3 zeta subunit; or (b) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9.

[0083] In some embodiments, the cell is an engineered immune cell comprising a modified TCR or fusion protein described herein. The immune cell may be a T cell (e.g., a regulatory T cell, a CD4 + T cells, CD8 + The cell may be a T cell, a gamma-delta T cell, a NK cell, an invariant NK cell, a NKT cell, a stem cell (e.g., a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPSC) cell), or a tumor infiltrating lymphocyte (TIL). In some embodiments, the cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0084] Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject of interest, for example, a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing therapy for a particular disease or condition. Immune cells can be enriched / purified from any tissue in which they are present, including, but not limited to, blood (including blood collected by a blood bank or umbilical cord blood bank), spleen, bone marrow, tissues removed and / or exposed during a surgical procedure, and tissues obtained via a biopsy procedure. The tissues / organs from which immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, with the non-living subject being an organ donor. The isolated immune cells can be used directly or they can be stored for a period of time, for example, by freezing. In some embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have enhanced immunomodulatory capabilities, as measured by CD4+ or CD8+ T cell suppression. In certain embodiments, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for enhanced immune-modulating capacity.Pooled blood can be from two or more sources, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0085] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells can be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor. The immune cell population can be taken from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, for example, from pooled umbilical cord blood. The population of immune cells can be derived from induced pluripotent stem cells (iPSCs) and / or any other stem cells known in the art. In some embodiments, the iPSCs and / or stem cells used to obtain the population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity, and thus, these iPSCs and / or stem cells will be autologous to the subject in need of therapy. Alternatively, the iPSCs and / or stem cells can be obtained from a donor and thus, allogeneic to the subject in need of therapy.

[0086] When the population of immune cells is obtained from a donor different from the subject, the donor is preferably allogeneic, provided that the obtained cells are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To make them subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0087] The cells can be modified to express the modified receptor or fusion protein described herein by any suitable method known in the art or described herein, for example, electroporation or lipofection. In some embodiments of the methods of the present disclosure, the introduction of the nucleic acid sequence and / or genome editing construct into the immune cells ex vivo, in vivo, in vitro, or in situ comprises a viral vector. In some embodiments of the methods of the present disclosure, the introduction of the nucleic acid sequence and / or genome editing construct into the immune cells ex vivo, in vivo, in vitro, or in situ comprises a combination of vectors. Exemplary non-limiting vector combinations include viral and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Exemplary but non-limiting vector combinations include DNA-derived and RNA-derived vector combinations, RNA and reverse transcriptase combinations, transposon and transposase combinations, non-viral vector and endonuclease combinations, and viral vector and endonuclease combinations.

[0088] In some embodiments of the methods of the present disclosure, the genome modification comprises introducing a nucleic acid sequence and / or a genome editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ to stably integrate the nucleic acid sequence, to transiently integrate the nucleic acid sequence, to generate site-specific integration of the nucleic acid sequence, or to generate biased integration of the nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0089] In some embodiments of the disclosed methods, the genome modification comprises introducing a nucleic acid sequence and / or a genome editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ, stably integrating the nucleic acid sequence. In some embodiments, the stable chromosomal integration can be random integration, site-specific integration, or biased integration. In some embodiments, the site-specific integration can be unassisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site-specific nuclease. In some embodiments, the site-specific nuclease comprises a transgene having 5' and 3' nucleotide sequence extensions that include a percentage of homology to the upstream and downstream regions of the genome integration site. In some embodiments, the transgene having the homologous nucleotide extensions allows for genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. In some embodiments, the site-specific integration is performed at a safe harbor site. Genomic safe harbor sites can accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective levels of expression) and does not introduce deleterious changes to the host genome that pose a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1), the naturally occurring AAV viral integration site on chromosome 19, the site of the chemokine (CC motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0090] In some embodiments, site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at an intron, exon, promoter, genetic element, enhancer, suppressor, start codon, stop codon, and response element. In some embodiments, exemplary target genes targeted by site-specific integration include, but are not limited to, any immunosuppressive gene and genes involved in allogeneic rejection.

[0091] In some embodiments, site-specific transgene integration occurs at a site that results in enhanced expression of the target gene, hi some embodiments, enhanced target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0092] In addition to viral delivery of nucleic acids encoding antigen receptors, the following are additional methods of recombinant gene delivery to a given cell (eg, NK cell) and thus are contemplated in this disclosure.

[0093] Introduction of nucleic acid such as DNA or RNA into immune cells of the present disclosure may use any suitable method for nucleic acid delivery for cell transformation, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection, by injection, including microinjection, by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome-mediated transfection and receptor-mediated transfection, by microprojectile bombardment, by stirring with silicon carbide fibers, by Agrobacterium-mediated transformation, by desiccation / inhibition-mediated DNA uptake, and any combination of such methods. Through the application of such techniques, organelles, cells, tissues, or organisms may be stably or transiently transformed.

