Chimeric antigen receptors comprising BCMA nanoantibodies linked to chimeric intracellular signaling domains

Abstract

The present application relates to an improved function third generation BCMA-CAR comprising a modified intracellular co-stimulatory domain, which can be used in adoptive cell therapy, for example in the treatment of diseases and conditions such as cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 358,399, filed on July 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence listing (NEOM_005_001WO_SeqList_ST26.xml; size 38,729 bytes; and creation date: July 5, 2023, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides a third generation chimeric antigen receptor (CAR) combined with B cell maturation antigen (BCMA). Such BCMA-CAR effectively induces T cell activation and proliferation, and provides a cytotoxic effect that responds to target cell enhancement.

[0006] background

[0007] Although adoptive cell therapy has shown efficacy in cancer treatment, the effectiveness of these therapies can be further improved by genetic modification of T cells for better expansion and persistence. In the development of genetically modified T cells for such adoptive cell therapy, it is necessary to introduce chimeric T cell co-stimulatory molecules that can be locally activated when T cells engage pathological antigens to effectively enhance T cell activation and increase therapeutic efficacy. The second generation of chimeric co-stimulatory molecules that incorporate a co-stimulatory signaling domain of a protein from the CD28 family or the TNFR family may not be optimal for the induction of durable tumor remission. Combining two co-stimulatory signaling domains from members of the CD28 family and the TNFR family to further enhance the therapeutic potential of T cells, the third generation of chimeric molecules that utilize the non-overlapping functions of the two co-stimulatory molecule families often suffers from reduced cell surface expression of the chimeric protein combining the two co-stimulatory signaling domains, which hinders the realization of the functional potential of the chimeric protein.

[0008] The present application discloses a chimeric antigen receptor (CAR) (BCMA-CAR) bound to a B cell maturation antigen (BCMA), comprising a BCMA nanobody and a third-generation chimeric T cell co-stimulatory molecule, the co-stimulatory molecule being incorporated with a CD28 family signaling domain and a TNFR family signaling domain for enhanced T cell function. These enhanced functions include enhanced proliferation, target cell killing, and reduced adverse events associated with the current leading clinical CAR. The present application discloses exemplary BCMA-CAR proteins for treating cancers including but not limited to multiple myeloma, as well as methods for preparing and using these BCMA-CARs.

[0009] Overview

[0010] The present disclosure provides a chimeric antigen receptor (BCMA-CAR) that binds to a B cell maturation antigen, comprising: (a) an extracellular domain comprising an amino acid sequence according to SEQ ID NO: 8; (b) a transmembrane domain of ICOS; and (c) a chimeric intracellular domain comprising a first signal transduction domain, a second signal transduction domain, and at least a third signal transduction domain, wherein the first signal transduction domain comprises an ICOS intracellular domain, the second signal transduction domain comprises a truncated CD137 (4-1BB) intracellular domain, and the at least third signal transduction domain comprises a truncated CD3 zeta domain.

[0011] In some embodiments, the BCMA-CAR comprises: (a) an extracellular domain comprising an amino acid sequence according to SEQ ID NO: 8; (b) a transmembrane domain of ICOS; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and a third signaling domain, wherein the first signaling domain comprises the ICOS intracellular domain, the second signaling domain comprises a truncated CD137 (4-1BB) intracellular domain, and the third signaling domain comprises a truncated CD3 zeta domain.

[0012] The present disclosure provides a BCMA-CAR comprising: (a) an extracellular domain comprising an amino acid sequence according to SEQ ID NO: 1; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and at least a third signaling domain, wherein the first signaling domain comprises an ICOS intracellular domain according to SEQ ID NO: 2, the second signaling domain comprises a truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the at least third signaling domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0013] In some embodiments, the BCMA-CAR comprises: (a) an extracellular domain comprising an amino acid sequence according to SEQ ID NO: 1; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and a third signaling domain, wherein the first signaling domain comprises an ICOS intracellular domain according to SEQ ID NO: 2, the second signaling domain comprises a truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the third signaling domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0014] In some embodiments, the extracellular domain further comprises a signal peptide, a hinge, or an ICOS extracellular stalk, or a combination thereof. In some embodiments, the extracellular domain further comprises a CD8a signal peptide, a CD8a hinge, an ICOS extracellular stalk, or a combination thereof. In some embodiments, the extracellular domain further comprises a CD8a signal peptide according to SEQ ID NO: 6, a CD8a hinge according to SEQ ID NO: 7, an ICOS extracellular stalk according to SEQ ID NO: 9, or a combination thereof.

[0015] In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO:1.

[0016] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:10.

[0017] The present disclosure also provides a BCMA-CAR comprising the amino acid sequence of SEQ ID NO: 5. The present disclosure also provides a nucleic acid encoding the BCMA-CAR of the present disclosure. The present disclosure also provides a vector comprising a nucleic acid of the present disclosure. The present disclosure also provides a cell comprising a nucleic acid or vector of the present disclosure. Compositions, such as pharmaceutical compositions, comprising BCMA-CAR, nucleic acids, vectors and / or cells of the present disclosure are also provided herein.

[0018] The present disclosure also provides a modified T cell comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR; and (b) a BCMA-CAR disclosed herein.

[0019] The present disclosure also provides a method for producing a variety of modified T cells, wherein the method comprises: a) providing a variety of primary T cells; b) providing a composition comprising the BCMA-CAR of the present disclosure, the nucleic acid of the present disclosure, or the vector of the present disclosure; and c) introducing the composition of (b) into the variety of primary T cells of (a) to produce a variety of modified T cells under the condition that BCMA-CAR is stably expressed in the variety of modified T cells.

[0020] The present disclosure also provides a composition comprising any one of the BCMA-CAR of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, or the modified T cell of the present disclosure.

[0021] The present disclosure also provides methods for treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective number of cells of the present disclosure, a therapeutically effective number of modified T cells of the present disclosure, a therapeutically effective amount of a composition of the present disclosure, or a therapeutically effective number of a plurality of modified T cells produced by the methods of the present disclosure.

[0022] In some embodiments, the disease or condition is cancer, autoimmune disease or condition, or inflammatory disease. In some embodiments, cancer is a blood cancer. In some embodiments, blood cancer is leukemia, lymphoma or myeloma. In some embodiments, cancer is selected from acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cells, T cells or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia (acute myelogenous leukemia), chronic myeloid leukemia (chronic myelocytic leukemia, CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma and multiple myeloma. In some embodiments, cancer is multiple myeloma. In some embodiments, cancer expresses BCMA. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a person. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A schematic diagram depicting the structure and orientation of the extracellular domain, transmembrane domain, and intracellular signaling domain of an exemplary BCMA-CAR disclosed herein ("NPB5005-ICOSBBtZt", also referred to as "Receptor 1") is shown. SP: signal peptide, BCMA: BCMA binding nanobody, H: hinge, S: handle, TM: transmembrane domain, ICOS: ICOS intracellular domain, 4-1BBt: 4-1BB intracellular domain with N-terminal truncation, CD3ζt: CD3ζ domain with C-terminal truncation of ITAM2 and ITAM3.

[0025] Figures 2A-2C Shown is a graph depicting increased CAR surface expression by the BCMA-CAR of the present disclosure ("NPB5005-ICOSBBtZt") compared to several exemplary second-generation CARs and third-generation CARs utilizing the same extracellular BCMA domain (see Table 2). Figure 2A A bar graph depicting surface expression of the CAR receptor is shown. Figure 2B Shows the Figure 2A Flow cytometry histogram of the data. Figure 2C The BCMA mean fluorescence intensity (MFI) of the transduced T cell populations is shown. Figure 2D Shows the Figure 2C Flow cytometry histogram of the data.

[0026] Figures 3A-3C Shown is a line graph depicting reduced cytokine production by a BCMA-CAR of the present disclosure ("NPB5005-ICOSBBtZt") compared to several exemplary second-generation CARs and third-generation CARs utilizing the same extracellular BCMA domain as controls (see Table 2). Figure 3A IL-2 production is shown. Figure 3B TNFα ("TNF") production is shown. Figure 3C IFNy production is shown. The x-axis depicts the ratio of effector CAR T cells to target tumor cells (E:T). The y-axis depicts cytokine production as measured by multiplex assays of 18-hour co-culture supernatants.

[0027] Figures 4A-4C Shown is a line graph depicting the killing of tumor cell lines by the BCMA-CAR of the present disclosure (“NPB5005-ICOSBBtZt”) during co-culture compared to effector CAR T cell therapy controls targeting BCMA (bb2121, FHVH33, and LCARB38M). Figure 4A The percent killing of MM.1S tumor cells is shown. Figure 4BThe percent killing of U266 tumor cells is shown. Figure 4C The percentage of killing of RPMI8226 tumor cells is shown. The x-axis depicts the ratio of effector CAR T cells to target tumor cells (E:T). The y-axis depicts the percentage of tumor cells killed. Effector CAR T cells are as indicated.

[0028] Figures 5A-5C The effects of multiple stimulations by the BCMA-CAR of the present disclosure ("NPB5005-ICOSBBtZt") are depicted. Figure 5A A schematic depiction of a method for testing the proliferation and function of T cells expressing an exemplary BCMA-CAR disclosed herein ("NPB5005-ICOSBBtZt") compared to T cells expressing a control CAR is shown. RPMI8226 myeloma cells expressing BCMA were co-cultured with BCMA-CAR T cells for a total of 16 days and stimulated with an additional 100,000 fresh RPMI8226 cells on days 4, 8, and 12. Figure 5B Shown are line graphs depicting the number of accumulated T cells after multiple stimulations. Figure 5C Line graphs depicting the number of target cells at the end of each stimulation are shown.

[0029] Figures 6A-6C A series of graphs are shown depicting surface expression of PD-1 on BCMA-CAR T cells of the present disclosure ("NPB5005-ICOSBBtZt") compared to control CAR T cells after the third stimulation with target cells in the exemplary model depicted in FIG. 5 . Fig. 6A are flow cytometry plots of PD1 using T cells from two separate donors (top and bottom panels). Figure 6B Shown is a bar graph depicting the percentage of cells expressing PD1 for RPMI-8226 cells. Figure 6C Shown is a bar graph depicting the percentage of cells expressing PD1 for U266 cells.

[0030] Figure 7A-7B is a line graph depicting the functionality of the BCMA-CAR ("NPB5005-ICOSBBtZt") of the present disclosure. Fig. 7A Depicted are line graphs of IL2 production following 18 hours of co-culture of CAR-transduced cells with RPMI-8226 cells at the indicated effector to target (E:T) ratios. Figure 7B Depicted is a line graph of CAR-positive cells after multiple stimulations with RPMI-8226 multiple myeloma cells.

[0031] Figures 8A-8DIt is depicted that the proliferative and cytolytic properties of the BCMA-CAR ("NPB5005-ICOSBBtZt") of the present disclosure are maintained in a clinical vector format lacking a eukaryotic selection marker. Fig. 8A Shown are flow cytometry plots of T cells transduced with NPB5005-ICOSBBtZt and reference CAR constructs and K562-BCMA engineered target cells after multiple repetitive stimulations. Figure 8B is a graph depicting the number of K562.GFP (left) and K562.BCMA.GFP (right) cells after 96 hours of co-culture with T cells transduced with the indicated CAR constructs. Figure 8C is a graph depicting the number of T cells after 96 hours of co-culture with K562.GFP (left) or K562.BCMA.GFP (right) cells. Fig.8D Depicted are bar graphs of target (left) and T cell numbers (right) for the indicated CAR-T cell populations and K562.BCMA.GFP target cell populations after multiple stimulations.

