Chimeric antigen receptors specific for b cell migration antigen (BCMA) and / or transmembrane activator and caml interaction factor (TACI)

Abstract

Genetically engineered immune cells (e.g., T cells or NK cells) expressing anti-BCMA, anti-TACI, or anti-BCMA / anti-TACI chimeric antigen receptors and their use in cancer therapy. In some embodiments, the genetically engineered immune cell may be an armored CAR-T or CAR-NK cell, which further expresses an armored polypeptide for enhancing the characteristic of the CAR-T or CAR-NK cell.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 376,530, filed on September 21, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on September 20, 2023, is named 112139-0073-7007WO00_SEQ.XML and is 197,654 bytes in size. BACKGROUND OF THE INVENTION

[0005] Chimeric antigen receptor (CAR)-T cells are genetically engineered T cells that express an artificial T cell receptor for immunotherapy. The artificial T cell receptor (called a chimeric antigen receptor) can specifically bind to disease cell antigens such as cancer antigens. Once bound to the disease cells, the CAR-T cells will be activated and eliminate the disease cells.

[0006] Although CAR-T cell therapy has shown efficacy in treating some hematological cancers, the efficacy of the treatment can be affected by various factors, such as tumor antigen escape. For example, the expression level of tumor antigens may be reduced to a level where CAR-T cells cannot participate and mediate cytotoxic activity. In some cases, tumor cells can escape killing by expressing alternative forms of target antigens that lack the binding epitope of the CAR. In other cases, tumor cells can escape killing by switching to a genetically related but phenotypically different disease (so-called lineage switching).

[0007] Therefore, it is of great significance to develop improved CAR-T methods to address these challenges. SUMMARY OF THE INVENTION

[0008] The present disclosure is at least partially based on the development of anti-BCMA monospecific, anti-TACI monospecific, and anti-BCMA / anti-TACI bispecific chimeric antigen receptors and CAR-T cells expressing such chimeric antigen receptors for successfully inhibiting tumor growth in animal models. Accordingly, there are provided such CAR constructs, such immunocyte expressions, and therapeutic uses in cancer therapy.

[0009] In some aspects, the present disclosure provides a bispecific chimeric antigen receptor (CAR) that is specific for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), the bispecific CAR comprising: (a) a first antigen-binding portion that is specific for TACI; (b) a second antigen-binding portion that is specific for BCMA; (c) a co-stimulatory signaling domain; and (d) a cytoplasmic signaling domain.

[0010] In some instances, the first antigen-binding portion of (a) that is specific for TACI comprises a heavy chain variable region (V H ) and a light chain variable region (V L ). The V H comprises heavy chain CDRs that are the same as the heavy chain CDRs in a reference antibody, and the V L comprises heavy chain CDRs that are the same as the heavy chain CDRs in the reference antibody, the reference antibody comprising TC-01, TC-02, TC-03, or TC-04. In some examples, the reference antibody is TC-01. In some examples, the V H and the V L of the first antigen-binding portion that is specific for TACI are the same as the V H and V L of the reference antibody.

[0011] In some examples, the first antigen-binding portion that is specific for TACI is a single-chain variable fragment (anti-TACI scFv). In a specific example, the anti-TACI scFv comprises the amino acid sequence of any one of SEQ ID NO:75, 84, 93, and 102 (e.g., SEQ ID NO:75).

[0012] In some embodiments, the second antigen-binding portion of (a) that is specific for BCMA comprises a heavy chain variable region (V H ) and a light chain variable region (V L ). The V H can comprise:

[0013] (hi) heavy chain complementarity-determining region (CDR) 1, the heavy chain CDR1 comprising X1YX2MH, wherein X1 is S or D, and X2 is A or G;

[0014] (hii) heavy chain CDR2, the heavy chain CDR2 comprising

[0015] (hii-a) X3IX4YDGSX5KYYADSVKG (SEQ ID NO:1), wherein X3 is V or F, X4 is S or R, and X5 is D or N;

[0016] (hii-b) FIRSKAYGGTTEYAASVKG (SEQ ID NO:27); or

[0017] (hii-c) GISWNSGSIGYADSVKG (SEQ ID NO:43); and

[0018] (hiii) heavy chain CDR3, said heavy chain CDR3 comprising

[0019] (hiii-a) DEHQVVPNYRFDF (SEQ ID NO:56),

[0020] (hiii-b) DWEDPLYYYDTPF (SEQ ID NO:35),

[0021] (hiii-c) DWDYYDSSGYYPDALGI (SEQ ID NO:16),

[0022] (hiii-d) DLWDGIVGAPAGY (SEQ ID NO:9),

[0023] (hiii-e) DLTTITPGY (SEQ ID NO:22),

[0024] (hiii-f) DLWEFGGDYADY (SEQ ID NO:63),

[0025] (hiii-g) GPHYDILTSNWFDP (SEQ ID NO:28), or

[0026] (hiii-h) VQX6PGAFDI (SEQ ID NO:181), where X6 is P or S; and

[0027] Alternatively or additionally, said V L may comprise:

[0028] (li) light chain CDR1, said light chain CDR1 comprising

[0029] (li-a) SGSGSNIGSNDVS (SEQ ID NO:58),

[0030] (li-b) QASQDIX7NYLN (SEQ ID NO:2), where X7 is N or S, or

[0031] (li-c) RX8X9X 10 ISSYLX 11(SEQ ID NO:3), where X8 is A or S, X9 is S or T, X 10 is G or S, and X11 is G or N;

[0032] (lii) The light chain CDR2, said light chain CDR2 comprising

[0033] (lii-a) WNDQRPS (SEQ ID NO:59),

[0034] (lii-b) DASNX 12 ET (SEQ ID NO:4), where X 12 is L or V, or

[0035] (lii-c) AX 13 SX 14 LQS (SEQ ID NO:5), where X 13 is A or T, and X 14 is S or T; and

[0036] (liii) The light chain CDR3, said light chain CDR3 comprising

[0037] (liii-a) AAWDDSLNGWV (SEQ ID NO:60),

[0038] (liii-b) QQYDX 15 LPX 16 T (SEQ ID NO:6), where X 15 is K or N, and X 16 is F, L or Y,

[0039] (liii-c) QHSYSTPHT (SEQ ID NO:32), or

[0040] (liii-d) QQLYS (SEQ ID NO:48).

[0041] In some cases, the V of the second antigen-binding portion specific for BCMA H comprises heavy chain CDRs identical to those of the heavy chain CDRs in a reference antibody, and / or the V of the second antigen-binding portion specific for BCMA L comprises heavy chain CDRs identical to those of the heavy chain CDRs in the reference antibody, said reference antibody comprising BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, BC-08 or BC-09. In some instances, the reference antibody is BC-06. In some instances, the V of the first antigen-binding portion specific for BCMAH and V L and V of said reference antibody H and V L are the same.

[0042] In some instances, the first antigen-binding portion specific for BCMA is a single-chain variable fragment (anti-BCMA scFv). In a specific instance, the anti-BCMA scFv comprises the amino acid sequence of any one of SEQ ID NO: 15, 20, 25, 34, 41, 50, 53, 62, and 66 (e.g., SEQ ID NO: 50).

[0043] Any bispecific CARs disclosed herein may include a co-stimulatory signaling domain. Examples include but are not limited to CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively or additionally, the bispecific CAR may include a cytoplasmic signaling domain, which may be from CD3ζ.

[0044] In some specific instances, the bispecific CAR comprises a fusion polypeptide that includes, from the N-terminus to the C-terminus, (i) the first antigen-binding portion, (ii) the second antigen-binding portion, (iii) the co-stimulatory signaling domain, and (iv) the cytoplasmic signaling domain. Alternatively, the bispecific CAR may include a fusion polypeptide that includes, from the N-terminus to the C-terminus, (i) the second antigen-binding portion, (ii) the first antigen-binding portion, (iii) the co-stimulatory signaling domain, and (iv) the cytoplasmic signaling domain. Any bispecific CARs disclosed herein may further include a hinge domain and a transmembrane domain, which are located between (ii) and (iii). In some cases, the bispecific CAR may further include a peptide linker that links the first antigen-binding portion and the second antigen-binding portion. Examples include GGGGS (SEQ ID NO: 104), GGGGSGGGGS (SEQ ID NO: 105), GGGGSGGGGSGGGGS (SEQ ID NO: 106), or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 107).