[0094] In some embodiments of the method of the present disclosure, the introduction of the nucleic acid sequence and / or genome editing construct into the immune cell ex vivo, in vivo, in vitro, or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises a plasmid DNA, a linear double-stranded DNA (dsDNA), a linear single-stranded DNA (ssDNA), DoggyBone™ DNA, a nanoplasmid, a minicircle DNA, a single-stranded oligodeoxynucleotide (ssODN), a DDNA oligonucleotide, a single-stranded mRNA (ssRNA), and a double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the present disclosure.

[0095] In some embodiments of the disclosed methods, an enzyme can be used to create a strand break in the host genome to facilitate delivery or integration of the transgene. In some embodiments, the enzyme creates a single-strand break. In some embodiments, the enzyme creates a double-strand break. In some embodiments, examples of cleavage-inducing enzymes include, but are not limited to, transposases, integrases, endonucleases, meganucleases, megaTALs, CRISPR-Cas9, CRISPR-CasX, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs). In some embodiments, the cleavage-inducing enzymes can be DNA-encoded, mRNA-encoded, delivered to cells as proteins, or as nucleoprotein complexes with guide RNAs (gRNAs).

[0096] Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising the cells described herein. The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human, as appropriate. It will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards, such as those required by the FDA Office of Biological Standards.

[0097] As used herein, a "pharmaceutically acceptable carrier" refers to a pharmaceutical composition, such as a lyophilized formulation or an aqueous solution, as known to those skilled in the art (see Remington's Pharmaceutical Sciences 22 ndedition, 2012), any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutritional supplements, such as similar materials, and combinations thereof. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0098] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight compounds (about 10 Examples of suitable surfactants include, but are not limited to, polypeptides (less than 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharma- ceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0099] How to use The MyD88 domains (e.g., MyD88 domains of SEQ ID NO: 1 or 2) and fusion proteins (e.g., fusion proteins of SEQ ID NO: 3 or 9) provided herein can be used to improve existing cell therapies, as will be understood by those of skill in the art. For example, a MyD88 domain can be fused or linked to a CD3ζ domain present in a CAR or TCR, and the fusion protein can be introduced into an immune cell.

[0100] Exemplary engineered antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing receptors into cells are described in, for example, PCT Publication Nos. WO 2000 / 14257, WO 2013126726, WO 2012 / 129514, WO 2014 / 031687, WO 2013 / 166321, WO 2013 / 071154, and WO 2013 / 123061; U.S. Patent Application Publication Nos. 2002 / 131960, 2013 / 287748, and 2013 / 0149337; and U.S. Patent Nos. 6,451,921, 6,451,925, and 6,512,726. 95, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,190, 7,446,191, 8,324,353, and 8,479,118; International Patent Application Publication No. 2014 / 055668(A1), and European Patent Application Publication No. 2 537 416, and / or Sadelain. et al., 2013, Davila et al., 2013, Turtle et al., 2012, and Wu et al., 2012.

[0101] Thus, in one aspect, provided herein is a method of improving a therapeutic cell or cell therapy, comprising transfecting a therapeutic cell with a modified receptor as described herein. In another aspect, provided herein is a method of improving a therapeutic cell or cell therapy, comprising transfecting a therapeutic cell with a fusion protein as described herein.

[0102] In some embodiments, the method of improving therapeutic cells results in increased expansion of therapeutic cells in vivo as determined by an increase in cell number over time. In some embodiments, the method of improving therapeutic cells results in an increase in expansion of therapeutic cells in vivo of at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the expansion of therapeutic cells prior to improvement or compared to therapeutic cells not comprising a modified TCR or fusion protein as described herein. Increased expansion can be determined, for example, by measuring the amount of cells per blood volume at a particular time point following administration of the cells.

[0103] In some embodiments, the method of improving a therapeutic cell results in increased cytokine production by the therapeutic cell. Immune cells are involved in the expression of interferon gamma (INFγ), CC motif chemokine ligand 11 (CCL-11), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), granzyme 9, interleukin (IL)-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-1b, IL-2, IL-21, IL-22, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, Monocyte Chemoattractant Protein (MCP)-1, MCP-4, Macrophage Inflammatory Protein (MIP)-1α, MIP-1β, perforin, and Regulated Upon Inflammation. The therapeutic cells produce a variety of cytokines, including Activation,Normal T Cell Expressed And Presumably Secreted (RANTES), Transforming Growth Factor (TGF)-β1, Tumor Necrosis Factor (TNF)-α, TNF-β, soluble Cluster of Differentiation (sCD) 137, and sCD40L. In some embodiments, the methods result in an increase in cytokine production in the therapeutic cell population of at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the therapeutic cell population prior to the improvement or compared to a therapeutic cell that does not comprise a modified TCR or fusion protein described herein.Cytokine production can be determined by measuring cytokine levels in the cell culture medium, for example by using ELISA or Western blotting.