[0032] Figures 9A-9B Depicting that the cytokine production and PD1 expression properties of the BCMA-CAR ("NPB5005-ICOSBBtZt") of the present disclosure are maintained in a clinical vector format lacking a eukaryotic selection marker. NPB5005-ICOSBBtZt maintains lower cytokine production and PD1 expression. Fig.9A Shown are line graphs evaluating cytokine production by CAR-transduced T cells at various E:T ratios in response to stimulation with K562.GFP and K562.BCMA.GFP target cells. Fig. 9B Shown are graphs of PD1 expression on the surface of T cells transduced with the indicated CAR constructs in response to K562.BCMA.GFP target cells.

[0033] Figures 10A-10C Depicted is the intermediate CAR intrinsic tonic signaling of the BCMA-CAR ("NPB5005-ICOSBBtZt") of the present disclosure. Fig. 10A Shown are flow cytometry graphs depicting T cell proliferation as labeled by dilution of the T cell's Tag-It Violet dye after 96 hours of treatment with the indicated stimulation conditions. Fig. 10B Describes the quantitative Fig. 10A Bar graph of cell numbers shown in , where the enlarged inset shows the number of T cells in the absence of exogenous stimulation. Fig. 10C Shown are bar graphs depicting cytokine production at the indicated E:T ratios in the absence of BCMA target protein.

[0034] Figures 11A-11D Depicted is the dose response of the BCMA-CAR of the present disclosure in a murine model. Fig.11A Shown are intravital imaging of MM.1S xenograft tumor burden in mice following treatment with increasing doses of NPB5005-ICOSBBtZt at days 4, 10, and 18 post-infusion. Fig. 11B Shown are measurements of tumor burden (using soluble luciferase) from MM.1S tumor-bearing mice treated with the indicated T cell populations. Fig. 11C Shown are survival curves for MM.1S tumor-bearing female mice treated with the indicated T cell populations. Fig.11D Shown are survival curves for MM.1S tumor-bearing male mice treated with the indicated T cell populations.

[0035] In certain figures and examples described herein, the following abbreviations are used to describe the domains of BCMA-CAR: "CD28" or "28" for CD28; "2" for CD2; "3" for CD3;

[0036] "4" is for CD4; "CD8" is for CD8a; "BB" is for 4-1BB; "BBt" is for truncated 4-1BB; "Z" is for CD3ζ; "Zt" is for truncated CD3ζ; "OX40" or "40" is for OX-40, and "40t" or "OX40t" is for truncated OX-40. The domains are ordered from the BCMA binding domain to the extracellular domain to the intracellular domain.

[0037] Details

[0038] Provided herein is an improved chimeric antigen receptor (BCMA-CAR) that binds to a B cell maturation antigen, comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and at least a third signaling domain, wherein the first signaling domain is based on an intracellular signaling domain of a CD28 family protein (e.g., ICOS), the second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein (e.g., 4-1BB), and the at least third intracellular signaling domain is derived from a CD3 signaling domain (e.g., CD3ζ).

[0039] Provided herein is a BCMA-CAR (e.g., NPB5005-ICOSBBtZt) showing high-target efficacy and low-level adverse phenotypic events. The BCMA-CAR of the present disclosure (e.g., NPB5005-ICOSBBtZt) provides increased effector cell (e.g., T cell) function, proliferation, and retains such phenotypes after multiple stimulations. Further, the BCMA-CAR of the present disclosure (e.g., NPB5005-ICOSBBtZt) produces low cytokine levels and low surface PD-1 expression. Such properties of the BCMA-CAR of the present disclosure (e.g., NPB5005-ICOSBBtZt) provide numerous technical advantages over the control BCMA-CAR tested.

[0040] In some embodiments, after multiple rounds of stimulation, such as two, three, or four rounds of stimulation with target cells, such as cancer cells, the BCMA-CAR (e.g., NPB5005-ICOSBBtZt) of the present disclosure retains increased effector cell function (e.g., T cell proliferation). In certain embodiments, the increase in effector cell function (e.g., T cell proliferation) is at least twice, at least three times, at least four times, or at least five times that of the control BCMA-CAR. In some embodiments, after multiple rounds of stimulation, such as two, three, or four rounds of stimulation with target cells, such as cancer cells, the BCMA-CAR (e.g., NPB5005-ICOSBBtZt) of the present disclosure retains increased effector cell function (e.g., target cell cell lysis, such as cancer cell cell lysis). In certain embodiments, the increase in effector cell function (e.g., target cell cell lysis, such as cancer cell cell lysis) is at least twice, at least three times, at least four times, or at least five times that of the control BCMA-CAR.

[0041] In some embodiments, after multiple rounds of stimulation with target cells, such as cancer cells, for example, two rounds, three rounds, and four rounds, the BCMA-CAR (e.g., NPB5005-ICOSBBtZt) of the present disclosure retains reduced adverse phenotypic events (e.g., surface PD-1 expression). In certain embodiments, the reduced adverse phenotypic events (e.g., surface PD-1 expression) are at least twice, at least three times, at least four times, or at least five times that of the control BCMA-CAR. In some embodiments, after multiple rounds of stimulation with target cells, such as cancer cells, for example, two rounds, three rounds, and four rounds, the BCMA-CAR (e.g., NPB5005-ICOSBBtZt) of the present disclosure retains reduced adverse phenotypic events (e.g., basal cytokine production, such as IL-2, IFNγ, or TNFɑ). In certain embodiments, the reduced adverse phenotypic events (e.g., basal cytokine production, such as IL-2, IFNγ, or TNFɑ) are at least twice, at least three times, at least four times, or at least five times that of the control BCMA-CAR.

[0042] In some aspects, the chimeric intracellular domain of the BCMA-CAR of the present disclosure enhances the activity and efficacy of BCMA-CAR.For example, when expressed in T cells, the BCMA-CAR of the present disclosure enhances T cell stimulation, proliferation, persistence and killing of target myeloma cells expressing BCMA.BCMA-CAR T cells can eliminate BCMA positive multiple myeloma cells, with the leading clinical CAR T cell therapy (e.g., bb2121, FHVH33 and LCARB38M) for BCMA equivalent efficacy and better efficacy, while producing significantly less cytokines.Accordingly, in some embodiments, the combination of improved T cell proliferation, persistence, tumor cell killing, reduced inflammatory cytokine production and lower PD-1 surface expression makes the BCMA-CAR protein of the present disclosure superior to the leading BCMA-CAR T cell therapy.

[0043] Tumor-associated antigens and tumor-specific antigens allow for immune targeting of tumors, with relatively minimal off-tumor, on-target side effects risk. Tumor cells can raise these antigens, which can then be targeted by human immune response. The disclosure herein provides a chimeric intracellular domain, which combines the ICOS intracellular domain and the 4-1BB intracellular domain to provide a co-stimulatory molecule, which exhibits superiority to other receptors based on CD28 or 4-1BB, to generate BCMA-CAR products that contribute to enhanced T cell proliferation, killing efficacy and resistance to the inhibitory function of the tumor microenvironment.

[0044] In some aspects, the present disclosure also provides nucleic acids encoding BCMA-CAR disclosed herein. In some embodiments, the nucleic acids encoding BCMA-CAR disclosed herein comprise a nucleotide sequence according to SEQ ID NO: 11.

[0045] In some embodiments, the nucleic acid disclosed herein comprises a nucleic acid sequence encoding a chimeric intracellular domain. In some embodiments, the BCMA-CAR disclosed herein is used for expression in T cells, wherein the T cells co-express at least one or a combination of endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8 and CD40L.

[0046] In some aspects, the disclosure also provides vectors comprising nucleic acids disclosed herein. In some embodiments, the vector disclosed herein is any one of a viral vector, a plasmid, a minicircle DNA, a cosmid, a yeast artificial chromosome, a bacterial artificial chromosome, or a transposon / transposase system. In some embodiments, the viral vector is an adenoviral vector or a lentiviral vector. In some embodiments, the vector is a lentiviral vector.

[0047] In some aspects, the disclosure also provides cells comprising nucleic acids or vectors disclosed herein. In some embodiments, cells disclosed herein are modified T cells. In some embodiments, modified T cells are allogeneic T cells. In some embodiments, modified T cells are autologous T cells. In some embodiments, modified T cells are naive T cells, early memory T cells, stem cell-like T cells, stem memory T cells (stemmemory T cell, TSCM), central memory T cells (TCM) and regulatory T cells (Treg) Any one.

[0048] In some embodiments, the BCMA-CAR comprises an extracellular domain of SEQ ID NO: 1. In some embodiments, the BCMA-CAR comprises a first signaling domain of SEQ ID NO: 2, a second signaling domain of SEQ ID NO: 3, and a third signaling domain of SEQ ID NO: 4. In some embodiments, the BCMA-CAR comprises a sequence of SEQ ID NO: 5.

[0049] definition

[0050] Unless otherwise specified, "B cell maturation antigen", "recombinant B cell maturation antigen" and "BCMA" are used interchangeably. B cell maturation antigen, also known as CD269, is a member of the tumor necrosis factor receptor superfamily 17 and is highly selectively expressed on the surface of multiple myeloma (MM) cells (Guo, R et al. Front Immunol Vol. 13: 839097). Under physiological conditions, BCMA is mainly expressed on plasmablasts and terminally differentiated plasma cells (PCs). Under pathological conditions, BCMA is expressed on almost all MM tumor cell lines (80%-100%), and the amount of BCMA on the surface of malignant PCs is much higher than that of regulatory PCs (Lee L, et al. Br J Haematol (2016) 174: 911–22). Its expression is limited to the B cell lineage and has been shown to be important for B cell development and autoimmune responses.

[0051] As used herein, "BCMA-specific" (e.g., a BCMA-specific T cell receptor or a BCMA-specific chimeric antigen receptor) refers to a binding agent that selectively binds to an antigen or epitope of BCMA, e.g., with high affinity, and does not significantly bind to other unrelated antigens or epitopes.

[0052] As described herein, "CD137" is a member of the tumor necrosis factor (TNF) receptor family, and is also referred to as 4-1BB, CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9) and induced by lymphocyte activation (ILA). As described herein, unless otherwise indicated, the terms "CD137", "4-1BB", "4-1BB wt", "4-1BB wild type", "BB", "BB wt" and "BB wild type" are used interchangeably throughout, for example, when describing the construct or costimulatory molecules of the application.

[0053] Unless otherwise indicated, the truncated CD137 intracellular domain as described herein is interchangeably referred to as "truncated CD137", "CD137t", "truncated 4-1BB", "4-1BBt", "truncated BB" or "BBt" throughout, for example, when describing the constructs or costimulatory molecules of the present application. In some embodiments, the mutant CD137 intracellular domain comprises a deletion of one, two, three or four lysine residues from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutations from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutations at amino acid positions 1, 5, 6 and 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutations at amino acid positions selected from amino acid positions 1, 5, 6 and 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the one or more lysine mutations are lysine to alanine mutations. In some embodiments, the CD137 intracellular domain comprises the amino acid sequence according to SEQ ID NO:3.

[0054] Unless otherwise indicated, the terms "co-stimulatory molecule", "co-stimulatory molecule", "co-stimulatory molecule", "co-stimulatory protein", "co-stimulatory protein", "co-stimulatory protein", "co-stimulatory receptor", "co-stimulatory receptor", "co-stimulatory receptor" and "switch receptor" are used interchangeably to refer to a recombinant T cell co-stimulatory receptor (RTCR) comprising a novel chimeric co-stimulatory intracellular domain of the present application. These terms can be used in combination with terms such as "recombinant T cell", "recombinant", "chimeric T cell" and "chimeric" to refer to the RTCR of the present application.

[0055] As described herein, the "recombinant T cell co-stimulatory receptor" or "switch receptor" of the present disclosure is a "co-stimulatory molecule", "co-stimulatory receptor" or "co-stimulatory protein" generated by operably linking an extracellular domain to an intracellular chimeric intracellular protein of the present disclosure.

[0056] The terms T cell, T-cell, t cell, t-cell and T lymphocyte are used interchangeably in this disclosure.