[0045] Any bispecific CARs disclosed herein may further include a signal peptide at the N-terminus.

[0046] In a specific example, the bispecific CAR disclosed herein may include an extracellular bispecific antigen-binding domain, and the extracellular bispecific antigen-binding domain may include the amino acid sequence of SEQ ID NO: 163 or 164. In one example, the bispecific CAR may include the amino acid sequence of SEQ ID NO: 165 or 166.

[0047] In some aspects, the present disclosure provides a nucleic acid or a set of nucleic acids that jointly encode any of the bispecific CARs disclosed herein. In some cases, the nucleic acid includes a first nucleotide sequence encoding the bispecific CAR. In some embodiments, the nucleic acid may further include a second nucleotide sequence encoding an armored polypeptide as disclosed herein, the armored polypeptide enhancing T cell functionality; and a third nucleotide sequence encoding a self-cleaving peptide, the third nucleotide sequence being located between the first nucleotide sequence and the second nucleotide sequence. The nucleic acid or set of nucleic acids may be an expression vector, optionally a viral vector.

[0048] Exemplary armored polypeptides include, but are not limited to, IL-2, IL-5, IL-15, co-stimulatory ligands, anti-PDL1 antibodies, or fusion polypeptides including the anti-PDL1 antibody. In some cases, the armored polypeptide is a fusion protein including an anti-PDL1 antibody (e.g., single-chain variable fragment (scFv)) and an IL-2 polypeptide. Exemplary armored polypeptides are provided in Table 11 below, and each of the armored polypeptides is within the scope of the present disclosure.

[0049] Additionally, the present disclosure provides a genetically engineered immune cell that expresses the bispecific CAR disclosed herein and optionally further expresses the armored polypeptide also disclosed herein. Such genetically engineered immune cells may include any of the nucleic acids encoding the bispecific CAR and optionally the armored polypeptide disclosed herein. In some embodiments, the genetically engineered immune cell may be a T cell, an NK cell, or a macrophage. In one example, the immune cell is a T cell. In a specific example, the genetically engineered immune cells provided herein (e.g., T cells or NK cells) may express the bispecific CAR of SEQ ID NO: 165 or 166 and an armored polypeptide, such as an anti-PDL1-IL2 fusion such as SEQ ID NO: 173 or 174, or an IL2 polypeptide such as SEQ ID NO: 177, 178, 179, or 180.

[0050] In other aspects, the present disclosure features an anti-TACI chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for TACI, a co-stimulatory signaling domain, and a cytoplasmic signaling domain; wherein the extracellular antigen-binding domain specific for BCMA is as disclosed herein. In some embodiments, the co-stimulatory signaling domain is from a co-stimulatory molecule selected from: CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively or additionally, the cytoplasmic signaling domain is from CD3ζ. In some instances, the anti-TACI CAR may further comprise a hinge domain and / or a transmembrane domain located between the extracellular antigen-binding domain specific for TACI and the co-stimulatory domain. In some instances, the anti-TACI CAR may further comprise both a hinge domain and a transmembrane domain. The anti-TACI CAR may further comprise a spacer located between the hinge domain and the transmembrane domain. In a specific instance, the anti-TACI CAR disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 143-162 (e.g., SEQ ID NO: 143 or 144).

[0051] In other aspects, the present disclosure features an anti-BCMA chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for BCMA, a co-stimulatory signaling domain, and a cytoplasmic signaling domain; wherein the extracellular antigen-binding domain specific for BCMA is as disclosed herein. In some embodiments, the co-stimulatory signaling domain is from a co-stimulatory molecule selected from: CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively or additionally, the cytoplasmic signaling domain is from CD3ζ. In some instances, the anti-BCMA CAR may further comprise a hinge domain and / or a transmembrane domain located between the extracellular antigen-binding domain specific for BCMA and the co-stimulatory domain. In some instances, the anti-BCMA CAR may comprise both the hinge domain and the transmembrane domain. The anti-BCMA CAR may further comprise a spacer located between the hinge domain and the transmembrane domain. In a specific instance, the anti-BCMA CAR disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 119-142 (e.g., SEQ ID NO: 127 or 128).

[0052] The present disclosure also provides a nucleic acid, which comprises a first nucleotide sequence encoding an anti-TACICAR as described herein or an anti-BCMACAR as also described herein. The nucleic acid may further comprise a second nucleotide sequence encoding an armored polypeptide as disclosed herein, which enhances T cell functionality; and a third nucleotide sequence encoding a self-cleaving peptide, which is located between the first nucleotide sequence and the second nucleotide sequence. In some cases, the nucleic acid is an expression vector, optionally a viral vector.

[0053] In addition, the present disclosure provides a genetically engineered immune cell, which expresses an anti-BCMACAR and / or an anti-TACI CAR as disclosed herein, and optionally any armored polypeptide among the armored polypeptides as disclosed herein. In some embodiments, the genetically engineered immune cell may be a T cell, an NK cell or a macrophage. In one example, the genetically engineered immune cell is a T cell.

[0054] In addition, the present disclosure provides a method for eliminating undesired cells in a subject, which comprises administering to a subject in need an effective amount of the genetically engineered immune cell as disclosed herein or a pharmaceutical composition comprising the genetically engineered immune cell, wherein the genetically engineered immune cell expresses any anti-BCMACAR, anti-TACICAR and anti-BCMA / anti-TACI bispecific CAR among the anti-BCMACAR, anti-TACICAR and anti-BCMA / anti-TACI bispecific CARs as disclosed herein. Such genetically engineered immune cells may be armored T cells, which further express one or more armored polypeptides, such as the armored polypeptides as disclosed herein.

[0055] In some embodiments, the undesired cells are cancer cells. In some embodiments, the subject is a human cancer patient. For example, the human cancer patient may comprise BCMA + and / or TACI + cancer cells. In some examples, the cancer cells are multiple myeloma cells, lung cancer cells, gastric cancer cells, breast cancer cells or testicular cancer cells.

[0056] Also within the scope of the present disclosure are genetically engineered immune cells for cancer treatment or pharmaceutical compositions comprising the genetically engineered immune cells as disclosed herein. In addition, the present disclosure provides the use of genetically engineered immune cells or pharmaceutical compositions comprising the genetically engineered immune cells for the preparation of a medicament for cancer treatment.

[0057] In addition, the present disclosure provides an anti-TACI antibody, which comprises a heavy chain variable region (V H ) and a light chain variable (VL ) region. V H and V L The chain includes complementarity determining regions (CDRs, including CDR1, CDR2, and CDR3) that are identical to one of the reference antibodies provided in Table 2 below (e.g., TC-01). In some cases, the anti-TACI antibody includes the same V H and V L as the reference antibody (such as TC-01). In some instances, the anti-TACI antibody is a single-chain antibody, such as the single-chain antibody provided in Table 2 (e.g., SEQ ID NO:75).

[0058] In addition, the present disclosure provides an anti-BCMA antibody, the anti-BCMA antibody including a heavy chain variable region (V H ) and a light chain variable (V L ) region. V H and V L The chain includes complementarity determining regions (CDRs, including CDR1, CDR2, and CDR3) that are identical to one of the reference antibodies provided in Table 1 below (e.g., BC-06). In some cases, the anti-BCMA antibody includes the same V H and V L as the reference antibody (such as BC-06). In some instances, the anti-BCMA antibody is a single-chain antibody, such as the single-chain antibody provided in Table 1 (e.g., SEQ ID NO:50).