[0104] In some embodiments, the methods of improving a therapeutic cell described herein result in the immune cell becoming multifunctional and expressing two or more cytokines. In some embodiments, the methods of improving a therapeutic cell result in an increase in the number of cytokine products by the cell. In some embodiments, the methods result in the production of at least one, at least two, at least three, at least four, or at least five additional cytokines produced by the cell compared to cytokine production by the therapeutic cell prior to improvement, or compared to cytokine production by a therapeutic cell that does not include a modified TCR or fusion protein described herein.

[0105] In some embodiments, the methods produced herein can be used to improve a therapeutic cell population, which results in a higher percentage of multifunctional cells in the therapeutic cell population compared to the cell population prior to improvement, or compared to a therapeutic cell population that does not include a modified TCR or fusion protein as described herein. In some embodiments, the methods result in an increase in the percentage of multifunctional cells in a therapeutic cell population of at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the therapeutic cell population prior to improvement, or compared to a therapeutic cell population that does not include a modified TCR or fusion protein as described herein.

[0106] In some embodiments, the method of improving therapeutic cells results in a reduction in exhaustion of therapeutic cells in vivo. Immune cell exhaustion is a progressive loss of cell function in vivo. Immune cell exhaustion is not immediately associated with a reduction in cell number, but rather is measured by a reduction in the expression of certain cytokines and a reduction in the capacity of cell effector functions. Molecular markers of cell exhaustion are known in the art, including, for example, elevated expression of inhibitory receptors (IR), little or no proliferation, high expression of PD-1, TIM3, and LAG3 inhibitory receptors, and loss of IL-2 (early) and TNF (late) production. Exhausted T cells may retain the ability to produce chemokines, including Mip1α, Mip1β, Rantes, and IL-100. Altered epigenetic, metabolic, and transcriptional profiles have also been described. See, e.g., McLane et al., Annu. Rev. Immunol. 2019.37:457-95).

[0107] In some embodiments, the methods result in a reduction in the percentage of exhausted cells in a therapeutic cell population by at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the therapeutic cell population before the improvement or compared to a therapeutic cell population that does not comprise a modified TCR or fusion protein described herein.

[0108] In some embodiments, the method of improving a therapeutic cell increases the proliferation capacity of the therapeutic cell. Increasing proliferation capacity can mean, for example, that the cell continues to proliferate even after multiple simulations. In some embodiments, the method results in an increase in the proliferation capacity of the therapeutic cell of at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the therapeutic cell prior to improvement or compared to a therapeutic cell that does not include a modified TCR or fusion protein as described herein.

[0109] In some embodiments, the method of improving therapeutic cells results in improved persistence of the therapeutic cells, e.g., persistence in the blood of a subject to which the cells are administered. Cell persistence can be measured by monitoring the number of cells in the subject's blood, e.g., using a flow cytometric hemocytometer count. In some embodiments, the method results in an increase in persistence of the therapeutic cells in the blood of a subject to which the cells are administered of at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the therapeutic cells prior to improvement or compared to therapeutic cells that do not comprise a modified TCR or fusion protein as described herein.

[0110] In some embodiments, the method of improving therapeutic cells results in beneficial changes in the tumor microenvironment. Examples of beneficial changes in the tumor environment are known in the art, including, for example, reduced hypoxia and reduced angiogenesis. See, for example, Benavente et al., Front. Oncol., 23 October 2020, which is incorporated herein by reference in its entirety.

[0111] Preferably, the method of improving therapeutic cells and therapeutic cell populations does not substantially affect the antigen specificity of the receptor contained in the cell. Thus, in a preferred embodiment, the method of improving therapeutic cells described herein results in improved cells having substantially the same antigen specificity. By "substantially the same antigen specificity" is meant that the type of antigen recognized by the receptor does not change, and the affinity of the receptor for the antigen is reduced by up to 10% compared to the receptor of the cell before improvement.

[0112] The cells and pharmaceutical compositions described herein can be used to treat a disorder or disease in a subject in need of such treatment. Thus, in another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject a cell comprising a modified receptor or fusion protein provided herein. In some embodiments, the disorder or disease is cancer.

[0113] Tumors for which the therapeutic methods of the present invention are useful include those found in any malignant cell type, such as solid tumors or hematological tumors. In some embodiments, the cancer is a CD22-positive cancer. In some embodiments, the cancer has low expression of CD22 (e.g., CD22-low cancer). In some embodiments, the cancer is a CD19-positive cancer. In some embodiments, the cancer has low expression of CD19 (e.g., CD19-low cancer).