[0057] The terms "NPB5005-ICOSBBtZt", "BCMA-ICOSBBtZt", "NPB5005-BCMA-ICOSBBtZt", "Receptor 1", "receptor 1", "Construct 1", "construct 1" and "V283" are used interchangeably in the present disclosure to refer to exemplary BCMACARs of the present disclosure or T cells expressing exemplary BCMACARs of the present disclosure.

[0058] The terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein.

[0059] The term "about" or "approximately" may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value. In some embodiments, "about" or "approximately" may be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value. In some embodiments, "about" or "approximately" may be understood as within 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value. In some embodiments, "about" or "approximately" may be understood as within 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value.

[0060] Throughout the disclosure, when compositions are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is additionally contemplated that there are compositions of the disclosure that consist essentially of, or consist of, those components, and that there are processes and methods according to the disclosure that consist essentially of, or consist of, those processing steps.

[0061] Extracellular domain

[0062] Provided herein are BCMA-CARs comprising an extracellular domain.

[0063] In some embodiments, the extracellular domain comprises a protein, peptide, glycoprotein, antibody, or fragment thereof, such as an antigen binding fragment, that binds to BCMA. In some embodiments, the antibody or fragment thereof is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, an sdAb, a Fv, a V H H fragment or single chain Fv fragment. In some embodiments, the antibody or its fragment is a nanobody. In some embodiments, the nanobody comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nanobody comprises the amino acid sequence of SEQ ID NO: 8.

[0064] In some embodiments, the extracellular domain is a B cell maturation antigen (BCMA) binding protein. In some embodiments, the BCMA binding protein is a BCMA-specific T cell receptor (TCR). In some embodiments, the BCMA binding protein is a nanobody. In some embodiments, the BCMA binding protein is a BCMA-specific chimeric antigen receptor (CAR).

[0065] In some embodiments, the extracellular domain comprises two or more binding sites for targeting two or more different target antigens. In some embodiments, the extracellular domain comprises two or more binding sites for targeting two or more different sites on the target antigen. In some embodiments, the extracellular domain comprises two antigen-binding domains or fragments of a bispecific antibody. In some embodiments, the extracellular domain comprises a F(ab)2 fragment of a bispecific antibody. In some embodiments, the extracellular domain comprises two or more antigen-binding domains or fragments of a multispecific antibody.

[0066] Signal peptide

[0067] In some embodiments, the extracellular domain includes a signal peptide, for example, at the N-terminus. In some embodiments, the signal peptide may be derived from a surface expressed protein or a secretory protein. In some embodiments, the signal peptide may be derived from a prolactin precursor (Preprolactin), HIV envelope protein precursor (HIV pre-Env), HCV polyprotein, CB virus polyprotein, pestivirus polyprotein, calreticulin precursor (Precalreticulin), VSV-G precursor (pre-VSV-G), HLA class I histocompatibility antigen or PD-1 signal peptide (PD-1SP), interleukin 12 (IL12), GM-CSF or CD8 alpha chain (CD8a) signal peptide.

[0068] In some embodiments, the signal peptide is derived from a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the CD8a signal peptide comprises an amino acid sequence of SEQ ID NO: 6.

[0069] Hinge

[0070] In some embodiments, the extracellular domain comprises a hinge region. In some embodiments, the hinge region is derived from CD8, PD-1, CD28, ICOS or IgG hinge domain. In some embodiments, the hinge region is derived from CD8 alpha chain (CD8a) hinge. In some embodiments, the CD8a hinge region comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, the CD8a hinge region comprises an amino acid sequence of SEQ ID NO:7.

[0071] handle

[0072] In some embodiments, the extracellular domain comprises a stalk region. In some embodiments, the stalk region is derived from CD8, PD-1, CD28, ICOS or IgG. In some embodiments, the hinge region is derived from ICOS. In some embodiments, the ICOS stalk region comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ICOS stalk region comprises an amino acid sequence of SEQ ID NO: 9.

[0073] In some embodiments, the extracellular domain further comprises one or more of a signal peptide, a hinge, and an ICOS extracellular handle, or a combination thereof. In some embodiments, the extracellular domain further comprises one or more of a CD8a signal peptide, a CD8a hinge, an ICOS extracellular handle, or a combination thereof.

[0074] In some embodiments, the CD8a signal peptide comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the CD8a hinge comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the ICOS extracellular stalk comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the extracellular domain further comprises one or more of a CD8a signal peptide according to SEQ ID NO: 6, a CD8a hinge according to SEQ ID NO: 7, and an ICOS extracellular stalk according to SEQ ID NO: 9, or a combination thereof.

[0075] In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO:1.

[0076] Transmembrane domain

[0077] In some embodiments, the transmembrane domain of the BCMA-CAR disclosed herein is derived from CD8, PD1, CD28, ICOS or IgG transmembrane domain. In some embodiments, the transmembrane domain is positioned between the extracellular domain and the first signal transduction domain. In some embodiments, the transmembrane domain is derived from the ICOS transmembrane domain. In some embodiments, the ICOS transmembrane domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ICOS transmembrane domain comprises an amino acid sequence of SEQ ID NO: 10.

[0078] Intracellular signaling domain

[0079] ICOS domain

[0080] In some embodiments, the BCMA-CAR of the present disclosure comprises a first signaling domain derived from an ICOS protein.

[0081] The CD28 family protein has a single extracellular immunoglobulin variable-like (IgV) domain followed by a short cytoplasmic tail. Members of the CD28 family protein include CD28, CD28H, inducible co-stimulatory factor (ICOS), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152), programmed death-1 (PD-1), and B and T lymphocyte attenuation factor (BTLA). CD28, CD28H, and ICOS are co-stimulatory proteins expressed on T cells and, for example, promote their activation, high levels of cytokine / chemokine expression, resistance to apoptosis, and proliferation of T cells. In some embodiments, the first signaling domain based on the intracellular signaling domain of the CD28 family protein is an ICOS protein.

[0082] As described herein, "ICOS protein" is an inducible T cell co-stimulatory protein, also known as AILIM, CD278, CCLP, CRP-1, H4, Ly115 and CVID1. In some embodiments, the ICOS intracellular domain can be from mammalian ICOS. In some embodiments, the mammalian ICOS can be human ICOS, mouse ICOS, rat ICOS or monkey ICOS. In some embodiments, the ICOS intracellular domain can be derived from human ICOS, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any of the human ICOS amino acid sequences according to the following GenBank Accession Numbers: AAH28006.1, NP_036224.1, AIC51287.1, AIC60036.1, NP_036224.1, Q9Y6W8.1, EAW70357.1, EAW70356.1, EAW70355.1, AAL40934.1, AAL40933.1, CAC06612.1, AAX93073.1, AAM00909.1, AAH28210.1, and CAD59742.1. In some embodiments, the ICOS intracellular domain can be derived from mouse ICOS, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any of the mouse ICOS amino acid sequences according to the following GenBank Accession Numbers: NP_059508.2, Q9WVS0.2, EDL00161.1, CAM13242.1, CAM13241.1, CAB71153.1, AAG48732.1, AAH34852.1, XP_006496203.1, XP_006496202.1, XP_006496201.1, ACX50464.1, ACX50463.1, AAH28006.1, XP_021052880.1, XP_029334968.1, and XP_021030282.1.In some embodiments, the ICOS intracellular domain can be derived from rat ICOS, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any of the rat ICOS amino acid sequences according to the following GenBank accession numbers: NP_072132.1, Q9R1T7.1, XP_008765358.1, XP_006245100.1, XP_006245099.1, EDL98922.1, EDL98921.1, XP_006245103.1, XP_006245105.1, XP_006245110.1, XP_0062451110.1, XP_006245112.1, XP_006245113.1, XP_006245114.1, XP_006245115.1, XP_006245116.1, XP_006245117.1, XP_006245118.1, XP_006245119.1, 038940099.1, XP_032755449.1, XP_017457364.1, P_029425757.1, XP_029425757.1, XP_021119236.1, XP_012929934.1, In some embodiments, the ICOS intracellular domain can be derived from non-human primate ICOS, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any of the monkey ICOS amino acid sequences according to the following GenBank accession numbers: XP_007964137.1, NP_001253918.1, XP_010350939.1, XP_012301785.1, XP_012301784.1, XP_017739861.1, XP_010334714.1, XP_003925677.1, AFH29328.1, XP_008997520.1, XP_023075107.1, XP_023075099.1, XP_012301785.1, XP_012301784.1, XP_017739861.1, XP_010334714.1, XP_003925677.1, AFH29328.1, XP_008997520.1, XP_023075107.1, XP_023075099.1, XP_012301785.1, XP_012301784.1 _021779593.1, XP_003907887.1, XP_025260988.1, XP_025260987.1, 986.1, XP_011716287.1, XP_011716285.1, P_011805288.1, XP_011805287.1, XP_011847867.1, XP_011847866.1, 2362.1, XP_033086489.1, XP_032134414.1, XP_032134413.1, and

[0083] In some embodiments, the chimeric intracellular domain comprises a first signal transduction domain derived from a protein of the CD28 family. In some embodiments, the first signal transduction domain is derived from any one of CD28, CD28H, ICOS, or a combination thereof.

[0084] In some embodiments, the first signaling domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the first signaling domain derived from ICOS comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0085] 4-1BB intracellular domain

[0086] Tumor necrosis factor receptor (TNFR) family proteins include TNFR1 (tumor necrosis factor receptor 1 / TNFRSF1A), TNFR2 (tumor necrosis factor receptor 2 / TNFRSF1B), lymphotoxin beta receptor / TNFRSF3, OX40 / TNFRSF4, CD40 / TNFRSF5, Fas / TNFRSF6, decoy receptor 3 / TNFRSF6B, CD27 / TNFRSF7, CD30 / TNFRSF8, 4-1BB / TNFRSF9, DR4 (death receptor 4 / TNFRSF10A), DR5 (death receptor 5 / TNFRSF10B), decoy receptor 1 / TNFRSF10C , decoy receptor 2 / TNFRSF10D, RANK (NF-κB receptor activator / TNFRSF11A), OPG (osteoprotegerin / TNFRSF11B), DR3 (death receptor 3 / TNFRSF25), TWEAK receptor / TNFRSF12A, TACI / TNFRSF13B, BAFF-R (BAFF receptor / TNFRSF13C), HVEM (herpes virus entry mediator / TNFRSF14), nerve growth factor receptor / TNFRSF16, BCMA (B cell maturation antigen / TNFRSF17, GITR (glucocorticoid-induced TNF receptor / TNFRSF18), TAJ (toxic and JNK inducer / TNFRSF19), RELT / TNFRSF19L, DR6 (death receptor 6 / TNFRSF21), TNFRSF22, TNFRSF23, exotropin A2 isoform receptor / TNFRS27, and exotropin 1-no sweat receptor. The interaction between tumor necrosis factor superfamily (TNFSF) ligands and TNF receptor superfamily (TNFRSF) receptors provides co-stimulatory signals that control the survival, proliferation, differentiation, and effector functions of immune cells. Depending on the specific intracellular signals induced by TNFRSF members, they can be divided into three groups – death domain (DD) containing receptors, decoy receptors, and TNF receptors. TNFRSFs such as TNFR-1, Fas, DR3, DR4, DR5 and DR6 contain their own DDs and / or interact with other adaptor molecules containing cytoplasmic DDs. Some other TNFRSFs, such as TNFR-2, CD27, CD30, CD40, glucocorticoid-induced TNFR family-related gene (GITR), Fn1, lymphotoxin beta receptor (LTβR), OX40, receptor activator of NF-κB (RANK) and XEDAR, lack DDs and contain a motif of four to six amino acids, called TRAF interaction motif (TIM), which recruits TRAF proteins.TRAF protein is an adaptor molecule that activates multiple downstream signaling pathways such as NF-κB, Janus kinase (JNK), ERK, p38MAPK and PI3K, which help cell survival, proliferation and cytokine production. In some embodiments, the second signaling domain is based on a mutant of the intracellular signaling domain of a TNFR family protein and is CD137 (4-1BB).