[0059] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and the detailed description of several embodiments, as well as from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure, which can be better understood by combining reference to the drawings with the detailed description of the specific embodiments presented herein.

[0061] Figure 1 is a graph showing that an exemplary anti-BCMA antibody (in scFv form) as indicated specifically binds to BCMA-expressing CHOK1 cells but does not specifically bind to parental CHOK1 cells.

[0062] Figures 2A - 2C includes a graph showing the binding of an exemplary anti-BCMA antibody (in scFv form) as indicated to cells expressing endogenous BCMA. Figure 2A : H929 cell line. Figure 2B : MM1.S cell line. Figure 2C: RPMI8226 cell line.

[0063] Figure 3 is a graph showing the binding activity of an exemplary anti-TACI antibody (in scFv form) to various cells as indicated.

[0064] Figures 4A - 4E contains a graph showing the cytotoxic T lymphocyte (CTL) activity of an exemplary monospecific anti-BCMACAR-T cell against various cells as indicated. Figure 4A : CAR-T cells expressing the anti-BCMA construct EPLV102 against various cell lines as indicated. Figure 4B : CAR-T cells expressing the anti-BCMA construct EPLV103 against various cell lines as indicated. Figure 4C : CAR-T cells expressing the anti-BCMA construct EPLV104 against various cell lines as indicated. Figure 4D : CTL activity of various anti-BCMACAR constructs against K562-GFP cells and K562-BCMA-TACI-GFP cells as indicated. Figure 4E : CAR-T cells expressing various anti-BCMACARs secrete IFNγ in the presence of target cells as indicated.

[0065] Figures 5A - 5B contains a graph showing the CTL activity of CAR-T cells expressing EPLV101, EPLV102, and EPLV103 against target cells as indicated. Figure 5A : Cell killing rate. Figure 5B : IFNγ secretion level.

[0066] Figures 6A - 6E contains a graph showing the CTL activity of CAR-T cells expressing EPLV102 against target cells as indicated. Figure 6A : E:T ratio is 10:1. Figure 6B : E:T ratio is 5:1. Figure 6C : Cell killing rate. Figure 6D : Percentage of CD4 / CD8 T cell subsets. Figure 6E : Percentage of effector T cell subsets.

[0067] Figures 7A - 7B contains a graph showing the CTL activity of CAR-T cells expressing EPLV102 upon re-challenge with target cells. Figure 7A : K562-GFP cells. Figure 7B : MM1R-GFP cells.

[0068] Figures 8A - 8EFigure showing the CTL activity of CAR-T cells expressing EPLV200, EPLV254, EPLV255, and EPLV256 against target cells as indicated. Figure 8A : Expression of anti-TACI CAR. Figures 8B - 8D : CTL activity against K562-GFP cells, H929-GFP cells, and MM1R-GFP cells, respectively. Figure 8E : Cell killing rate.

[0069] Figures 9A - 9C Figure showing the persistence of CAR-T cells expressing anti-TACI CAR constructs with different spacers upon re-challenge with target cells. Figure 9A : CAR expression level. Figures 9B - 9C : CTL activity against K562-GFP cells and MM1R-GFP cells, respectively.

[0070] Figures 10A - 10B Figure showing the expression of anti-BCMA / anti-TACI bispecific antibody in immune cells. Figure 10A : Expression of bispecific CAR construct EPLV217 in T cells from two donors. Figure 10B : Expression of bispecific CAR construct EPLV302 in T cells from two donors.

[0071] Figures 11A - 11E Figure showing the CTL activity of CAR-T cells expressing bispecific CAR EPLV217 against various target cells. Figure 11A : Against K562-GFP cells. Figure 11B : Against H929-GFP cells. Figure 11C : Against MM1R-GFP cells. Figure 11D : Cell killing rate. Figure 11E : IFNγ secretion.

[0072] Figures 12A - 12I Figure showing the CTL activity of CAR-T cells expressing bispecific CAR EPLV302 against various target cells. Figures 12A - 12B : CTL activity of bispecific CAR-T cells from donors 994 and 995 at a 1:1 E:T ratio upon re-challenge with K562-GFP cells. Figures 12C - 12D : CTL activity of bispecific CAR-T cells from donors 994 and 995 at a 1:1 E:T ratio upon re-challenge with MM1R-GFP cells. Figures 12E - 12F: CTL activity of bispecific CAR-T cells from donors 994 and 995 respectively under re-challenge with H929-GFP cells at an E:T ratio of 1:1. Figures 12G - 12H : Cytotoxicity rates of CAR-T cells derived from donors 994 and 995 respectively. Figure 12I : IFNγ secretion.

[0073] Figures 13A - 13C Graphs showing the in vivo activity of CAR-T cells expressing anti-BCMA monospecific CAR, anti-TACI monospecific CAR, and anti-BCMA / anti-TACI bispecific CAR in an animal model. Figure 13A : Radiation level. Figure 13B : Body weight. Figure 13C : Tumor growth.

[0074] Figures 14A - 14E Graphs showing the biological activity of engineered T cells expressing either the anti-BCMA / TACI bispecific CAR alone (non-armored CAR) or in combination with an armored polypeptide (armored CAR). Figure 14A : CAR expression level in engineered T cells. Figure 14B : Cytotoxicity of engineered T cells against BCMA / TACI-positive and BCMA / TACI-negative target cells. Figure 14C : IFNγ release at 48 hours. Figure 14D : IFNγ release at 120 hours. Figure 14E : IFNγ release at 168 hours.

[0075] Figures 15A - 15B Graphs showing the anti-tumor activity of engineered T cells expressing either the anti-BCMA / TACI bispecific CAR alone (non-armored CAR) or in combination with an armored polypeptide (armored CAR). Figure 15A : Photos showing tumor burden at different time points after CAR-T cell treatment. Figure 15B : Graphs showing the anti-tumor activity of armored CAR-T cells relative to non-armored CAR-T cells.

[0076] Figures 16A - 16D Graphs showing the biological activity of engineered NK cells expressing either the anti-BCMA / TACI bispecific CAR alone (non-armored CAR) or in combination with an armored polypeptide (armored CAR). Figure 16A : Purity of expanded NK cells. Figure 16B : Expression of CAR in transduced NK cells. Figure 16C : Cytotoxicity of engineered CAR-NK cells. Figure 16D : IFNγ release at different time points. Detailed implementation mode

[0077] B cell maturation antigen (BCMA), a member of the tumor necrosis factor-receptor superfamily, is also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17) and CD269. This receptor is highly expressed on hematopoietic cancer cells (e.g., on multiple myeloma (MM) cells), but not on other normal tissues except normal plasma cells. BCMA plays a role in regulating B cell proliferation and survival, as well as maturation and differentiation into plasma cells.

[0078] Transmembrane activator and CAML interactor (TACI), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), is a membrane protein of the TNF receptor superfamily mainly expressed on the surface of B cells. TACI has three ligands: a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), and calcium-modulating ligand (CAML). The binding of TACI to these ligands can trigger signal transduction pathways, leading to the regulation of cell activity.

[0079] This article provides anti-BCMA or anti-TACI monospecific chimeric antigen receptors (CARs) and anti-BCMA / anti-TACI bispecific CARs, as well as CAR-T cells expressing such CARs. In some embodiments, the CAR-T cells disclosed herein can further express armored polypeptides that enhance CAR-T cell characteristics (e.g., proliferation and expansion, persistence, and / or cytotoxicity). The CAR-T cells disclosed herein can be used to treat diseases involving BCMA and / or TACI (e.g., for cancer therapy). The bispecific CAR-T cells disclosed herein can activate CAR-T cells by bispecifically targeting either one or both of the two tumor antigens, BCMA and TACI. Such bispecific CAR-T cells are expected to be more effective in cancer treatment, for example, by preventing and / or treating target antigen escape. The armored CAR-T cells disclosed herein can further enhance CAR-T cell efficiency, activate immune cells, and / or mediate anti-tumor activity, overcoming the inhibitory tumor microenvironment, thereby enhancing cancer treatment.