[0114] Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include, but are not limited to, tumors of the bone marrow, T-cell or B-cell malignancies, myeloid malignancies, leukemia, lymphoma, blastoma, myeloma. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0115] The cancer may be of the following histological types, among others, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; alveolar epithelial adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma;eosinophilic carcinoma;eosinophilic adenocarcinoma;basophilic carcinoma;clear cell adenocarcinoma;granular cell carcinoma;follicular adenocarcinoma;papillary and follicular adenocarcinoma;nonencapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma;adnexal carcinoma;apocrine gland carcinoma;sebaceous gland adenocarcinoma;ceruminous gland carcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease of the breast;acinar cell carcinoma;adenosquamous carcinoma;squamous metaplasia adenocarcinoma;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granular cell tumor, malignant;male germinoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;angiocytoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;lentiginous malignant melanoma;acral lentiginous melanoma;nodular melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal Rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant malignant; lymphoma; T-lymphoblastic leukemia; T-lymphoblastic lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; lymphoma, small lymphocytic; lymphoma, large cell, diffuse; lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma lymphoma, NHL);small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade diffuse NHL;high-grade immunoblastic NHL;high-grade lymphoblastic NHL;high-grade small non-cleaved cell NHL;bulky disease NHL;mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphosarcoma cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;hairy cell leukemia;chronic lymphocytic leukemia chronic myeloid leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative tumor; chronic myeloid leukemia; diffuse large B-cell lymphoma peripheral T-cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL). ;

[0116] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by the abnormal proliferation (production by expansion) of blood cells, usually immature white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that encompasses a variety of diseases. Leukemia is clinically and pathologically divided into its acute and chronic forms and / or by cell type of origin (myeloid or lymphoid). In some embodiments, the leukemia is a low antigenic leukemia. In some embodiments, the leukemia is a low CD22 leukemia.

[0117] In some embodiments, the cancer is breast cancer, sarcoma, melanoma, or lung cancer. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject treated according to the methods described herein is a human patient, e.g., a human adult.

[0118] In some embodiments, cells are obtained from a patient, modified and expanded ex vivo, and re-infused into the patient. The cells can be allogeneic or autologous to the patient.

[0119] One of skill in the art would be able to determine appropriate treatment durations and dosages of cells containing the modified receptors or fusion proteins provided herein and / or pharmaceutical compositions provided herein.

[0120] The cells comprising the modified receptors or fusion proteins provided herein, or the pharmaceutical compositions provided herein, may be administered by any suitable route of administration, including, for example, intravenous, intrathecal, intraocular, subcutaneous, intraperitoneal, intramuscular, intracerebral, intraventricular, or intratracheal administration. In a preferred embodiment, the cells comprising the modified receptors or fusion proteins provided herein, or the pharmaceutical compositions provided herein, are administered intravenously.

[0121] In certain embodiments of the present disclosure, immune cells are delivered to an individual who needs immune cell delivery, for example, an individual with cancer or infectious disease. The cells then boost the individual's immune system to attack or directly attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with one or more doses of immune cells. When the individual is provided with two or more doses of immune cells, the period between administrations should be sufficient to allow time for reproduction in the individual, and in certain embodiments, the period between administrations is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more.

[0122] In some embodiments, subjects treated according to the methods described herein do not undergo lymphodepletion prior to administration of T cells. In some embodiments, subjects treated according to the methods described herein do not receive cytokines while receiving cells.

[0123] The immune cells may be administered in combination with one or more other therapeutic agents for the treatment of immune-mediated disorders. The immune cells may be administered before, during, after, or in various combinations with additional cancer therapies such as radiation therapy, chemotherapy, or immunotherapy (e.g., immune checkpoint therapy). Administration may be at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments in which the immune cell therapy is provided to the patient separately from the additional therapeutic agent, one will generally ensure that no significant period of time passes between the time of each delivery so that the two compounds can still exert their advantageously combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 hours or 72 hours of each other, more specifically, within about 6-12 hours of each other. In some situations, it may be desirable to significantly extend the duration of the treatment, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0124] Various combinations can be used. In the example below, the immune cell therapy is "A" and the anti-cancer therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0125] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0126] The cells comprising the modified receptors or fusion proteins provided herein, or the pharmaceutical compositions provided herein, can be administered for any suitable period of time, for example, until symptoms improve or for a predetermined period of time, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks.

[0127] The effectiveness of cancer treatment can be assessed by any suitable method known in the art or described herein, including, for example, monitoring tumor growth or measuring the time to tumor recurrence. In some embodiments, the treatment methods described herein induce tumor regression. In some embodiments, the tumor regresses by at least 10%, at least 20%, at least 30% within about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, or about 12 months after the first administration of the bispecific binding agent or functional fragment thereof, or pharmaceutical composition.