[0087] In some embodiments, the CD137 intracellular domain can be derived from mammalian CD137. In some embodiments, mammalian CD137 can be derived from human CD137, mouse CD137, rat CD137 or non-human primate CD137. In some embodiments, the CD137 intracellular domain can be derived from human CD137, or an isoform or variant thereof, for example, comprising an amino acid sequence identical to any one of the human CD137 amino acid sequences according to the following GenBank accession numbers: U03397, AAA62478, NP_001552, Q07011, AAH06196 and XP_006710681. In some embodiments, the CD137 intracellular domain can be derived from mouse CD137, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any one of the mouse CD137 amino acid sequences according to the following GenBank accession numbers: NP_001070977.1, NP_001070976.1, NP_035742.1, NP_033430.1, P20334.1, XP_011248530.1, XP_011248530.1, ABI30213.1, BAE32724.1, and AAH28507.1. In some embodiments, the CD137 intracellular domain can be derived from rat CD137, or an isoform or variant thereof, e.g., comprising an amino acid sequence identical to any one of the rat CD137 amino acid sequences according to the following GenBank accession numbers: NP_852049.1, NP_001020944.1, BAD99404.1, XP_008762504.1, XP_006239534.1, EDL81196.1, AAH97483.1, EHB16663.1, EHB16663.1, KFO38282.1, XP_010618177.1, XP_029414155.1, XP_029414154.1, XP_021099219.1, and XP_012888584.1. In some embodiments, the CD137 intracellular domain can be from non-human primate CD137, or an isoform or variant thereof comprising an amino acid sequence identical to any of the non-human primate CD137 amino acid sequences according to the following GenBank accession numbers: ABY47575.1, ABI30212.1, ABY47577.1, ABY47576.1, and ABY47578.1.

[0088] In some embodiments, the CD137 intracellular domain as described herein comprises an amino acid sequence starting from amino acid position 214 to the last amino acid at the C-terminal end of the amino acid sequence of the human CD137 protein as described herein.

[0089] In some embodiments, the CD137 intracellular domain as described herein comprises an amino acid sequence starting from amino acid position 215 to the last amino acid at the C-terminal end of the amino acid sequence of the mouse CD137 protein as described herein.

[0090] In some embodiments, the mutant CD137 intracellular domain described herein is derived from any of the CD137 proteins as described herein, comprising one or more mutations, wherein the mutation can be the addition / insertion, deletion / truncation, or substitution / replacement of one or more amino acids within the amino acid sequence of the CD137 protein. In some embodiments, the mutant CD137 intracellular domain described herein is any of the CD137 intracellular domain sequences as described herein, comprising one or more mutations, wherein the mutation can be the addition / insertion, deletion / truncation, or substitution / replacement of one or more amino acids within the amino acid sequence of the CD137 intracellular domain. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 intracellular domain as described herein, comprising the deletion or substitution of one or more amino acids within the amino acid sequence of the CD137 intracellular domain, and the amino acid can be a target for ubiquitination. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising the deletion or substitution of one or more lysine residues in the amino acid sequence of the CD137 intracellular domain, and the lysine residues can be a target for ubiquitination. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising the deletion or substitution of one, two, three or four lysine residues in the amino acid sequence of the CD137 intracellular domain, and the lysine residues can be a target for ubiquitination. In some embodiments, the lysine residues that can be deleted or substituted in the amino acid sequence of the CD137 intracellular domain described herein are at amino acid positions 214, 218, 219 and / or 225 of the CD137 intracellular domain. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising the deletion or substitution of K214. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising a deletion or substitution of K218. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising a deletion or substitution of K219. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising a deletion or substitution of K225.

[0091] In some embodiments, the mutant CD137 intracellular domain can be a truncated CD137 intracellular domain. The truncated CD137 intracellular domain as described herein can be any of the CD137 proteins described herein, wherein more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, fifty, one hundred, two hundred or more amino acids of the continuous segment from the N-terminus of the CD137 protein as described herein is missing. The truncated CD137 intracellular domain as described herein can be any of the CD137 intracellular domain sequences described herein, wherein more than one, two, three, four, five, six, seven, eight, nine, ten or more amino acids of the continuous segment from the N-terminus of the CD137 intracellular domain as described herein is missing. In some embodiments, the amino acids deleted from the N-terminus of the CD137 intracellular domain include one or more proximal polybasic amino acids of the CD137 intracellular domain.

[0092] In some embodiments, the mutant CD137 intracellular domain can be a truncated CD137 intracellular domain. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence from amino acid position 13 to amino acid position 42 of the CD137 intracellular domain according to the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a continuous segment of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids missing from the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain of the present disclosure. In some embodiments, the CD137 intracellular domain comprises the amino acid sequence according to SEQ ID NO:3.

[0093] In some embodiments, the mutant CD137 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminal of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations from amino acid position 1 to amino acid position 12 of the N-terminal of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations at amino acid positions 1, 2, 3, 4, 5 and 6 of the N-terminal of the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprising one or more proximal basic amino acid mutations at amino acid positions selected from the CD137 intracellular domain of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprising one or more proximal basic amino acid mutations further comprises a lysine mutation at amino acid position 12 of the N-terminal of the CD137 intracellular domain of the present disclosure. In some embodiments, the lysine mutation is a lysine to alanine mutation, such as K12A. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 3.

[0094] In some embodiments, the second signal transduction domain of the BCMA-CAR disclosed herein comprises a truncated CD137 intracellular domain comprising an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the second signal transduction domain of the BCMA-CAR disclosed herein is a truncated CD137 intracellular domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 3.

[0095] CD3ζ domain

[0096] In some embodiments of the BCMA-CAR disclosed herein, the third signaling domain is derived from a CD3 zeta domain.

[0097] In some embodiments, the third signaling domain of the BCMA-CAR disclosed herein is a truncated CD3 zeta domain comprising an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the third signaling domain of the BCMA-CAR disclosed herein, the third signaling domain of the BCMA-CAR disclosed herein is a truncated CD3 zeta domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 4.

[0098] Ribosome skipping sequence

[0099] In some embodiments, the BCMA-CAR of the present disclosure is polycistronic, that is, it contains more than one different polypeptide produced by a single mRNA transcript. The sequence can be polycistronic by using various linkers, for example, a polynucleotide sequence encoding a first molecule can be connected to a nucleotide sequence encoding a second molecule, for example, 5' first gene: linker: second gene 3'. The linker can encode a 2A ribosomal jumping element, such as P2A. In some embodiments, the P2A linker comprises an amino acid sequence of SEQ ID NO: 16. In certain embodiments, the P2A linker is encoded by a nucleotide sequence comprising SEQ ID NO: 18. Other 2A ribosomal jumping elements include, but are not limited to, E2A, T2A, and F2A. Such 2A ribosomal jumping elements allow the production of separate polypeptides encoded by the first and second genes produced during translation.

[0100] Exemplary BCMA-CAR

[0101] The amino acid sequences of exemplary BCMA-CARs and domains of the present disclosure are shown in Table 1.

[0102] Table 1. Receptor 1 BCMA-CAR domain and amino acid sequence

[0103]

[0104]

[0105] In some embodiments, the BCMA-CAR of the present disclosure is encoded by the following nucleotide sequence ("clinical vector format").

[0106]

[0107] Table 2. Control BCMA-CAR domains and corresponding amino acid sequences

[0108]

[0109]

[0110]

[0111]

[0112] In some aspects, provided herein is a BCMA-CAR comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signal transduction domain, a second signal transduction domain, and at least a third signal transduction domain, wherein the first signal transduction domain comprises the ICOS intracellular domain according to SEQ ID NO: 2, the second signal transduction domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the at least third signal transduction domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0113] In some embodiments, the BCMA-CAR comprises (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and a third signaling domain, wherein the first signaling domain comprises the ICOS intracellular domain according to SEQ ID NO: 2, the second signaling domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the third signaling domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0114] In some embodiments, the BCMA-CAR comprises: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; (b) a transmembrane domain, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and at least a third signaling domain, wherein the first signaling domain comprises the ICOS intracellular domain according to SEQ ID NO: 2, the second signaling domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the at least third signaling domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0115] In some embodiments, the BCMA-CAR comprises: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; (b) a transmembrane domain, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and a third signaling domain, wherein the first signaling domain comprises the ICOS intracellular domain according to SEQ ID NO: 2, the second signaling domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the third signaling domain comprises a truncated CD3 zeta domain according to SEQ ID NO: 4.

[0116] In some embodiments, the BCMA-CAR comprises an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the BCMA-CAR comprises a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the BCMA-CAR comprises a first signaling domain comprising the amino acid sequence of SEQ ID NO: 2, a second signaling domain comprising the amino acid sequence of SEQ ID NO: 3, and a third signaling domain comprising the amino acid sequence of SEQ ID NO: 4.

[0117] In some embodiments, the BCMA-CAR extracellular domain comprises a signal peptide domain comprising an amino acid sequence of SEQ ID NO: 6. In some embodiments, the extracellular domain comprises a hinge domain comprising an amino acid sequence of SEQ ID NO: 7. In some embodiments, the extracellular domain comprises a BCMA binding domain comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments, the extracellular domain comprises an ICOS handle comprising an amino acid sequence of SEQ ID NO: 9. In some embodiments, the BCMA-CAR extracellular domain comprises a signal peptide domain of an amino acid sequence of SEQ ID NO: 6. In some embodiments, the extracellular domain comprises a hinge domain of an amino acid sequence of SEQ ID NO: 7. In some embodiments, the extracellular domain comprises a BCMA binding domain of an amino acid sequence of SEQ ID NO: 8. In some embodiments, the extracellular domain comprises an ICOS handle of an amino acid sequence of SEQ ID NO: 9. In some embodiments, BCMA-CAR comprises an extracellular domain comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, BCMA-CAR comprises an extracellular domain of SEQ ID NO: 1.

[0118] In some embodiments, the BCMA-CAR comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 1.

[0119] In some embodiments, the BCMA-CAR comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the BCMA-CAR comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 5. In some embodiments, the BCMA-CAR comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 5, wherein the BCMA binding domain comprises an amino acid sequence of SEQ ID NO: 8.

[0120] Therapeutic applications

[0121] Also provided herein are methods of using the BCMA-CAR of the present disclosure. The therapeutic method can be characterized according to the disease or condition to be treated, such as cancer. The BCMA-CAR of the present disclosure is suitable for cancer, such as cancer expressing BCMA.

[0122] In some embodiments, the cancer is a soft tissue sarcoma or a sarcoma of the bone (osteosarcoma). In some embodiments, the cancer is a sarcoma selected from the group consisting of: bullous rhabdomyosarcoma, bullous soft tissue sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, bright tissue sarcoma, dedifferentiated liposarcoma, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma; sensitive neuroblastoma (olfactory neuroblastoma), Ewing sarcoma, extrarenal rhabdomyosarcoma, extraosseous myxoid chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi's sarcoma, leiomyosarcoma of bone, liposarcoma, osteosarcoma, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH), fibroids, myxoid liposarcoma, myxoid inflammatory fibroblastic sarcoma, multiple neoplasms with perivascular epithelioid cell differentiation, osteosarcoma, extraperiosteal osteosarcoma, neoplasms with perivascular epithelial cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing's tumor, rhabdomyosarcoma, small cell osteosarcoma, single fibroids, synovial sarcoma, or telangiectatic osteosarcoma.

[0123] In some embodiments, the cancer is a cancer selected from basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. In some embodiments, the cancer is a cancer selected from adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, or small cell carcinoma.