[0080] I. Chimeric antigen receptor

[0081] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor capable of binding to an antigen expressed by an undesired cell, such as a target antigen of interest (TAA) (here BCMA and / or TACI). Generally, a CAR can comprise a fusion polypeptide that includes an extracellular antigen-binding domain (e.g., a single-chain variable fragment or scFv derived from an antibody specific for the target antigen), a co-stimulatory domain, and an intracellular signaling domain. In some cases, the fusion polypeptide can further include a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. In some embodiments, the CARs disclosed herein are T cell receptors. In other embodiments, the CARs disclosed herein can be NK cell receptors.

[0082] An antibody (used interchangeably in the plural) is an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" (e.g., anti-BCMA antibody or anti-TACI antibody) encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., only V H antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising the antigen recognition site of the desired specificity, said modified configurations comprising glycosylation variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies. An antibody (e.g., anti-galectin-9 antibody) encompasses any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and the antibody does not need to belong to any particular class. Immunoglobulins can be classified into different classes according to the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0083] A typical antibody molecule includes a heavy-chain variable region (V H ) and a light-chain variable region (V L ), which are typically involved in antigen binding. V H and VL The region can be further subdivided into hypervariable regions, also known as "complementary determining regions" ("CDRs"), which are interspersed with more conserved regions called "framework regions" ("FRs"). Each V H and V L is typically composed of three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The ranges of the framework regions and CDRs can be precisely identified using methods known in the art, such as by Kabat definition, Chothia definition, AbM definition, and / or contact definition, all of which are well-known in the art. See, for example, Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0084] In some embodiments, the antibody portions disclosed herein can share the same heavy chain and / or light chain complementary determining regions (CDRs) or the same V H and / or V L chains with a reference antibody. Two antibodies having the same V H and / or V L CDRs means that their CDRs are the same when determined by the same method (e.g., the Kabat method, Chothia method, AbM method, contact method, or IMGT method known in the art). See, for example, bioinf.org.uk / abs / . Such anti-BCMA or anti-TACI antibodies can have the same V H , the same V L or both, compared to the exemplary antibodies described herein.

[0085] In some embodiments, the antibody portions disclosed herein may share a certain level of sequence identity compared to a reference sequence. The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. The BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecule of interest. In cases where there are gaps between two sequences, Gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997 can be utilized. When using the BLAST program and the Gapped BLAST program, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used.

[0086] In some embodiments, the antibody portions disclosed herein can have one or more amino acid variations relative to a reference antibody. The amino acid residue variations disclosed in the present disclosure (e.g., in framework regions and / or in CDRs) can be conservative amino acid residue substitutions. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those of ordinary skill in the art, such as those found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 or Current Protocols in Molecular Biology, edited by F.M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0087] The anti-BCMA monospecific CARs, anti-TACI monospecific CARs, and anti-BCMA / anti-TACI bispecific CARs disclosed herein each include an anti-BCMA portion and / or an anti-TACI portion in the extracellular antigen-binding domain.

[0088] (a) Anti-BCMA binding portion

[0089] The anti-BCMA binding portion in any of the CARs disclosed herein (e.g., any anti-BCMA monospecific CAR or anti-BMCA / anti-TACI bispecific CAR in the anti-BCMA monospecific CARs or anti-BMCA / anti-TACI bispecific CARs disclosed herein) can be in the form of a scFv, which is a fusion polypeptide comprising the heavy chain variable domain (V H ) and the light chain variable domain (V L ) of an anti-BCMA antibody linked by a peptide linker. In the scFv fragment, the V H and V L fragments can be in any orientation. In some cases, the scFv can include the V L fragment, the peptide linker, and the V HFragment. Alternatively, the scFv can include V from the N-terminus to the C-terminus H fragment, a peptide linker, and V L fragment. In some instances, the scFv can further include an N-terminal signal peptide for directing the CAR comprising the scFv to the cell surface.

[0090] Exemplary anti-BCMA antibodies are provided in Table 1 below, and any of the anti-BCMA antibodies are within the scope of the present disclosure. In one instance, for example, the anti-BCMA antibody used to construct the monospecific or bispecific CAR provided herein can be Clone BC-06.

[0091] Table 1: Anti-BMCA Antibodies

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] *CDRs determined by the Chothia scheme

[0098] The anti-BCMA binding portion (and the anti-TACI binding portion disclosed below) derived from a reference antibody refers to a binding portion having substantially similar structural and functional characteristics to the reference antibody. Structurally, the binding portion can have the same heavy and / or light chain complementarity determining regions or the same V H and / or V L chains as the reference antibody. Alternatively, the binding portion can have only a limited number of amino acid variations in one or more framework regions and / or in one or more CDRs without significantly affecting its binding affinity and binding specificity relative to the reference antibody. See the description below.

[0099] In some embodiments, the anti-BCMA binding portion can include one or more heavy and light chain CDR motifs provided in the heavy and light chain complementarity determining region (CDR) motifs provided in Table 1, such as all six CDR motifs provided in Table 1. In some instances, the anti-BCMA binding portion can be derived from the anti-BCMA antibody BC-05. In some instances, the anti-BCMA binding portion can be derived from the anti-BCMA antibody BC-06. In some instances, the anti-BCMA binding portion can be derived from the anti-BCMA antibody BC-08.

[0100] In some instances, the anti-BCMA binding portion can include a heavy chain CDR that is the same as the heavy chain CDR in any of the reference antibodies provided in Table 1 above (e.g., BC-05, BC-06, or BC-08). Alternatively or additionally, the anti-BCMA binding portion can have a light chain CDR that is the same as the light chain CDR in any of the reference antibodies provided in Table 1 above (e.g., BC-05, BC-06, or BC-08). Such anti-BCMA binding portions can include the same V H and / or V L chains as the reference antibody. Alternatively, the anti-BCMA binding portion can include amino acid variations in one or more framework regions relative to the corresponding framework regions in the reference antibody. For example, relative to the corresponding framework regions in the reference antibody, the anti-BCMA binding portion can collectively include up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions.

[0101] In some embodiments, the anti-BCMA portion can include a certain level of variation in one or more CDRs relative to those in any of the reference antibodies provided in Table 1 above (e.g., BC-05, BC-06, or BC-08). For example, the anti-BCMA portion can include a heavy chain CDR that has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, either individually or collectively, compared to the V H CDR of the reference antibody. Alternatively or additionally, the anti-BCMA antibody can include a light chain CDR that has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, either individually or collectively, compared to the V L CDR of the reference antibody. As used herein, "individually" means that one CDR of the antibody shares the indicated sequence identity with the corresponding CDR of a reference antibody (e.g., a reference antibody in Table 1, such as any of BC-05, BC-06, or BC-08 or any anti-TACI reference antibody disclosed below). "Collectively" means that the combined three V H or V L CDRs of the antibody share the indicated sequence identity with the corresponding three V H or V L CDRs of the combined reference antibody.

[0102] In some cases, relative to those in the CDRs of the reference antibodies provided in Table 1 (e.g., BC-05, BC-06, or BC-08), the anti-BCMA portion can collectively include up to 10 amino acid variations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more of the heavy and light chain CDRs. In some cases, the anti-BCMA portion can include the same heavy chain CDR3 as the reference antibody and include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0103] In some instances, the anti-BCMA portions disclosed herein can be any of the anti-BCMA scFv fragments provided in Table 1 above. In a specific instance, the anti-BCMA scFv can include the amino acid sequence of SEQ ID NO:41, 50, or 62. Alternatively, the anti-BCMA portion can include an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to the sequences provided in Table 1 (e.g., SEQ ID NO:41, 50, or 62). In other instances, the anti-BCMA portions disclosed herein can include the same V H and V L sequences as those listed in Table 1, but with the opposite V H and V L fragment orientation.