[0128] In some embodiments, the treatment methods described herein result in regression of the tumor to undetectable and delay tumor recurrence, hi some embodiments, tumor recurrence is delayed for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years, or at least 5 years after the tumor becomes undetectable.

[0129] The efficacy of the treatment methods described herein can be evaluated in comparison to untreated subjects with a comparable diagnosis, or subjects with a comparable diagnosis receiving standard treatment therapy. In some embodiments, the efficacy of the methods described herein is compared to subjects treated according to the methods described herein prior to the first administration.

[0130] kit Also provided herein are articles of manufacture or kits that include the modified receptor, fusion protein, or immune cells. The articles of manufacture or kits may further include a package insert that includes instructions for using the immune cells to treat or delay the progression of cancer in an individual, or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the articles of manufacture or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The articles of manufacture or kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts that include instructions for use. In some embodiments, the articles of manufacture further include one or more of another agent (e.g., chemotherapeutic agents, and anti-neoplastic agents). Suitable containers for one or more pharmaceutical agents include, for example, bottles, vials, bags, and syringes. EXAMPLES

[0131] The examples in this section are provided for illustrative purposes only and are not intended to limit the invention.

[0132] Example 1: Expression of TCR:CD3z:MyD88 constructs in human T cells Human T cells collected from peripheral blood mononuclear cells (PBMCs) were activated for 48-72 hours using soluble anti-CD3 (OKT3 clone 50ng / ml / 5E6 cells) and IL-2 (100U / ml). Cells were genetically engineered using gamma retroviral particles expressing each of the indicated DMF5 TCR variants. MHC I-MART1(27-35) pentamers were used to detect expression of DMF5 TCR approximately 72 hours after transduction using flow cytometry. Results are shown in Table 2. Wild type, DMF5 alpha:MyD88 and DMF5 beta:MyD88 were expressed on the surface of T cells. DMF5 TCRs containing MyD88 in both the alpha and beta TCR chains were weakly detected on the surface of T cells. Rhe MHC I-MART1(27-35) pentamer bound to TCR, demonstrating that the TCR retained specificity for peptide antigens and MHC I molecules.

[0133] Example 2: T cells engineered with DMF5 CD3z:MyD88 appended to the TCR beta or TCR alpha chain increase effector cytokine expression by intracellular staining. In this experiment, it was determined whether appending the intracellular domain of CD3z with or without MyD88 to the TCR was functional and improved IL-2 secretion in Jurkat T cells. Jurkat cells were engineered to express the DMF5 TCR:CD3z:MyD88 construct using a gamma-retroviral vector. Transduced Jurkat cells were stimulated with increasing concentrations of plate-bound anti-CD3 antibodies or decreasing numbers of human melanoma HLA-A2+ A375 cells expressing MART-1(27-35) for 20 hours, at which time supernatants were collected and tested for the presence of IL-2 by ELISA. A375 cells were incubated with the indicated concentrations of peptide. The results are shown in Figure 3A, where each point represents the mean of three technical replicates, with error bars = 1 standard deviation. * indicates significant differences (p<0.05) between the DMF5α:CD3z:MyD88 and DMF5β:CD3z:MyD88 groups with wild-type DMF5 but not with each other, ** indicates significant differences (p<0.05) between each other, the α:CD3z:MyD88 group, the β:CD3z:MyD88 group, and wild type by one-way ANOVA.

[0134] 624 melanoma cells (naturally expressing HLA-A2+MART1+) were sorted based on various levels of HLA-A2 expression and then used to stimulate engineered Jurkat cells as described above. The results are shown in Figure 3B, where each point represents one technical replicate, each bar represents the mean of the replicates, and error bars = 1 standard deviation. p values ​​are as indicated on the graph and were determined by one-way ANOVA.

[0135] Jurkat T cells engineered with DMF5 TCR containing the CD3z:MyD88 construct performed better than wild-type TCR. (See FIG. 3A). Jurkat T cells engineered with all DMF5 TCR variants recognized antigen in a concentration-dependent manner when stimulated with A375 melanoma cells. (Right panel of FIG. 3A). Jurkat cells expressing TCR:CD3z:MyD88 variants outperformed WT TCR when stimulated with A375 cells. (Right panel of FIG. 3A). Jurkat T cells engineered with DMF5 TCR containing alpha or beta CD3z:MyD88 outperformed WT TCR. However, DMF5 beta:CD3z:MyD88 appeared to outperform DMF5 alpha:CD3z:MyD88 when stimulated with tumor cells expressing low, medium, or high MHC I levels.