[0124] In some embodiments, the cancer is selected from anal cancer, appendix cancer; bile duct cancer (i.e., biliary tract cancer), breast cancer, bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal cancer, colon polyps, undetermined primary cancer (cup), esophageal cancer, eye cancer, fallopian tube cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0125] In some embodiments, breast cancer is invasive ductal carcinoma, ductal carcinoma in situ, invasive lobular carcinoma, or lobular carcinoma in situ. In some embodiments, pancreatic cancer is adenocarcinoma or islet cell carcinoma. In some embodiments, colorectal cancer is adenocarcinoma. In some embodiments, colon polyps are associated with familial adenomatous polyposis. In some embodiments, bladder cancer is transitional cell bladder cancer, squamous cell bladder cancer, or adenocarcinoma. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, non-small cell lung cancer is adenocarcinoma, squamous cell lung cancer, or large cell lung cancer. In some embodiments, non-small cell lung cancer is large cell lung cancer. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, prostate cancer is adenocarcinoma or small cell carcinoma. In some embodiments, ovarian cancer is epithelial ovarian cancer. In some embodiments, bile duct cancer is proximal bile duct cancer or distal bile duct cancer.

[0126] In some embodiments, the cancer is any one of the hematological cancers selected from leukemia, myeloma or lymphoma. In some embodiments, the cancer is a leukemia selected from acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute promyelocytic leukemia (APL), mixed lineage leukemia (MLL) or myelodysplastic syndrome (MDS).

[0127] In some embodiments, the cancer is a myeloma, such as multiple myeloma. In some embodiments, the cancer is a multiple myeloma selected from a hyperdiploid (HMM) or non-hyperdiploid or hypodiploid subtype of multiple myeloma. In some embodiments, the multiple myeloma is selected from a light chain myeloma, a non-secretory myeloma, a solitary plasmacytoma, an extramedullary plasmacytoma, a monoclonal gammopathy of undetermined significance (MGUS), a smoldering multiple myeloma (SMM), an immunoglobulin D (IgD) myeloma, or an immunoglobulin E (IgE) myeloma.

[0128] In some embodiments, cancer is a lymphoma, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma. In some embodiments, cancer is a non-Hodgkin's lymphoma. In some embodiments, cancer is a non-Hodgkin's lymphoma selected from the following non-Hodgkin's lymphoma: small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphoma, diffuse large cell lymphoma, follicular center cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, mantle cell lymphoma or marginal zone B cell lymphoma. In some embodiments, cancer is a lymphoma, such as Hodgkin's lymphoma. In some embodiments, cancer is a Hodgkin's lymphoma selected from the following: nodular sclerosis typical Hodgkin's lymphoma, lymphocyte-rich typical Hodgkin's lymphoma or lymphocyte depletion of typical Hodgkin's lymphoma.

[0129] In some embodiments, the cancer is any one of the following: acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis cancer, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, and hemangioma.

[0130] In some embodiments, the extracellular domain of the B cell maturation antigen (BCMA) chimeric antigen receptor (BCMA-CAR) described herein binds to the target with a binding affinity of 1fM to 100μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1pM to 100μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1pM to 10pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10pM to 50pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10pM to 100pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 100pM to 500pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 500pM to 1nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1nM to 10nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10nM to 100nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 100nM to 500nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 500nM to 1μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1μM to 10μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1μM to 5μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 5μM to 7.5μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 7.5μM to 10μM.

[0131] In some aspects, BCMA CAR is also provided herein, comprising: (a) an extracellular domain, wherein the extracellular domain has an amino acid sequence according to SEQ ID NO: 1; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signal transduction domain, a second signal transduction domain, and at least a third signal transduction domain, wherein the first signal transduction domain comprises an ICOS intracellular domain according to SEQ ID NO: 2, the second signal transduction domain comprises a truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO: 3, and the at least third signal transduction domain comprises a truncated CD3 ζ domain according to SEQ ID NO: 4. In some embodiments, BCMA-CAR comprises an amino acid sequence according to SEQ ID NO: 5.

[0132] In some aspects, the disclosure also provides nucleic acids encoding BCMA-CAR of the disclosure. The disclosure also provides vectors, such as expression vectors, comprising nucleic acids of the disclosure. In some embodiments, the nucleic acid or vector comprises SEQ ID NO: 11. The disclosure also provides cells, such as isolated cells, comprising nucleic acids or vectors of the disclosure. In some embodiments, the cell comprises a nucleic acid or vector containing SEQ ID NO: 11.

[0133] Expression on T cells

[0134] In some embodiments, the BCMA-CAR disclosed herein is used for expression in T cells, wherein the T cells co-express at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, or a combination thereof.

[0135] In some embodiments, BCMA-CAR disclosed herein is co-expressed with a T cell receptor (TCR) in a T cell. In some embodiments, TCR is an endogenous TCR. In some embodiments, TCR is an artificial TCR. In some embodiments, artificial TCR is an affinity-enhanced TCR. In some embodiments, when co-expressed with a TCR in a T cell, BCMA-CAR provides a second activation signal for inducing activation and proliferation of T cells, wherein the first activation signal is provided by antigen binding via TCR.

[0136] In some embodiments, the BCMA-CAR disclosed herein is expressed in T cells as a component of an artificial receptor for a target. In some embodiments, the artificial receptor is a chimeric antigen receptor (CAR), a receptor for a ligand or a component thereof, an antibody or a fragment thereof. In some embodiments, the BCMA-CAR disclosed herein is expressed as a component of a CAR. In some embodiments, the BCMA-CAR disclosed herein is expressed as a component of an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, an sdAb, a Fv, a V H H fragment or single-chain Fv fragment. In some embodiments, BCMA-CAR is expressed as a component of an artificial receptor in T cells as disclosed herein, and induces activation and / or proliferation of T cells after target binding through the artificial receptor.

[0137] In some embodiments, the modified T cells disclosed herein co-express at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, or a combination thereof.

[0138] In some embodiments, the methods disclosed herein further comprise modification of an endogenous sequence encoding a component of the major histocompatibility complex (MHC) class I (MHC-I), wherein the modification reduces or eliminates the expression or activity level of MHC-I. In some embodiments, the modification reduces or eliminates the expression or activity of β2-macroglobulin.

[0139] The present disclosure also provides a composition comprising a BCMA-CAR disclosed herein. The present disclosure also provides a composition comprising a nucleic acid encoding a BCMA-CAR disclosed herein. The present disclosure also provides a composition comprising a vector, the vector comprising a nucleic acid disclosed herein. The present disclosure also provides a composition comprising a cell disclosed herein. The present disclosure also provides a composition comprising a T cell disclosed herein, such as a modified T cell.

[0140] The present disclosure also provides a composition comprising a cell colony, wherein the colony comprises a variety of cells, which comprises a nucleic acid encoding a BCMA-CAR disclosed herein or a vector comprising a nucleic acid encoding a BCMA-CAR disclosed herein. The present disclosure also provides a composition comprising a cell colony, wherein the colony comprises a variety of modified T cells disclosed herein.

[0141] The present disclosure also provides a method for producing a variety of modified T cells, wherein the method includes: a) providing a variety of primary T cells disclosed herein; b) providing a composition comprising BCMA-CAR disclosed herein, a nucleic acid encoding BCMA-CAR disclosed herein, or a vector encoding a nucleic acid of BCMA-CAR disclosed herein; and c) introducing the composition of (b) into a variety of primary T cells of (a) to produce a variety of modified T cells under conditions of stably expressing BCMA-CAR in a variety of modified T cells. In some embodiments, the method for producing a variety of modified T cells disclosed herein further includes modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of endogenous TCR. In some embodiments, the method for producing a variety of modified T cells disclosed herein further includes modifying an endogenous sequence, wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I).

[0142] In some embodiments, the endogenous sequence encoding T cell receptor (TCR) is modified using a nucleic acid modification system. In some embodiments, the endogenous sequence that modifies the expression or activity level is modified using a nucleic acid modification system. In some embodiments, the nucleic acid modification system includes one or more of CRISPR / Cas protein, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN) and endonuclease. In some embodiments, the modification of the endogenous sequence is completed by non-homologous end joining repair. In some embodiments, the non-homologous end joining repair is generated by, for example, zinc finger nuclease introduced into the cell via physical means, viral vectors or non-viral vectors. In some embodiments, the non-homologous end joining repair is generated by TALE nuclease (i.e., TALEN) introduced into the cell via physical means, viral vectors or non-viral vectors. In some embodiments, the endogenous sequence encoding T cell receptor (TCR) is modified to reduce or eliminate the expression level of the alpha chain of TCR. In some embodiments, the endogenous sequence encoding T cell receptor (TCR) is modified to reduce or eliminate the expression level of the beta chain of TCR. In some embodiments, modification of the endogenous sequence encoding a T cell receptor (TCR) reduces or eliminates the expression level of both the α and β chains of the TCR α chain.

[0143] In some embodiments, modification of the endogenous sequence reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I), wherein modification of the endogenous sequence reduces or eliminates the expression or activity level of MHC-I. In some embodiments, modification of the endogenous sequence reduces or eliminates the expression or activity of β2-macroglobulin.

[0144] In some embodiments, the methods of generating a plurality of modified T cells disclosed herein further comprise: d) maintaining or expanding the plurality of modified T cells in a suitable cell culture medium; and e) either: i) cryopreserving the plurality of modified T cells in a suitable cell freezing medium; or ii) preparing the plurality of modified T cells for administration to a subject having a disease or disorder.

[0145] Compositions intended for administration to subjects, comprising cells or modified T cells of the present disclosure, and various modified T cells produced by the methods of the present disclosure may need to meet one or more "release criteria", which indicate that the composition is safe and effective for preparation and / or administration to subjects as a drug product. Release criteria may include such requirements: compositions of the present disclosure (e.g., cells or modified T cells of the present disclosure) contain a specific percentage of cells or modified T cells, which express the BCMA-CAR of the present disclosure on their cell surfaces. The amplification process should continue until specific criteria have been met (e.g., reaching a certain total number of cells or modified T cells of the present disclosure or a certain total percentage of cells or modified T cells expressing the BCMA-CAR of the present disclosure).

[0146] Some standards can send the signal of the point that the amplification process should end under it.For example, once cells reach the cell size of 300fL, they should be prepared, reactivated or cryopreserved (otherwise, cells reaching the size above this threshold value may begin to die). Once the average cell size less than 300fL is reached by cell colony, immediate cryopreservation can produce better cell recovery when thawing and cultivating, because cells have not yet reached a complete quiescent state (complete quiescent size is about 180fL) before cryopreservation.Before amplification, the T cell of the present disclosure may have a cell size of about 180fL, but after amplification for 3 days, its cell size may increase more than four times (for example, to about 900fL).In the cultivation of the next 6-12 days, the T cell colony may slowly reduce the cell size to the complete quiescence when 180fL.

[0147] The method for preparing a cell colony for preparation may include, but is not limited to, the following steps: concentrating cells of the cell colony, washing cells and / or further selecting cells via magnetic bead sorting for drug resistance or for specific surface expression markers. The method for preparing a cell colony for preparation may further include a sorting step to ensure the safety and purity of the final product. For example, if tumor cells from a patient have been used to stimulate the modified T cells of the present disclosure, or have been modified to stimulate the modified T cells of the present disclosure being prepared for preparation, it is critical that the tumor cells from the patient are not included in the final product.

[0148] In some embodiments, a cell disclosed herein or a modified T cell disclosed herein expresses a BCMA-CAR comprising a mutant CD137 intracellular signaling domain disclosed herein on the cell surface at a level of at least about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, or 20×, respectively, compared to the expression level of a costimulatory molecule comprising a wild-type CD137 intracellular domain.