[0104] Any of the anti-BCMA portions in the anti-BCMA portions disclosed herein (e.g., those provided in Table 1, such as SEQ ID NO:41, 50, or 62 or their counterparts with the opposite V H and V L orientation) can be used to construct anti-BCMA monospecific CAR constructs and / or anti-BCMA / anti-TACI bispecific CAR constructs as disclosed herein.

[0105] (b) Anti-TACI binding portion

[0106] The anti-TACI binding portion in any of the CARs disclosed herein (e.g., any anti-TACI monospecific CAR and / or anti-BCMA / anti-TACI bispecific CAR in the anti-TACI monospecific CARs and / or anti-BCMA / anti-TACI bispecific CARs disclosed herein) can be in the form of an scFv, which is a heavy chain variable domain (V H ) of an anti-TACI antibody and a light chain variable domain (V Lfusion polypeptide. In the scFv fragment, V H and V L fragments can be in any orientation. In some cases, the scFv can include, from the N-terminus to the C-terminus, V L fragment, a peptide linker, and V H fragment. Alternatively, the scFv can include, from the N-terminus to the C-terminus, V H fragment, a peptide linker, and V L fragment. In some instances, the scFv can further include an N-terminal signal peptide for directing the CAR comprising the scFv to the cell surface.

[0107] In some embodiments, the anti-TACI binding portion can be derived from any of those provided in Table 2 below. The heavy and light chain complementarity determining regions provided in Table 2 (and in Table 1) are based on the Chothia definition.

[0108] Table 2: Anti-TACI antibodies

[0109]

[0110]

[0111]

[0112] In some instances, the anti-TACI binding portion can include a heavy chain CDR that is the same as the heavy chain CDR in any of the reference antibodies listed in Table 2 above (e.g., TC-01). Alternatively or additionally, the anti-TACI binding portion can have a light chain CDR that is the same as the light chain CDR in a reference antibody (e.g., TC-01). Such anti-TACI binding portions can include the same V H and / or V L chains as the reference antibody. Alternatively, the anti-TACI binding portion can include amino acid variations in one or more framework regions relative to the corresponding framework regions in the reference antibody. For example, relative to the corresponding framework regions in the reference antibody, the anti-TACI binding portion can collectively include up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions.

[0113] In some embodiments, relative to those of the reference antibody (e.g., TC-01) provided in Table 2 above, the anti-TACI binding portion can include a certain level of variation in one or more CDRs. For example, the anti-TACI binding portion can include a V HCDRs that have at least 80% (e.g., 85%, 90%, 95% or 98%) sequence identity, either alone or in combination, to the heavy chain CDRs. Alternatively or additionally, the anti-TACI antibody can comprise V L CDRs that have at least 80% (e.g., 85%, 90%, 95% or 98%) sequence identity, either alone or in combination, to the light chain CDRs.

[0114] In some cases, relative to those in the CDRs of the reference antibody (e.g., TC-01) provided in Table 2, the anti-TACI binding portion can comprise, in one or more of the heavy and light chain CDRs, a total of up to 10 amino acid variations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variations), either alone or in combination. In some cases, the anti-TACI binding portion can comprise the same heavy chain CDR3 as the reference antibody and one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0115] In some instances, the anti-TACI binding portions disclosed herein can comprise the amino acid sequence of any of the anti-TACI scFv fragments provided in anti-TACI scFv fragments in Table 2 above (e.g., SEQ ID NO:75). Alternatively, the anti-TACI portion can comprise an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98% or more) identical to the amino acid sequence of the reference antibody provided in Table 2 (e.g., SEQ ID NO:75). In other instances, the anti-TACI binding portions disclosed herein can comprise V H and V L sequences that are identical to the V H and V L sequences, but have the opposite V H and V L fragment orientation compared to the reference antibody.

[0116] Any of the anti-TACI binding portions disclosed herein (e.g., those provided in Table 2) can be used to construct anti-TACI monospecific and / or anti-BCMA / anti-TACI bispecific CARs as disclosed herein. In some instances, the anti-TACI portion can comprise the amino acid sequence of SEQ ID NO:75 or its counterpart with the opposite V H and V L orientation.

[0117] (c) Other components of the chimeric antigen receptor construct

[0118] In addition to the extracellular antigen-binding domains disclosed herein, any CAR in a CAR (including an anti-BCMA CAR, an anti-TACI CAR, or an anti-BCMA / anti-TACI bispecific CAR) can further include one or more intracellular signaling domains (e.g., a costimulatory signaling domain and a cytoplasmic signaling domain), and optionally a hinge domain, a transmembrane domain, an N-terminal signal peptide, or a combination thereof. In some cases, the CAR can be co-expressed with an armored polypeptide in a host immune cell, which can enhance the physical and / or biological characteristics of the host immune cell. See, e.g., the disclosure herein. For example, the CAR coding sequence and a suicide gene can be configured in a bicistronic expression cassette, where the CAR coding sequence and the armored gene can be linked by a self-cleaving peptide (e.g., P2A or T2A) coding sequence. Examples are provided in Table 3 below.

[0119] Table 3: Exemplary components of chimeric antigen receptor constructs.

[0120]

[0121]

[0122] Signaling Domain

[0123] Any CAR construct in the CAR constructs disclosed herein (including an anti-BCMA CAR, an anti-TACI CAR, or an anti-BCMA / anti-TACI bispecific CAR) includes one or more intracellular signaling domains, and the one or more intracellular signaling domains generally contain a costimulatory domain and a cytoplasmic signaling domain. A "costimulatory signaling domain" refers to at least a fragment of a costimulatory signaling protein that mediates intracellular signal transduction to induce an immune response such as effector function (secondary signal). The cytoplasmic signaling domain can be any signaling domain that is involved in triggering cell signaling (primary signaling) leading to the proliferation and / or activation of immune cells. The cytoplasmic signaling domain as described herein is not a costimulatory signaling domain, which, as is known in the art, transmits a costimulatory or secondary signal to fully activate immune cells.

[0124] In some embodiments, the co-stimulatory signaling domain and the cytoplasmic signaling domain are for the CAR constructs to be introduced into T cells as disclosed herein. In some cases, the co-stimulatory signaling domain can be derived from co-stimulatory proteins involved in T cell responses, e.g., members of the B7 / CD28 family, members of the TNF superfamily, members of the SLAM family, or any other co-stimulatory molecule. Examples include but are not limited to 4-1BB, CD28, OX40, ICOS, CD40, CD40L, CD27, GITR, HVEM, TIM1, LFA1 (CD11a), or CD2. In a specific example, the co-stimulatory signaling domain is the 4-1BB signaling domain (e.g., SEQ ID NO:116 in Table 3 above). In other specific examples, the co-stimulatory signaling domain is the CD28 signaling domain (e.g., SEQ ID NO:117 in Table 3 above).

[0125] The cytoplasmic signaling domain can include an immunoreceptor tyrosine-based activation motif (ITAM) domain or can be ITAM-free. As used herein, "ITAM" is a conserved protein motif typically present in the tails of signaling molecules expressed in many immune cells. Exemplary cytoplasmic signaling domains include the signaling domain of CD3ζ, e.g., SEQ ID NO:118.

[0126] Hinge Domain and Transmembrane Domain

[0127] In some cases, the CAR constructs disclosed herein (e.g., any anti-BCMA CAR, anti-TACI CAR, or anti-BCMA / anti-TACI bispecific CAR among the anti-BCMA CARs, anti-TACI CARs, or anti-BCMA / anti-TACI bispecific CARs disclosed herein) can contain a transmembrane domain, which can be a hydrophobic α-helix spanning the membrane. A "transmembrane domain" can be a peptide fragment that is thermodynamically stable in the cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability to the CAR containing such a domain. Exemplary transmembrane domains can be the CD8 transmembrane domain or the CD28 transmembrane domain. In one example, the transmembrane domain can include SEQ ID NO:109, 110, 111, or 112 shown in Table 3 above.