[0136] Example 3: T cells engineered with TCRB:CD3z:MyD88 show enhanced responses and are more polyfunctional than cells engineered with wild-type TCR or control TCR:CD3z. The purpose of this experiment was to determine whether adding the intracellular domain of CD3z with or without MyD88 to the TCR altered polyfunctionality based on cytokine production. T cells engineered with the indicated TCR constructs were preincubated with Golgi stop for 1 hour and stimulated with A375 melanoma cells pulsed with either no peptide antigen or 10ug / mL MART-1(27-35). After 2 hours of co-culture, stimulated T cells were harvested and stained for intracellular cytokines TNFα, IFNγ, IL-2, and GM-CSF. The results are shown in Figure 4A and Figure 4B. In the absence of stimulation, none of the engineered T cells produced effector molecules. This indicates that MHC antigen is required to initiate the TCR response and therefore the TCR is not overtly activated (left column of Figure 4A and Figure 4B, respectively). CD3z and MyD88 improved cytokine production in T cells when added to the B chain of the TCR, but CD3z alone did not. A higher percentage of TCRB:CD3z:MyD88 engineered T cells demonstrated the ability to produce at least two cytokines in response to stimulation with melanoma cells than the control group: more TCRB:CD3z:MyD88 T cells produced GM-CSF than control T cells (Figure 4B).

[0137] Example 4: T cells engineered with TCRB:CD3z:MyD88 show enhanced cytokine production over cells engineered with wild-type TCR, TCRA:CD3z:MyD88, or TCR:CD3z. T cells extracted from human PBMCs and engineered with various DMF5 TCR constructs were stimulated with A375 melanoma cells pulsed with increasing concentrations of MART-1(27-35) peptide. After 6 hours, supernatants were collected and levels of TNFa, IFNg, or IL-2 were determined by ELISA. Figure 5A shows a comparison between DMF5 WT, TCRB:CD3z, and TCRB:CD3z:MyD88, and Figure 5B shows a comparison between DMF5 WT, TCRB:CD3z, and TCRA:CD3z:MyD88, and TCRB:CD3z:MyD88. Each dot represents the mean of three technical replicates, error bars = 1 standard deviation.

[0138] TCRB:CD3z:MyD88 engineered T cells showed an enhanced ability to produce cytokines over control T cells. Cytokine secretion occurred in an antigen concentration-dependent manner. In the absence of antigen, T cells did not produce cytokines, highlighting that the engineered TCR retained the requirement to recognize MHC antigens. TCRB:CD3z:MyD88 were more responsive than control WT or TCR:CD3z based on their ability to be stimulated by approximately 10-fold less antigen. Figure 5A shows that the levels of cytokines produced by TCRB:CD3z:MyD88 T cells continued to rise in response to elevated levels of antigen stimulation. In sharp contrast, the ability of control T cells to produce cytokines plateaued at approximately 0.1 μg / ml of antigen. This suggests that TCRB:CD3z:MyD88 overrides the natural ability of T cells to suppress cytokine responses.

[0139] In summary, the ability of T cells to respond to suboptimal antigen levels is a beneficial feature because cancer cells can evade T cell detection by reducing the expression levels of antigen or MHC I, or by expressing weakly immunogenic antigens. The ability of T cells to produce higher levels of effector molecules is a beneficial feature because T cells eventually become exhausted and reduce or stop cytokine production.

[0140] Example 5: TCRB:CD3z:MyD88 confers improved sustained expansion of T cells over repeated stimulation. T cells were engineered with the indicated DMF5 TCR and stimulated with HLA-A2+MART-1+624mel cells (2 tumors: 1 T cell) for 7 days, at which point they were collected, counted, and restimulated. The results are shown in Figures 6A-C. TCRB:CD3z:MyD88 T cells expanded at a faster rate than control T cells. Control T cell expansion plateaued after one stimulation and reached terminal exhaustion (loss of ability to persist) after two stimulations. TCRB:CD3z:MyD88 conferred upon T cells the ability to expand over repeated stimulations.

[0141] Example 6: TCRB:CD3z:MyD88 confers sustained antitumor activity and effector molecule production to T cells. T cells from three donors were engineered with the indicated DMF5 TCR or DMF5 TCR with CD3z:MyD88 appended to the TCR beta chain. T cells were stimulated with HLA-A2+MART-1+624mel cells (2 tumors: 1 T cell) every 7 days, at which time their ability to kill melanoma cells was determined by flow cytometry. Their ability to produce IFNg and GM-CSF was also assessed every 7 days by ELISA. Data are shown in Figure 7. TCRB:CD3z:MyD88 T cells maintained their ability to kill cancer cells for at least 5 stimulations compared to control T cells. TCRB:CD3z:MyD88 T cells sustained their ability to produce IFNγ over several stimulations with cancer cells. Furthermore, TCRB:CD3z:MyD88 conferred on T cells the ability to improve cytokine production as well as sustain GM-CSF production over repeated stimulations.