[0149] In some embodiments, the cells disclosed herein further comprise sequences encoding artificial antigen receptors, therapeutic polypeptides, immune cell regulatory proteins, or combinations thereof. In some embodiments, the artificial antigen receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the artificial antigen receptor comprises a recombinant T cell receptor (rTCR). In some embodiments, the artificial antigen receptor comprises a TCR that enhances affinity. In some embodiments, the artificial antigen receptor binds to a tumor-associated antigen (TAA), a pathogen-associated protein, or an antigen associated with a disease or condition, and the disease or condition is cancer, an autoimmune disease or condition, an infectious disease, an inflammatory disease, a kidney disease or condition, a lung disease or condition, a liver disease or condition, a neurodegenerative condition or condition, or a metabolic condition or condition.

[0150] In some embodiments, the artificial antigen receptor binds to a TAA associated with a solid tumor or a hematological cancer. In some embodiments, the artificial antigen receptor binds to a TAA associated with a cancer selected from any of the following: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, B cell, T cell, or FAB ALL, Acute Myeloid Leukemia (AML), Acute Myeloid Leukemia, Chronic Myeloid Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), Hairy Cell Leukemia, Myelodysplastic Syndrome (MDS), Lymphoma, Hodgkin's Disease, Malignant Lymphoma, Non-Hodgkin's Lymphoma, Burkitt's Lymphoma, Multiple Myeloma, Kaposi's Sarcoma, Colorectal Cancer, Pancreatic Cancer, Nasopharyngeal Cancer, Malignant Histiocytosis, Paraneoplastic Syndrome / Hypercalcemia of Malignancy, Solid Tumors, Bladder Cancer, Breast Cancer, Colorectal Cancer, Endometrial Cancer, Head Cancer, Neck Cancer, Hereditary Non-polyposis Cancer, Hodgkin's Lymphoma, Liver Cancer, Lung Cancer, Non-small Cell Lung Cancer, Ovarian Cancer, Pancreatic Cancer, Prostate Cancer, Renal Cell Carcinoma, Testicular Cancer, Adenocarcinoma, Sarcoma, Malignant Melanoma, and Hemangioma.

[0151] In some embodiments, the artificial antigen receptor binds to a TAA selected from the group consisting of kallikrein 4, papilloma virus binding factor (PBF), melanoma preferentially expressed antigen (PRAME), Wilms tumor-1 (WTI), hydroxysteroid dehydrogenase-like 1 (HSDLI), mesothelin, testicular cancer antigen (NY-ES0-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2 / neural receptor tyrosine kinase (Her2 / Neu), cancer-associated epithelial cell adhesion molecule (EpCAM), ovarian and uterine cancer antigen (CAI25), Folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), L1 cell adhesion molecule (LICAM), mannan-MUC-1, human endogenous retrovirus K (HERV-K-MEL), Kitakyushu lung cancer antigen-I (KK-LC-1), human cancer / testis antigen (KM-HN-1), testicular cancer antigen (LAGE-I), melanoma antigen-Al (MAGE-Al), sperm surface zona pellucida binding protein (Spl 7), synovial sarcoma, X breakpoint 4 (SSX-4), transient axonal glycoprotein-1 (TAG-I), transient axonal glycoprotein-2 (TAG-2), Enabled Homolog (ENAH), mammoglobin-A, NY-BR-I, breast cancer antigen, (BAGE-1), B melanoma antigen, melanoma antigen-Al (MAGE-Al), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X breakpoint 2 (SSX-2), paclitaxel resistance-associated gene-3 (TRAG-3), avian myelocytic tumor virus oncogene (c-myc), cyclin B1, mucin I (MUCI), p62, survivin, lymphocyte common antigen (CD45), Dickkopf WNT signaling pathway inhibitor I (DKKI), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxylesterase, alpha-fetoprotein, macrophage colony stimulating factor (M-CSF), prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), cytochrome C oxidase assembly factor I homolog (COA-1), 0-linked-N-acetylglucosamine transferase (OGT), osteosarcoma amplification 9, endoplasmic reticulum lectin (OS-9), transforming growth factor beta receptor 2 (TGF-βRII), murine leukemia glycoprotein 70 (gp70), calcitonin-related polypeptide alpha (CALCA), programmed cell death 1 ligand 1 (CD274), mouse double minute 2 homolog (mdm-2), alpha-actinin-4, elongation factor 2, malic enzyme 1 (MEI), Nuclear transcription factor gamma subunit C (NFYC), G antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), testicular cancer antigen XAGE-lb, prostate six transmembrane epithelial antigen 1 (STEAPl), PAP, prostate specific antigen (PSA), fibroblast growth factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTCl), B-Raf proto-oncogene (B-RAF), serine / threonine kinase, β-catenin, cell division cycle 27 homolog (Cdc27), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 12 (CDK12), cyclin-dependent kinase inhibitor 2A (CDKN2A), casein kinase 1α1 (CSNKlA1), fibronectin 1 (FNl), Gruwih Anest specific 7 (GAS7), glycoprotein non-metastatic melanoma protein B (GPNMB), HAUSAugmin-like complex subunit 3 (HAUS3), LDLR-fucosyltransferase, melanoma antigen recognized by T cells 2 (MART2), myostatin (MSTN), melanoma-associated antigen (mutated) 1 (MUM-1-2-3), poly (A) polymerase gamma (neo-PAP), myosin class I, protein phosphatase 1 regulatory subunit 3B (PPP1R3B), peroxiredoxin-5 (PRDX5), receptor-type tyrosine protein phosphatase kappa (PTPRK), transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPD1, triosephosphate isomerase, ocular albinism type 1 protein (OAl), RAS oncogene family member (RAB38), tyrosine tyrosinase, melanin-A (MART-1), glycoprotein 100 melanoma antigen (gplOO), N-acetylglucosamine transferase V gene (GnTVf), lymphocyte antigen 6 complex locus K (LY6K), melanoma antigen-A10 (MAGE-A10), melanoma antigen-A12 (MAGE-A12), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, paclitaxel resistance-associated protein 3 (TRAG-3), BDZ binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), breakpoint cluster region-Abelson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, elongation factor Tu GTP binding domain 2 (EFTUD2), ETS variant gene 6 / acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase-3 internal tandem repeat (FLT3-ITD), cyclin-Al, fibronectin type III domain-containing 3B (FDNC3B), promyelocytic leukemia / retinoic acid receptor alpha fusion protein (pml-RARalpha), melanoma antigen-Cl (MAGE-Cl), membrane protein alternative splicing isoform (D393-CD20), melanoma antigen-A4 (MAGE-A4) and melanoma antigen-A3 (MAGE-A3).

[0152] In some embodiments, the artificial antigen receptor binds an antigen associated with an autoimmune condition or disorder selected from any one of type 1 diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or multiple sclerosis (MS). In some embodiments, the artificial antigen receptor binds an antigen associated with an autoimmune condition or disorder selected from any one of the following: carboxypeptidase H, chromogranin A, glutamate decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), proinsulin, α-enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, S100-β, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly (ADP-ribose) polymerase, Sm antigen of U-1 small nuclear ribonucleoprotein complex.

[0153] In some embodiments, the artificial antigen receptor binds a pathogen-associated antigen from a bacterial, fungal, or parasitic protein or fragment thereof. In some embodiments, the artificial antigen receptor binds an antigen associated with HIV infection, human cytomegalovirus infection, hepatitis B infection, hepatitis C infection, Ebola virus infection, dengue fever, yellow fever, listeriosis, tuberculosis, cholera, malaria, leishmaniasis, or trypanosoma infection, or a combination thereof.

[0154] In some embodiments, the artificial antigen receptor binds to an antigen associated with a neurodegenerative disorder or condition selected from Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, prion disease, motor neuron disease (MND), Huntington's disease (HD), spinocerebellar ataxia (SCA), or spinal muscular atrophy (SMA). In some embodiments, the antigen associated with a neurodegenerative disorder or condition is any one or a combination of amyloid β (Aβ), tau, α-synuclein (α-syn), mHTT, or prion PrPsc.

[0155] In some embodiments, the therapeutic polypeptide is a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, an immunogenic polypeptide, or a cell surface protein that binds to a target on the surface of another cell. In some embodiments, the immune cell regulatory protein is a cytokine, a chemokine, a transcription factor, a protein kinase, a protease, a component, or an adaptor protein of a cell signaling pathway.

[0156] In some embodiments, the cells disclosed herein express BCMA-CAR disclosed herein. In some embodiments, the cells disclosed herein stably or transiently express BCMA-CAR disclosed herein. In some embodiments, the cells disclosed herein stably express BCMA-CAR disclosed herein. In some embodiments, the cells disclosed herein transiently express BCMA-CAR disclosed herein.

[0157] In some embodiments, the cells disclosed herein co-express at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, or a combination thereof.

[0158] The present disclosure also provides modified T lymphocytes (T cells), which include: (a) modification of endogenous sequences encoding T cell receptors (TCR), wherein the modification reduces or eliminates the expression or activity level of TCR, and / or (b) recombinant T cell co-stimulatory receptors (RTCR) disclosed herein. In some embodiments, modification of endogenous sequences encoding T cell receptors (TCR) is performed using a nucleic acid modification system. In some embodiments, the nucleic acid modification system is one or more of CRISPR / Cas proteins, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases. In some embodiments, modification of endogenous sequences encoding T cell receptors (TCR) is completed by non-homologous end joining repair. In some embodiments, non-homologous end joining repair is generated by zinc finger nucleases introduced into cells via physical means, viral vectors, or non-viral vectors. In some embodiments, non-homologous end joining repair is generated by TALE nucleases introduced into cells via physical means, viral vectors, or non-viral vectors. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of the α chain of the TCR. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of the β chain of the TCR. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of both the α chain and the β chain TCR α chain.

[0159] Pharmaceutical composition or preparation

[0160] In some embodiments, the compositions disclosed herein and the modified T cell colonies produced using the methods disclosed herein are in the form of pharmaceutical preparations (or compositions). In some embodiments, the pharmaceutical preparations disclosed herein include a pharmaceutically acceptable carrier. The pharmaceutical preparations of the present disclosure can be distributed in bags for infusion, cryopreservation and / or storage.

[0161] The pharmaceutical preparations of the present disclosure can be cryopreserved using standard protocols and an optional infusible cryopreservation medium. For example, a cryoprotectant that does not contain DMSO (e.g., CryoSOfree TM DMSO-free Cryopreservation Medium) can be used to reduce freezing-related toxicity. The cryopreserved pharmaceutical preparations of the present disclosure can be stored for infusion to patients at a later date. Effective treatment may require multiple administrations of the pharmaceutical preparations of the present disclosure, and therefore, the pharmaceutical preparations can be packaged in pre-aliquoted "doses" that can be stored frozen but thawed separately for each dose.

[0162] The pharmaceutical formulations of the present disclosure can be stored at room temperature. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and therefore, the pharmaceutical formulations can be packaged in pre-aliquoted "doses" that can be stored together but separated for administration of individual doses.

[0163] The pharmaceutical formulations of the present disclosure can be archived for subsequent re-expansion and / or selection for generating additional doses for the same patient in the context of allogeneic therapy, who may require administration at a future date, for example, following remission and relapse of a condition.

[0164] As noted above, the present disclosure provides stable formulations that can include phosphate buffers with saline or selected salts, and preservative solutions and formulations containing preservatives, or multi-purpose preservative formulations suitable for pharmaceutical and / or veterinary use, comprising at least one modified cell in a pharmaceutically acceptable formulation. The preservative formulation contains at least one known preservative in an aqueous diluent or is optionally selected from at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, polymers or mixtures thereof. Any suitable concentration or mixture as known in the art can be used, such as about 0.0015%, or any range, value or fraction therein. Non-limiting examples include, preservative-free, about 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., 0.05, 0 .25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkyl paraben (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%) and the like.

[0165] As noted above, in some aspects, the disclosure provides an article of manufacture comprising packaging material and at least one vial comprising a solution of at least one modified cell, optionally in an aqueous diluent, with a specified buffer and / or preservative, wherein the packaging material comprises a label indicating that such solution can be stored for a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours, or longer.