[0128] Alternatively or additionally, the CAR constructs disclosed herein may also include a hinge domain, which may be located between the extracellular antigen-binding domain and the transmembrane domain or between the transmembrane domain and the intracellular signaling domain. The hinge domain may be used to provide flexibility to the CAR or its domains, or to prevent steric hindrance of the CAR or its domains. The hinge domain may contain 5-20 amino acid residues. In some embodiments, the hinge domain may be a CD8 hinge domain or an IgG hinge. Other hinge domains may be used. In one example, the hinge domain may include SEQ ID NO:106 shown in Table 3 above.

[0129] (d) Anti-BCMA / anti-TACI bispecific CAR

[0130] In some aspects, the present disclosure provides anti-BCMA / anti-TACI bispecific CARs, each of which includes an anti-BCMA binding portion (e.g., an anti-BCMA scFv, such as the anti-BCMA scFv disclosed herein; see Table 1 above, such as SEQ ID NO:50), an anti-TACI portion (e.g., an anti-TACI scFv, such as the anti-TACI scFv disclosed; see Table 2 above, such as SEQ ID NO:75), one or more intracellular signaling domains (such as a co-stimulatory signaling domain and a cytoplasmic signaling domain), and optionally a hinge domain and a transmembrane domain as disclosed herein. In some cases, the anti-BCMA / anti-TACI bispecific CAR may be a single polypeptide that includes both an anti-BCMA portion and an anti-TACI portion. In other cases, the anti-BCMA / anti-TACI bispecific CAR may be a multi-chain (e.g., 2-chain) molecule. The anti-BCMA portion and the anti-TACI portion may be located on separate polypeptides.

[0131] In some embodiments, the anti-BCMA / anti-TACI bispecific CARs disclosed herein may include an anti-BCMA binding portion (e.g., scFv) derived from BC-06 and an anti-TACI binding portion (e.g., scFv) derived from TC-01.

[0132] The anti-BCMA binding portion (e.g., scFv) derived from BC-06 may be any of the anti-BCMA portions in the anti-BCMA portions related to BC-06 disclosed above. In some cases, it may include the same heavy chain and / or light chain CDRs as BC-06. In a specific example, the scFv may include the same V H and / or the same V L . In some cases, the scFv may include V LFragment (e.g., SEQ ID NO:49), peptide linker (e.g., SEQ ID NO:104), and V H Fragment (e.g., SEQ ID NO:45). Alternatively, the scFv can comprise, from the N-terminus to the C-terminus, V H Fragment (e.g., SEQ ID NO:45), peptide linker (e.g., SEQ ID NO:104), and V L Fragment (e.g., SEQ ID NO:49). In a specific instance, the anti-BCMA binding portion can comprise SEQ ID NO:50.

[0133] The anti-TACI binding portion (e.g., scFv) derived from TC-01 can be any of the anti-TACI binding portions in the anti-TACI binding portions related to TC-01 disclosed above. In some cases, it comprises the same heavy-chain and / or light-chain CDRs as TC-01. In a specific instance, the scFv can comprise the same V H and / or the same V L . In some cases, the scFv can comprise, from the N-terminus to the C-terminus, V L Fragment (e.g., SEQ ID NO:74), peptide linker (e.g., SEQ ID NO:104), and V H Fragment (e.g., SEQ ID NO:70). Alternatively, the scFv can comprise, from the N-terminus to the C-terminus, V H Fragment (e.g., SEQ IDNO:70), peptide linker (e.g., SEQ ID NO:104), and V L Fragment (e.g., SEQ ID NO:74). In a specific instance, the anti-TACI portion can comprise SEQ ID NO:75.

[0134] In some embodiments, the bispecific anti-BCMA / anti-TACI binding portion can be located on a single polypeptide. In some instances, the single polypeptide contains a spacer (peptide linker) between the anti-BCMA binding portion and the anti-TACI binding portion. Examples are provided in Table 3. See the examples below.

[0135] Any fusion polypeptide in the fusion polypeptide comprising an anti-BCMA and an anti-TACI portion can further comprise a costimulatory signaling domain and a cytoplasmic signaling domain, such as the costimulatory signaling domain and the cytoplasmic signaling domain disclosed herein. Optionally, the fusion polypeptide can further comprise a hinge domain and a transmembrane domain also as disclosed herein. In some instances, the bispecific CAR can be included in a polycistronic expression cassette with an armored gene (e.g., those listed in Table 11 below) via a self-cleaving peptide linker.

[0136] (e) Anti-BCMA or anti-TACI monospecific CAR

[0137] Also within the scope of the present invention are anti-BCMA or anti-TACI monospecific CARs comprising any anti-BCMA binding portion or anti-TACI binding portion of the anti-BCMA binding portions or anti-TACI binding portions disclosed herein.

[0138] In some aspects, provided herein are anti-BCMA CARs, nucleic acids encoding the anti-BCMA CARs, and host cells expressing the anti-BCMA CARs. The anti-BCMA CARs can comprise (a) an extracellular binding domain, which can be any anti-BCMA binding portion of the anti-BCMA binding portions, e.g., an anti-BCMA scFv derived from any of the reference antibodies provided in Table 1 above (e.g., BC-05, BC-06, or BC-08); (b) a co-stimulatory signaling domain, such as the co-stimulatory signaling domains disclosed herein; and (c) a cytoplasmic signaling domain, such as the cytoplasmic signaling domains disclosed herein. The anti-BCMA CARs can further comprise a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. In one example, the anti-BCMA CAR comprises any of the amino acid sequences of SEQ ID NOs: 119-142 (e.g., SEQ ID NO: 127 or 128).

[0139] In some aspects, provided herein are anti-TACI CARs, nucleic acids encoding the anti-TACI CARs, and host cells expressing the anti-TACI CARs. In some examples, the anti-TACI CARs can comprise (a) an extracellular binding domain, which can be any anti-TACI binding portion of the anti-TACI binding portions, e.g., an anti-TACI scFv derived from any of the reference antibodies provided in Table 2 above; (b) a co-stimulatory signaling domain, such as the co-stimulatory signaling domains disclosed herein; and (c) a cytoplasmic signaling domain, such as the cytoplasmic signaling domains disclosed herein. The anti-TACI CARs can further comprise a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. In one example, the anti-TACI CAR comprises any of the amino acid sequences of SEQ ID NOs: 143-164 (e.g., SEQ ID NO: 143 or 144).

[0140] Exemplary anti-BCMA monospecific CARs, anti-TACI monospecific CARs, and anti-BCMA / anti-TACI bispecific CARs are provided in Table 4 below, all of which are within the scope of the present disclosure.

[0141] Table 4: Exemplary Monospecific and Bispecific CAR Constructs

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] II.CAR-Expressing Immune Cells

[0152] In some aspects, the present disclosure provides genetically engineered immune cells, such as T cells, NK cells, or macrophages, that have surface expression of any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs disclosed herein. In some cases, the genetically engineered immune cells are T cells that express any of the anti-BCMA / TACI bispecific CARs provided in Table 4 above (e.g., SEQ ID NO: 165 or 166).

[0153] (a) Armored CAR-T Cells

[0154] Any of the CAR-expressing immune cells disclosed herein can be engineered to further have additional mechanisms to reprogram the CAR-expressing cells in order to enhance their bioactivity and / or persistence, thereby enhancing the overall therapeutic effect. For example, the CAR-expressing immune cells can be further engineered to express armored polypeptides, thereby enhancing the physical and / or biological characteristics of the CAR-T cells. Such CAR-T cells are referred to as armored CAR-T cells, which co-express one or more CAR constructs and armored polypeptides or combinations thereof that can enhance CAR-T cell characteristics (e.g., improve growth and / or persistence, enhance efficacy, reduce toxicity, etc.).