[0142] Example 7: CD3z:MyD88 enhances effector molecules and promotes the development of polyfunctional T cells. Tumor infiltrating lymphocytes (TILs) were either engineered with control CD3z or CD3z:MyD88, or left unmodified. TILs were then stimulated with plate-bound CD3 antibody (clone OKT3) for 6 hours, at which time intracellular levels of cytokines were determined by flow cytometry. Results are shown in FIG. 8A and show that intracellular interferon gamma (INFγ) increased in a dose-dependent manner. Based on the increased levels of polyfunctional TILs (those producing two or more cytokines), Isoplexus (measurement of the indicated cytokines on a per-cell basis) was used to assess the functional ability of each individual T cell to co-produce multiple cytokines. Results are shown in FIG. 8B and FIG. 8C and show that both CD3z:MyD88 increased the levels of effector molecules and promoted the development of polyfunctional T cells.

[0143] Example 8: CD3z:MyD88 confers a strong proliferative advantage to TILs. TILs from breast cancer or sarcoma patients were extracted and either engineered with control CD3z or CD3z:MyD88 or left unmodified. TILs were cultured in the presence of anti-CD3, anti-CD28, and anti-4-1BB antibodies (1:20 bead:TIL ratio) and beads coated with IL-2 (500 U / ml) and restimulated every 15 days. Cells were counted every 5-10 days (as shown in Figure 9A and Figure 9F). Results for breast cancer TILs (Figure 9A and Figure 9C), sarcoma TILs (Figure 9B and Figure 9D), and melanoma TILs (Figure 9E and Figure 9F) showed that TILs transfected with CD3z:MyD88 persisted for up to 100 days (somewhat longer), an unexpectedly long survival compared to control TILs or unmodified TILs, which died within a few weeks. Furthermore, the rate of cell expansion was more rapid for CD3z:MyD88 TILs compared to CD3z control and non-transfected TILs. This data demonstrates a remarkable ability to maintain TILs over long periods of time, which may confer a survival advantage to TILs in living hosts.

[0144] Example 9: TCR diversity and repertoire is maintained in CD3z- and CD3z:MyD88-engineered TILs. TILs from melanoma, sarcoma, breast cancer, and lung cancer samples were extracted and engineered with control CD3z or CD3z:MyD88, or left unmodified (before transduction). The diversity of the TCR variable (V)β repertoire was determined in pre-engineered TILs (before expansion) or after 15 days of expansion (with coated antibody beads as described in Example 2) and IL-2, and assessed by flow cytometry. The results are shown in Figure 10, where each population represents one TCR Vβ family member. These data indicate that TCR diversity and repertoire are maintained in CD3z and CD3z:MyD88 engineered TILs.

[0145] Example 10: CD3z:MyD88 sustains T cell polyfunctionality over long periods of time. T cells were engineered to express control CD3z or CD3z:MyD88. T cells were cultured in the presence of anti-CD3, anti-CD28, and anti-4-1BB antibodies (1:20 bead:TIL ratio) and beads coated with IL-2 (500 U / ml) and restimulated every 15 days. Just prior to expansion (days 15, 30, and 45), T cells were stimulated with plate-bound anti-CD3 antibody (clone OKT3) for 6 hours, at which point intracellular levels of cytokines were determined by flow cytometry. The results of IFNγ expression over time are shown in Figure 11A, and the percentage of T cells capable of co-producing various effector molecules at different time points is shown in Figure 11B. Most T cells from the control group were exhausted by day 30. In sharp contrast, CD3z:MyD88 T cells not only retained their ability to produce at least one cytokine, but most remained polyfunctional.

[0146] Example 11: CD3z:MyD88 enhances T cell activity (based on IFNg production). TILs from the indicated tumor types were left unmanipulated or manipulated to express control CD3z or CD3z:MyD88. T cells were stimulated with the indicated concentrations of plate-bound anti-CD3 antibody (clone OKT3) for 6 hours, at which time cytokine levels were determined by ELISA.

[0147] Results for breast cancer (FIG. 12A), sarcoma (FIG. 12B), and melanoma (FIG. 12C and FIG. 12D) samples are shown in FIG. 12A-D. CD3z:MyD88 amplified TCR signals across different TCR stimulation strengths, resulting in enhanced effector molecule production compared to CD3z and untransfected controls.

[0148] Example 12: CD3z:MyD88 confers a proliferative advantage to T cells. T cells from healthy donors were engineered with control CD3e, CD3z, CD3e:MyD88, or CD3z:MyD88. T cells were stained with proliferation dyes and stimulated with anti-CD3 (OKT3 clone) and IL 2 (100 U / ml) and proliferation was assessed after 5 days. Results are shown in Figures 13A and 13B.

[0149] CD3z:MyD88 conferred a proliferation advantage to both CD4 and CD8 T cells compared with controls, however, CD3ε:MyD88 did not appear to provide enhanced proliferation over CD3ε alone.