[0166] The articles of manufacture of the present disclosure can be used for administration over a period ranging from immediately to twenty-four hours or longer. Accordingly, the articles of manufacture currently claimed provide significant advantages for patients. The formulations of the present disclosure can optionally be safely stored at a temperature of about 2°C to about 40°C and retain the biological activity of the protein for an extended period of time, thus allowing the package label to indicate that the solution can be stored and / or used within a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or longer.

[0167] The products of the present disclosure may include packaging materials. In addition to the information required by regulatory agencies, the packaging materials also provide the conditions under which the product can be used.

[0168] In another aspect, the disclosure also provides a method for treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective number of cells comprising a nucleic acid encoding a CAR disclosed herein or a vector containing a nucleic acid encoding a CAR disclosed herein, a therapeutically effective number of any modified T cells disclosed herein, a therapeutically effective amount of any composition disclosed herein, or a therapeutically effective number of multiple modified T cells produced by the methods disclosed herein. BCMA-CAR as provided herein can be used to treat a disease or condition expressing BCMA, such as a method for treating a cancer expressing BCMA.

[0169] In some embodiments, the subject is a mammal. In some embodiments, the mammal is any one of humans, primates, rodents, canines, felines, ungulates, equines, and swine. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human primate. In some embodiments, the disease or condition is any one of cancer, autoimmune disorders, infectious diseases, inflammatory diseases or conditions, kidney diseases or conditions, lung diseases or conditions, liver diseases or conditions, cardiovascular diseases or conditions, neurodegenerative disorders or conditions, or metabolic disorders or conditions. In some embodiments, cancer is a solid tumor or a hematologic cancer. In some embodiments, the infectious disease is caused by bacteria, viruses, fungi, protozoa, or parasites. In some embodiments, the neurodegenerative disorder or condition is any of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, prion disease, motor neuron disease (MND), Huntington's disease (HD), spinocerebellar ataxia (SCA), or spinal muscular atrophy (SMA).

[0170] The following examples are provided to better illustrate the present disclosure, but should not be construed as limiting the scope of the present disclosure. When specific materials are mentioned, they are only used for illustrative purposes and are not intended to limit the present disclosure. Those skilled in the art can develop equivalent means or reactants without exerting creative ability and without departing from the scope of the present disclosure. Example

[0171] Example 1: Materials and Methods

[0172] Culture media and cell lines

[0173] DMEM was supplemented with Pen / Strep / glutamine, 20 mM HEPES, 10 μg / mL gentamicin, and 10% FBS to prepare complete DMEM. RPMI was supplemented with Pen / Strep / glutamine, 20 mM HEPES, 10 μg / mL gentamicin, 10% FBS, and 50 μM 2-ME to prepare complete RPMI. T cell growth medium was prepared by supplementing complete RPMI with 50 ng / ml IL2, 10 ng / ml IL7, and 10 ng / mL IL15 (Peprotech). X-Vivo 15 TM Cytokine medium was prepared by supplementing with 1% human serum, 20 mM HEPES, Pen / Strep / glutamine, and 10 μg / mL gentamicin. Human peripheral blood mononuclear cells (PBMC) were purchased from The cells were cultured in complete RPMI. 293FT was purchased from K562 and A375 cells were purchased from The cells were cultured in complete DMEM.

[0174] Plasmids and cloning

[0175] Lentiviral plasmids were ordered from VectorBuilder containing the PGK promoter driving the truncated human EGFR receptor (huEGFRt), followed by the MSCV promoter and subsequent WPRE sequence driving GFP. The co-stimulatory molecule was followed by the P2A sequence and ordered as a single gene block (Invitrogen) and used The builder is placed in frame with the huEGFRt sequence based on homology-based recombination. The CAR sequence consists of a gene block fragment was constructed and after GFP excision was used builder was cloned downstream of the MSCV promoter. PD-L1_P2A and HLA-A2 were cloned in frame with huEGFRt and replaced GFP.

[0176] P2A amino acid sequence

[0177] GSGATNFSLLKQAGDVEENPGP(SEQ ID NO:16)

[0178] Human EGFRt amino acid sequence (other name: huEGFRt (AA112))

[0179]

[0180] P2A nucleic acid sequence

[0181]

[0182] Human EGFRt nucleic acid sequence

[0183]

[0184]

[0185] HLA-A2 signal peptide nucleic acid sequence

[0186]

[0187] huGMCSF signal peptide, nucleic acid sequence

[0188]

[0189] Lentiviral production and preparation of Retronectin plates

[0190] VSV pseudotyped lentivirus was produced in 6-well plates. Briefly, 293FT was cultured at 0.9 x 10 6 or 1.4x10 6 Once the cells have attached and reached at least 80% confluence, lipofectamine 3000 was used according to the manufacturer's protocol. Transfect a mixture of lentiviral plasmids, packaging vectors (psPAX2) and VSV-G envelope expression plasmids (PMD2.G). After 18 hours, replace the culture medium with 3mL fresh DMEM. The viral supernatant is harvested 48 hours after replacing the culture medium, and is spun down at 1500RPM to remove 293FT cells / debris. Retronectin is coated on a 24-well non-tissue culture treated plate at 37°C for 2 hours or overnight at 4°C at 20μg / well in PBS- / -. Before adding lentiviral supernatant (2mL), take out Retronectin and wash once with PBS. The plate is then rotated at 1500G for 90 minutes at 32°C to concentrate the viral particles onto retronectin. Remove the lentiviral supernatant immediately before transducing primary T cells or tumor cells. Alternatively, transduce T cells with 8μg / mL of polybrene, accompanied by a spin infection (spinfection) of 800G at 32°C for 2 hours.

[0191] T cell culture, transduction, and isolation

[0192] In complete RPMI (100 μl beads / 50x10 6 PBMC) with CD3 / CD28 microbeads Human PBMCs were activated in T cell growth media. At 48 hours post-activation, activated PBMCs were transferred to lentivirus-coated Retronectin plates for 48 hours and then transferred to 6-well plates containing fresh T cell growth media. After an additional 24 hours of culture, cell transduction was determined by flow cytometry and transduced cells were enriched based on huEGFRt expression. To separate cells based on EGFR expression, T cell cultures were collected and activation beads removed. Cells were then stained with anti-EGFR-APC antibody at 1:100 in MACS buffer for 30 minutes at 4°C. Cells were then washed and incubated with anti-APC microbeads at 4°C. Incubate together for 15-30 minutes. Unbound microbeads are then removed by centrifugation, and huEGFRt cells are isolated by positive selection on a mini-MACS column. Cells are eluted from the mini-MACS column and placed back into culture in T cell growth medium and used in experiments within 2 weeks. To create a stable cell line, cells are collected and transduced like primary T cells. EGFR selection is performed twice, two weeks apart.

[0193] T cell stimulation

[0194] In cases where T cells are stimulated with plate-bound antibodies, Maxisorp TM Flat-bottom plates (Invitrogen TM ) were treated with the indicated amount of anti-human CD3 antibody (HIT3a- ) was coated at 37°C for 2 hours. Plates were washed twice in basic RPMI before use. For myeloma cell line stimulation, RPMI18226 and U266 cell lines were collected and resuspended in cytokine medium and dispensed into U-bottom plates. Similarly, A375 cells were plated in DMEM in 96-well flat-bottom plates 1 day before the addition of T cells. The medium was replaced before the addition of homologous T cells. After EGFR+ selection, T cells were collected, counted and resuspended in cytokine medium at appropriate concentrations and distributed into wells containing antibodies or APCs. For RPMI18226 and U266 experiments, anti-CD3 (HIT3a / Biolegend) was added at the indicated doses after RPMI18226 and U266 / T cell interaction for 1-2 hours at 37°C. TM ). In cases where T cell proliferation is to be followed, prior to addition to the stimulation plate, the cells were plated according to Biolegend TM T cells were labeled with Violet Tracking Dye (CTV) according to the protocol. Supernatants were collected 18-36 hours after stimulation to evaluate cytokine secretion and proliferation / T cell killing was evaluated after 96 hours of stimulation.

[0195] Cytokine multiplex assay

[0196] After collecting T cell supernatants, Legendplex for human Th or Th1 cytokines was used. TM Multi-Analyte Flow Assay Kit(Biolegend TM ) Measure cytokines. Follow the manufacturer's protocol with the following exceptions: 75 μL T cell supernatants are used to measure cytokines, and 2 μL of each reagent / well is used. Secreted cytokines are measured by flow cytometry, and values ​​are standardized for the maximum response of the control group to combine and analyze multiple experiments, and for standardized variability between experiments and donors.

[0197] Lentiviral vector manufacturing

[0198] 10 mg of GMP-like transfer plasmid will be produced. The transfer plasmid works with the third generation packaging system in adherent or suspension HEK293 cells for virus production. 7 20-30 liters of GMP lentiviral vector were produced at a titer of 10 / ml and concentrated to 9 / ml.

[0199] Mouse experiments

[0200] NSG mice (8-10 weeks old) were injected intravenously (tail vein) with 3e6MM.1S cells stably expressing Gaussia luciferase-GFP. After 18-20 days, mice were randomly divided into groups containing roughly equal average luciferase counts, and T cells were injected with indicated cell counts through the tail vein. From the time of tumor cell injection, blood samples were collected once a week, and the Gaussia luciferase level in serum was determined by in vitro luciferase assay. In short, 5 μL of clarified plasma was added to 96-well plates in duplicate. According to the manufacturer's protocol, the sample was mixed with Gaussia substrate (Coelenterazine, GoldBio#CZ25) and measured immediately. For in vivo imaging, after IP administration of coelenterazine, animals were imaged. In vitro luciferase measurements were standardized for measurements obtained at day 0 after T cell injection. Animal studies were completed at the Institute of Human Virology at the University of Maryland.

[0201] Example 2: Construction of BCMA-CAR

[0202] The disclosure herein provides the design of BCMA-CAR comprising a potent BCMA nanobody (Kd=1 nM) driven by a proprietary third generation ICOS and modified 4-1BB co-stimulatory signaling domain and a modified CD3ζ domain (modified third generation BCMA-CAR). Figure 1 Shown is a schematic diagram of the structure of an exemplary chimeric antigen receptor comprising a BCMA Nanobody linked to a chimeric intracellular signaling domain described herein.

[0203] Example 3: Targeted elimination of BCMA-positive multiple myeloma cell lines using modified third-generation BCMA-CAR-T cells results in reduced cytokine production

[0204] T cells expressing exemplary BCMA-CARs of the present disclosure are described herein. The disclosure herein provides the design of BCMA CAR molecules and the validation of their function in vitro for CAR T cell killing of tumor cell lines.

[0205] like Figure 1 The BCMA-CAR described in ("receptor 1" or "NPB5005 BCMA-ICOSBBtZt" or "modified third generation BCMA-CAR") is introduced into T cells via transduction. Figures 2A-2C As shown in , truncation of the CD3ζ intracellular signaling domain in the BCMA-CAR of the present disclosure increases the surface expression of the CAR receptor, improving the surface expression gain produced by using a modified third-generation CAR receptor ( Figure 2A A bar graph of expression is shown, and Figure 2B The corresponding flow chart is shown). Figure 2C BCMA MFI is shown, demonstrating that surface expression of both second and third generation receptors benefited from truncation of CD3ζ, and Figure 2D The corresponding flow chart is shown.

[0206] Cytokine production was then assessed. Figures 3A-3C As shown in , ζ chain truncation results in reduced pro-inflammatory cytokine production. IL2 ( Figure 3A )、TNFɑ( Figure 3B ) and IFNγ( Figure 3C ) levels were all reduced, indicating that the NPB5005-ICOSBBtZtCAR receptor produced less key inflammatory cytokines relative to constructs with the full-length ζ intracellular signaling domain and the reference construct.