[0155] Exemplary armored polypeptides include, but are not limited to, suitable cytokines (such as IL-2, IL-5, and / or IL-15), co-stimulatory ligands (e.g., CD80 or CD86), checkpoint inhibitors (e.g., anti-PD1 or anti-PDL1 antibody fragments), soluble receptors (such as soluble PD1, TGFR2 trap, or VEGFR2 trap), and / or immune cell activation ligands (e.g., 4-1BBL). In some embodiments, the armored polypeptide can be a fusion polypeptide that includes, for example, a cytokine or a fragment thereof (e.g., IL2 or IL15 or a fragment thereof) and a checkpoint inhibitor (e.g., an anti-PDL1 fragment). Specific examples of armored polypeptides are provided in Table 11 below, and each of the armored polypeptides is within the scope of the present disclosure.

[0156] Table 11: Exemplary Armored Polypeptides

[0157]

[0158]

[0159] Additional information on armored polypeptides can be found, for example, in WO2021 / 030633 and WO2022 / 159771, and the relevant disclosures of each of these documents are incorporated herein by reference for the subject matter and purposes cited herein.

[0160] In some cases, the coding sequences of the CAR construct and the armored polypeptide can be located in the same expression cassette. The two coding sequences can be separated by a coding sequence of an internal ribosome entry site (IRES) or a self-cleaving peptide (e.g., P2A or T2A) to produce two separate polypeptides (the CAR and the armored polypeptide). In other cases, two separate expression cassettes can be used to express the CAR construct and the armored polypeptide in armored CAR-T cells.

[0161] In some instances, the armored polypeptide contains an N-terminal signal peptide such that the polypeptide can be secreted from the CAR-T cell. Alternatively, the armored polypeptide can be expressed as an intracellular protein or a membrane-bound protein.

[0162] (a) Preparation of CAR-Expressing Immune Cells

[0163] The genetically engineered immune cells disclosed herein can be prepared by introducing one or more expression cassettes encoding any of the CAR constructs disclosed herein (e.g., any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs disclosed herein, such as those provided in Table 4), optionally one or more armored polypeptides (such as the armored polypeptides disclosed herein) into a suitable immune cell and collecting the resulting engineered immune cells that express the CAR on the cell surface.

[0164] A population of immune cells as the starting parental cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissue (such as spleen, lymph node, thymus, stem cells, or tumor tissue). Sources suitable for obtaining the desired host cell type will be apparent to those skilled in the art. In some embodiments, the immune cell population is derived from PBMCs. The desired host cell type (e.g., T cells, NK cells, macrophages, or a combination thereof) can be expanded in a cell population obtained by co-incubating the cells with a stimulating molecule. As a non-limiting example, anti-CD3 and anti-CD28 antibodies can be used to expand T cells. In some embodiments, a specific type of cell (e.g., T cells, NK cells, or macrophages) can be enriched from the immune cell population. Such enriched cell subsets can be amplified and / or activated in vitro prior to genetically engineering the introduction of the expression cassette encoding the CAR and / or the expression cassette encoding the armored polypeptide (which can be the same expression cassette).

[0165] To construct immune cells expressing any of the CAR polypeptides described herein (e.g., any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs in the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs disclosed herein, such as those provided in Table 4), optionally one or more armored polypeptides (such as the armored polypeptides disclosed herein) (e.g., those provided in Table 11), expression vectors for stable or transient expression of the CAR polypeptide and optionally the armored polypeptide can be generated by conventional methods and introduced into immune host cells. For example, nucleic acids encoding the CAR polypeptide and optionally the armored polypeptide can be cloned into one or more suitable expression vectors, such as viral vectors operably linked to a suitable promoter. Non-limiting examples of useful vectors in the present disclosure include viral vectors, such as, for example, retroviral vectors (including γ-retroviral vectors), adeno-associated virus vectors (AAV vectors), and lentiviral vectors. The nucleic acid and the vector can be contacted with a restriction enzyme under suitable conditions to generate complementary ends on each molecule that can pair with each other and be ligated with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the ends of the nucleic acids encoding the CAR polypeptide and optionally the armored polypeptide. The synthetic linker can contain a nucleic acid sequence corresponding to a specific restriction site in the vector. The choice of expression vector / plasmid / viral vector will depend on the type of host cell expressing the CAR polypeptide and optionally the armored polypeptide, but should be suitable for integration and replication in eukaryotic cells. Any such nucleic acids encoding the CAR and optionally the armored polypeptide and expression vectors comprising said nucleic acids are also within the scope of the present disclosure.

[0166] A variety of promoters can be used to express the CAR polypeptides described herein and optionally the armored polypeptides, including but not limited to the cytomegalovirus (CMV) immediate early promoter, viral long terminal repeats (LTRs) such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, etc., the simian virus 40 (SV40) early promoter, or the herpes simplex tk virus promoter. Additional promoters for expressing the CAR polypeptide and optionally the armored polypeptide include any constitutively active promoter in immune cells. Alternatively, any inducible promoter can be used such that its expression can be regulated within the immune cell. In some embodiments, the promoter can be the pEF1α promoter.

[0167] In addition, the vector can contain, for example, some or all of the following: selectable marker genes for the selection of stable or transient transfectants in host cells, such as the neomycin gene or the kanamycin gene; enhancer / promoter sequences from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; the SV40 polyomavirus origin of replication and ColE1 for appropriate episomal replication; internal ribosome entry sites (IRES); universal polylinker sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; "suicide switches" or "suicide genes" that cause the death of cells carrying the vector when triggered (e.g., HSV thymidine kinase or inducible caspases such as iCasp9); and reporter genes for assessing CAR polypeptide expression.

[0168] In one specific embodiment, such vectors can also contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene can be a gene that confers sensitivity of cells expressing the gene to an agent (e.g., a drug) and causes cell death when the cells are contacted or exposed to the agent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

[0169] The nucleic acids disclosed herein can include two coding sequences, one coding sequence for any of the CAR constructs disclosed herein (e.g., any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs disclosed herein, such as those provided in Table 4), and another coding sequence for an armored polypeptide. The two coding sequences can be configured such that the polypeptides encoded by the two coding sequences can be expressed as separate (and physically distinct) polypeptides. To achieve this, the nucleic acids described herein can contain a third nucleotide sequence located between the first coding sequence and the second coding sequence. This third nucleotide sequence can, for example, encode a ribosomal skipping site. A ribosomal skipping site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of an additional open reading frame from one messenger RNA. This third nucleotide sequence can, for example, encode a self-cleaving peptide, such as a P2A, T2A, or F2A peptide (see, e.g., Kim et al., PLoS One. 2011;6(4):e18556). See also Figure 3 。

[0170] Also within the scope of the present disclosure are any vectors containing nucleic acid sequences encoding the ACTR polypeptides described herein and optionally an armored polypeptide.

[0171] Such vectors or the sequences contained therein encoding the CAR polypeptides and optionally an armored polypeptide can be delivered into host cells, such as host immune cells (e.g., T cells, NK cells, or macrophages), by any suitable method. Methods for delivering vectors to immune cells are well known in the art and can include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction, such as lentiviral transduction).

[0172] After introducing into a host cell a vector encoding any CAR polypeptide provided herein (e.g., an anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR construct, such as those provided in Table 4), optionally also an armored polypeptide as provided herein, the cells can be cultured under conditions that permit expression of the CAR polypeptide and optionally the armored polypeptide. When expression of the CAR polypeptide and / or armored polypeptide is regulated by an inducible promoter, the host cells can be cultured under conditions in which the inducible promoter is activated. In some embodiments, the promoter is an inducible promoter, and immune cells are cultured in the presence of an inducing molecule or under conditions that result in the production of an inducing molecule. It will be apparent to those skilled in the art to determine whether the CAR polypeptide and / or armored polypeptide is expressed, and it can be evaluated by any known method, e.g., by detecting the mRNA encoding the CAR polypeptide and / or encoding the armored polypeptide by quantitative reverse transcriptase PCR (qRT-PCR) or by detecting the CAR or armored polypeptide protein by methods including Western blotting, fluorescence microscopy, and flow cytometry. Alternatively, expression of a functional CAR can be determined by binding activity and / or CTL activity against cells expressing a target antigen, e.g., BCMA and / or TACI.