[0150] Example 13: CD3z:MyD88 T cells provide potent antitumor effects against established melanoma tumors. Transgenic mice reactive to antigens contained within the gp100 protein were left unmanipulated or engineered to express mCD3z or mCD3z:MyD88. Three days after transduction, T cells were stimulated with B6 splenocytes pulsed with various concentrations of gp100 antigen or with B16 tumor cells, and IFNg production was assessed by ELISA approximately 12 hours later. Results are shown in FIG. 14A. TNFa and IFNg production and percentages were determined by intracellular FACS after 6 hours of stimulation with antigen-pulsed splenocytes. Results are shown in FIG. 14B and FIG. 14C. These data indicate that CD3z:MyD88 T cells provide a potent antitumor effect against established melanoma tumors.

[0151] Example 14: CD3z:MyD88 T cells provide potent antitumor effects against established melanoma tumors. Mice were subcutaneously injected with B16 tumor cells (approximately 200,000 cells). Once tumors were palpable, mice were preconditioned with 400 cGy radiation and injected with vehicle control (PBS) or tumor-specific T cells engineered to express control CD3z or CD3z:MyD88 2 days (3E6 cells) and 5 days (4.8E6 cells) after radiation. IL-2 (10KU) was delivered via intraperitoneal injection on the day of the first T cell infusion and 2 days after the first T cell infusion. Tumor growth was monitored using electronic calipers.

[0152] Results from one representative experiment of three are shown in Figure 15A (n=4-5 mice / group). Figure 15B shows the average results for each group, and Figure 17C shows the percentage of surviving mice (statistical significance in Figure 15C was determined by a mixed-effects ANOVA model with Geisser Greenhouse correction and Tukey's multiple comparison test). *** p<0.001).

[0153] These data show that TILs expressing CD3z:MyD88 are able to prevent tumor growth over the period studied (50 days), which is in stark contrast to TILs expressing control CD3z.

[0154] Because immunotherapy can be associated with severe immune-related adverse events, including cachexia, a hallmark of this in mice and humans, weight loss was monitored in all treatment groups.

[0155] Data for three different experiments are presented in Figures 16A-C. These results show that the rate of weight regain in tumor-bearing mice after treatment was comparable between groups in three different experiments.

[0156] Example 15: CD3z:MyD88 awakens neoantigen-reactive TILs. CD8 TILs were extracted from B16 F10 tumors to express CD3z or CD3z:MyD88. Dendritic cells were engineered to express plasmids containing tandem mini genes (TMG) expressing 10 neo-antigens each. DCs were co-cultured with engineered TILs after about 24 hours. Figure 17A shows a schematic of the experimental setup. The levels of IFNg were determined by ELISA, and the number of IFNg-producing CD8 T cells as well as the use of TCRvB family were determined by flow cytometry. The results are shown in Figures 17B and 17C. These data indicate that CD3z:MyD88 can awaken neo-antigen-reactive TILs.

Claims

1. A cell, (a) a modified T cell receptor (TCR) comprising an α chain, a β chain, a CD3ζ intracellular domain, and a MyD88 polypeptide comprising an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2, wherein the MyD88 polypeptide is fused to the CD3ζ subunit; or (b) a cell comprising a fusion protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9.

2. The cell of claim 1 , wherein the modified TCR comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

11.

3. The cell of claim 1 or 2, wherein the cell is an immune cell.

4. The cell according to any one of claims 1 to 3, wherein the immune cell is a T cell or a tumor infiltrating lymphocyte (TIL).

5. A pharmaceutical composition comprising the cell according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. 13. A method of treating cancer in a subject in need thereof, comprising administering to the subject a cell according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5.

7. 7. The method of claim 6, wherein the cancer is breast cancer, sarcoma, melanoma, or lung cancer.

8. A method for improving a therapeutic cell, comprising modifying a receptor of said therapeutic cell to fuse a MyD88 domain to a CD3ζ domain present in said receptor of said therapeutic cell.

9. The method of claim 8, wherein the MyD88 domain comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1 or 2.

10. 1. A method for improving a therapeutic cell, comprising introducing into said cell a fusion protein comprising a CD3 ζ intracellular domain and MyD88 DD.

11. The method of claim 10, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 or 9.

12. The method according to any one of claims 8 to 11, wherein the cell is an immune cell.

13. The method of any one of claims 8 to 11, wherein the cells are derived from a patient.

14. 14. The method of any one of claims 8 to 13, wherein the method results in an increase in expansion of the therapeutic cells in vivo of at least 50% compared to the therapeutic cells before the improvement.

15. The method of any one of claims 8 to 13, wherein the method results in an increase in cytokine expression in the therapeutic cells in vivo of at least 50% compared to the therapeutic cells before improvement.

16. The method of any one of claims 8 to 13, wherein the method results in a reduction in exhaustion of the therapeutic cells in vivo of at least 20% compared to the therapeutic cells before the improvement.

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