[0207] In an overnight killing assay, T cells expressing "receptor 1" were co-cultured with multiple myeloma cell lines expressing BCMA, RPMI-8226, and U266 to evaluate the effectiveness of modified third-generation BCMA-CAR T cells in killing target tumor cells compared to exemplary clinical BCMA CART cell therapies known in the art (bb2121, FHVH33, and LCARB38M) and GFP controls. Target tumor cells include myeloma cell lines expressing BCMA. NPB5005 and control CAR T effector cells (E) were co-cultured with target cancer cells (T) at multiple ratios (E:T) of 1:01 to 1:32. The results disclosed herein show that at each ratio tested between 1:01 and 1:32, modified third-generation BCMA-CAR T cells killed similar percentages of MM.1S, U266, and RPMI-8225 target cells (respectively 1:01, ... Figures 4A-4C ).

[0208] like Figure 1 As described in, the exemplary "Receptor 1" or "NPB5005 BCMA-ICOSBBtZt" was compared with the reference second generation CAR FHVH33 and CAR in terms of cytokine production (IL-2, IFNγ and TNF), wherein the CAR is composed of the following: the same CD8 leader peptide (SEQ ID NO:6), BCMA binding nanobody domain (SEQ ID NO:8), and CD8 hinge (SEQ ID NO:7) and CD3 zeta domain driven by various second generation signaling domains or third generation signaling domains (NPB5005-28Z (SEQ ID NO:12), NPB5005-BBZ (SEQ ID NO:13), NPB5005-ICOSBBZ (SEQ ID NO:14), NPB5005-ICOSBBtZ (SEQ ID NO:15)). BCMA-CAR T cells were co-cultured with the BCMA-positive cell line RPMI-8226 at various E:T ratios (1:32, 1:16, 1:08, 1:04, 1:02, 1:01, and 2:01). The amounts of IL-2, IFNγ, and TNFα released when T cells expressing "NPB5005-ICOSBBtZ" were co-cultured with target cells were lower than the cytokine levels released by T cells expressing the second-generation BCMA-CAR (with the same or different BCMA-binding nanobodies) ( Figures 3A-3CCompared with the third-generation BCMA-CAR with the same extracellular domain (SEQ ID NO: 1), T cells expressing "NPB5005-ICOSBBtZ" produced similar or slightly higher cytokines compared with the GFP control, but produced significantly lower cytokines compared with T cells expressing the control CAR ( Figures 3A-3C ).

[0209] Results of the studies described herein show that exemplary BCMA-CAR T cells of the present disclosure eliminated a variety of BCMA-positive cancer cells with equivalent efficacy compared to leading clinical CAR T cells ( Figure 4A -4c), while producing significantly less cytokine expression (see Figures 3A-3C ), reducing toxicity and adverse side effects for patients.

[0210] Example 4: BCMA-CAR eliminates BCMA-positive myeloma cell lines with excellent efficacy during long-term repeated co-culture

[0211] In vitro studies described herein show increased T cell proliferation and persistence by T cells transduced with a modified third-generation BCMA-CAR ("Receptor 1" or "NPB5005-ICOSBBtZt") compared to T cells with a control clinical CAR when co-cultured with the BCMA-expressing myeloma cell line RPMI-8226, based on an exemplary long-term repeated co-culture assay ( Figure 5A ). 100,000 CAR T cells were cultured with 500,000 RPMI-8266 cell targets. The cultures were stimulated with an additional 250,000 fresh RPMI-8226 target cells every 4 days (on days 4, 8, and 12) and cultured for a total of 16 days. Figure 5B and 5C The number of T cells and target cells at the end of each stimulation are shown, respectively, confirming the enhanced long-term functional cytolytic potential of the NPB5005-ICOSBBtZt CAR construct relative to the reference CAR.

[0212] NPB5005-ICOSBBtZt leads to lower PD-1 expression in antigen-stimulated CAR-T cells, such as Figures 6A-6C In RPMI-8226 ( Figure 6B ) and U266( Figure 6C ) cell lines, reduced PD-1 expression on the surface of T cells was observed. It was further observed that the third generation co-stimulatory domain is essential for the full functionality of the truncated CD3ζ chain-based CAR receptor of the present disclosure, as shown by Fig. 7A The IL2 in the comparison and Figure 7BThis was confirmed by the CAR-positive cells in the

[0213] The BCMA-CAR of the present disclosure was cloned into a clinical vector format lacking a eukaryotic selection marker. The functionality of the BCMA-CAR was evaluated from the new vector, including proliferation and cytolytic phenotypes ( Figures 8A-8D ) and decreased cytokine production and PD1 expression ( Figures 9A-9B Intermediate CAR-intrinsic basal signaling was also identified ( Figures 10A-10C ), where NPB5005-ICOSBBtZt demonstrated basal signaling levels intermediate between the two reference CAR-T cells; in the absence of IL2 or CAR ligand, cell survival was comparable to LCAR-B38M ( Figures 10A-10B ), but basal cytokine production was significantly reduced ( Fig. 10C ).

[0214] Example 5: BCMA-CAR shows dose-dependent efficacy in xenograft mouse model

[0215] To evaluate the in vivo efficacy, MM.1S cells were injected via tail vein at 1×10 6 , 3x10 6 and 5x10 6 Injected into mice. In vivo imaging of tumor burden on days 4, 10, and 18 ( Fig.11A ) and quantification of secreted Gaussia luciferase ( Fig. 11B ) demonstrated significantly reduced tumor burden and a dose-dependent effect at all doses tested. Female ( Fig. 11C ) and male ( Fig.11D ) mice demonstrated enhanced survival of treated subjects, with mice receiving NPB5005-ICOSBBtZt demonstrating approximately 100% survival within 90 days post-dose.

[0216] In general, the results described herein show that T cells expressing the modified third-generation BCMA-CAR (e.g., NPB5005-ICOSBBtZt) of the present disclosure can be used to treat cancer with high efficacy and significantly reduced adverse events. Specifically, the expression of NPB5005-ICOSBBtZt leads to increased effector function, proliferation and persistence, while minimizing cytokine release. As shown above, NPB5005-ICOSBBtZt kills multiple myeloma cell lines expressing BCMA, with comparable efficacy to leading BMCA-CAR T cell therapies, produces reduced toxic cytokines and expresses lower levels of surface PD-1. Therefore, NPB5005-ICOSBBtZt is more resistant to negative signaling from cells in the tumor microenvironment. After repeated stimulation with target cells, NPB5005-ICOSBBtZt proliferates to a higher number than alternative BCMA-CAR T cells.

Claims

1. A chimeric antigen receptor that binds to a B cell maturation antigen (BCMA-CAR), comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 8; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and at least a third signaling domain, wherein the first signal transduction domain comprises the ICOS intracellular domain according to SEQ ID NO:2, the second signal transduction domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO:3, and the at least third signal transduction domain comprises the truncated CD3 zeta domain according to SEQ ID NO:

4.

2. The BCMA-CAR of claim 1, wherein the extracellular domain further comprises a signal peptide, a hinge, an ICOS extracellular handle, or a combination thereof.

3. The BCMA-CAR according to claim 1 or 2, wherein the extracellular domain further comprises a CD8a signal peptide, a CD8a hinge, an ICOS extracellular handle or a combination thereof.

4. The BCMA-CAR of any one of claims 1-3, wherein the extracellular domain further comprises a CD8a signal peptide according to SEQ ID NO: 6, a CD8a hinge according to SEQ ID NO: 7, an ICOS extracellular handle according to SEQ ID NO: 9, or a combination thereof.

5. The BCMA-CAR according to any one of claims 1-4, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO:

1.

6. The BCMA-CAR of any one of claims 1-5, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

10.

7. The BCMA-CAR of claim 1, wherein the BCMA-CAR comprises the amino acid sequence of SEQ ID NO:

5.

8. A chimeric antigen receptor that binds to a B cell maturation antigen (BCMA-CAR), comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10; and (c) a chimeric intracellular domain comprising a first signaling domain, a second signaling domain, and at least a third signaling domain, wherein the first signal transduction domain comprises the ICOS intracellular domain according to SEQ ID NO:2, the second signal transduction domain comprises the truncated CD137 (4-1BB) intracellular domain according to SEQ ID NO:3, and the at least third signal transduction domain comprises the truncated CD3 zeta domain according to SEQ ID NO:

4.

9. A chimeric antigen receptor that binds to B cell maturation antigen (BCMA-CAR), comprising the amino acid sequence of SEQ ID NO:

5.

10. A nucleic acid encoding the BCMA-CAR according to any one of claims 1-9. A vector comprising the nucleic acid according to claim 10 . The vector according to claim 11 , wherein the vector is a lentiviral vector.

13. A cell comprising the nucleic acid according to claim 10 or the vector according to claim 11 or 12.

14. The cell of claim 13, wherein the cell is a modified T cell or a modified NK-T cell.

15. The cell of claim 13, wherein the cell is an allogeneic T cell.

16. The cell of claim 13, wherein the cell is an autologous T cell.

17. The cell of claim 14, wherein the modified T cell is a naive T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (TSCM), a central memory T cell (TCM), or a regulatory T cell (Treg).

18. The cell of any one of claims 13-17, wherein the cell transiently or stably expresses the BCMA-CAR of any one of claims 1-7.

19. The cell of any one of claims 13-18, wherein the cell co-expresses BCMA-CAR and at least one endogenous co-stimulatory molecule selected from CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, and a combination thereof.

20. A modified T cell comprising: (a) modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR; and (b) The BCMA-CAR according to any one of claims 1 to 9.

21. The modified T cell of claim 20, further comprising a modification of an endogenous sequence encoding a component of major histocompatibility complex (MHC) class I (MHC-I), wherein the modification reduces or eliminates the expression or activity level of MHC-I.

22. The modified T cell of claim 20 or 21, wherein the modified T cell co-expresses at least one endogenous co-stimulatory molecule selected from CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, and a combination thereof.

23. A composition comprising the BCMA-CAR according to any one of claims 1-9, the nucleic acid according to claim 10, the vector according to claim 11 or 12, the cell according to any one of claims 13-19, or the modified T cell according to any one of claims 20-22.

24. A method for producing a plurality of modified T cells, wherein the method comprises: a) providing a variety of primary T cells; b) providing a composition comprising the BCMA-CAR according to any one of claims 1 to 9, the nucleic acid according to claim 10, or the vector according to claim 11 or 12; and c) introducing the composition of (b) into the multiple primary T cells of (a) to produce multiple modified T cells under the condition that BCMA-CAR is stably expressed in the multiple modified T cells.

25. The method of claim 24, wherein the method further comprises the step of modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the endogenous TCR.

26. The method of claim 24 or 25, wherein the method further comprises the step of modifying endogenous sequences of a plurality of primary T cells, wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I).

27. The method according to any one of claims 24-26, wherein the method further comprises: d) maintaining or expanding the various modified T cells in a suitable cell culture medium; and e) Any of the following: i) cryopreserving the plurality of modified T cells in a suitable cell freezing medium; or ii) preparing the plurality of modified T cells for administration to a subject in need thereof.

28. A method of treating a disease or condition comprising administering to a subject in need thereof a therapeutically effective number of cells according to any one of claims 13-19, a therapeutically effective number of modified T cells according to any one of claims 20-22, a therapeutically effective amount of a composition according to claim 21, or a therapeutically effective number of a plurality of modified T cells produced by the method according to any one of claims 24-27.

29. The method of claim 28, wherein the disease or disorder is cancer, an autoimmune disease or disorder, or an inflammatory disease.

30. The method of claim 29, wherein the cancer is a hematological cancer.

31. The method of claim 30, wherein the hematological cancer is leukemia, lymphoma, or myeloma.

32. The method of any one of claims 29-31, wherein the cancer is selected from acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma and multiple myeloma.

33. The method of any one of claims 29-31, wherein the cancer is multiple myeloma.

34. The method of any one of claims 29-33, wherein the cancer expresses BCMA.

35. The method of any one of claims 29-34, wherein the subject is a mammal.

36. The method of claim 35, wherein the mammal is a human.

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