[0173] A method for preparing a host cell that expresses any CAR polypeptide provided herein and optionally an armored polypeptide can further include activating the host cell ex vivo. Activating the host cell means stimulating the host cell into an activated state in which the cell may be capable of performing effector functions. The method for activating the host cell will depend on the type of host cell used for expressing the CAR polypeptide and optionally the armored polypeptide. For example, T cells can be activated ex vivo in the presence of one or more molecules including, but not limited to: anti-CD3 antibody, anti-CD28 antibody, IL-2, and / or phytohemagglutinin. In other examples, NK cells can be activated ex vivo in the presence of one or more molecules such as 4-1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL12, IL-21, and / or K562 cells. In some embodiments, host cells expressing any CAR polypeptide provided herein (CAR-expressing cells) and optionally an armored polypeptide (armored CAR cells) are activated ex vivo prior to administration to a subject. It will be apparent to those skilled in the art to determine whether the host cell is activated, and it can include evaluating the expression of one or more cell surface markers associated with cell activation, cytokine expression or secretion, and cell morphology.

[0174] Methods for preparing host cells for expressing any of the CAR polypeptides described herein and optionally an armored polypeptide may include ex vivo expansion of the host cells. Expansion of the host cells may involve any method that causes an increase in the number of cells expressing the CAR polypeptide and optionally the armored polypeptide, such as allowing the host cells to proliferate or stimulating the host cells to proliferate. The method for stimulating host cell expansion will depend on the type of host cell used for expressing the CAR polypeptide and optionally the armored polypeptide and will be apparent to those skilled in the art. In some embodiments, the host cells expressing any of the CAR polypeptides described herein, optionally the armored polypeptide, are expanded ex vivo prior to administration to a subject.

[0175] In some embodiments, prior to administering the cells to a subject, the host cells expressing the CAR polypeptide and optionally the armored polypeptide are expanded ex vivo and activated. Host cell activation and expansion can be used to allow integration of a viral vector into the genome and expression of the gene encoding the CAR polypeptide and optionally the armored polypeptide as described herein. If mRNA electroporation is used, activation and / or expansion may not be required, although electroporation on activated cells may be more efficient.

[0176] In some cases, the CAR polypeptide and / or the armored polypeptide is transiently expressed (e.g., for 3 - 5 days) in a suitable host cell. Transient expression may be advantageous if there is potential toxicity and should facilitate the initial stages of clinical testing for possible side effects.

[0177] (b) Pharmaceutical compositions

[0178] Any of the genetically engineered immune cells expressing a CAR and optionally an armored polypeptide as disclosed herein (e.g., any of the anti - BCMA, anti - TACI, or anti - BCMA / TACI bispecific CAR constructs, such as those provided in Table 4 above) can be admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0179] The phrase "pharmaceutically acceptable" as used in connection with the compositions of the present disclosure refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and generally produce no adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in mammals and, more particularly, in humans. "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, carriers, cells, or therapeutic antibodies) and will not have a negative effect on the subject to which the composition is administered. Any pharmaceutical composition for use in the methods of the invention may include a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution.

[0180] Pharmaceutically acceptable carriers (including buffers) are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, edited by K.E. Hoover.

[0181] For additional examples of medicaments used, see also: Physician's Desk Reference, 59th Edition, (2005), Thomson P D R, Montvale N.J.; edited by Gennaro et al., Remington: The Science and Practice of Pharmacy, 20th Edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; edited by Braunwald et al., Harrison's Principles of Internal Medicine, 15th Edition, (2001), McGraw Hill, New York; edited by Berkow et al., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0182] IV. Therapeutic Applications

[0183] Any genetically engineered immune cell (e.g., T cell, NK cell or macrophage) expressing a CAR as disclosed herein (e.g., any anti-BCMA, anti-TACI or anti-BCMA / TACI bispecific CAR construct provided in Table 4) and optionally also an armored polypeptide as disclosed herein (e.g., those provided in Table 11) can be used for therapeutic purposes, e.g., for eliminating undesired cells expressing BCMA and / or TACI. In some examples, the genetically engineered immune cell is an armored CAR-T cell that expresses any anti-BCMA, anti-TACI or anti-BCMA / TACI bispecific CAR construct (such as those provided in Table 4 above) and an armored polypeptide (such as those provided in Table 11 above).

[0184] To practice the methods described herein, an effective amount of immune cells (NK cells, T lymphocytes, or macrophages) or a pharmaceutical composition thereof that express any of the CARs described herein (e.g., any anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR construct, such as those provided in Table 4 above) and optionally an armored polypeptide (e.g., those provided in Table 11 above) can be administered to a subject in need of treatment by a suitable route (such as intravenous administration). As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NK cells, T lymphocytes, or macrophages expressing a CAR and optionally an armored polypeptide) that confers a therapeutic effect upon administration to the subject. Determining whether the amount of the cells or compositions described herein achieves a therapeutic effect will be apparent to those skilled in the art. As recognized by those skilled in the art, the effective amount varies depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, gender / sex, and weight, the duration of treatment, the nature of concurrent therapies (if any), the specific route of administration, and similar factors within the knowledge and expertise of a health practitioner. In some embodiments, the effective amount alleviates, slows, reduces, improves, or delays the progression of the symptoms of any disease or disorder in the subject. In some embodiments, the subject is human. In some embodiments, the subject in need of treatment is a human cancer patient.

[0185] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce a desired activity upon administration to a subject in need thereof. Note that when combinations of active ingredients are administered, the effective amount of the combination may or may not include the amounts of each ingredient that would be effective if administered alone. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a compound or pharmaceutical composition sufficient to delay the manifestation of a condition being treated by the methods of the present disclosure, prevent its development, slow or alleviate at least one of its symptoms.

[0186] In some embodiments, the methods of the present disclosure can be used to eliminate or inhibit disease cells expressing BCMA and / or TACI. Accordingly, any of the immune cells disclosed herein can be used to treat diseases associated with BCMA + and / or TACI + disease cells (such as BCMA + and / or TACI + cancer cells). The methods disclosed herein can be used to treat diseases involving BCMA + and / or TACI +Cancers of cancer cells, such as multiple myeloma, lung cancer, gastric cancer, breast cancer, testicular cancer, etc.

[0187] In some embodiments, an effective amount of a genetically engineered immune cell expressing a CAR as disclosed herein (e.g., any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs provided in Table 4) and optionally an armored polypeptide (e.g., those provided in Table 11) can be administered to a subject in need of treatment by a suitable route (e.g., intravenous infusion). The subject can be a human patient suffering from a disease associated with BMCA + and / or TACI + disease cells such as BCMA + and / or TACI + cancer cells. In some cases, the human patient has a cancer involving BCMA + and / or TACI + cancer cells. In some cases, the human patient may have multiple myeloma, lung cancer, gastric cancer, breast cancer, or testicular cancer.

[0188] In some embodiments, the immune cells (e.g., NK and / or T cells) used for the treatment disclosed herein can be autologous to the subject, i.e., the immune cells can be obtained from the subject in need of treatment, genetically engineered to express a CAR polypeptide, and then administered to the same subject. In a specific embodiment, autologous immune cells (e.g., T lymphocytes, NK cells, or macrophages) are activated and / or expanded ex vivo before reintroduction into the subject. Administering autologous cells to a subject may result in reduced rejection of the host cells compared to administering non-autologous cells.

[0189] Alternatively, the genetically engineered immune cells (e.g., T cells, NK cells, or macrophages) can be allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered to express a CAR polypeptide, and administered to a second subject different from the first subject but of the same species. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor. In a specific embodiment, the T lymphocytes are allogeneic T lymphocytes in which the expression of the endogenous T cell receptor is inhibited or eliminated. In a specific embodiment, allogeneic T lymphocytes are activated and / or expanded ex vivo before introduction into the subject. The T lymphocytes can be activated by any method known in the art, such as in the presence of anti-CD3 / CD28, IL-2, and / or phytohemagglutinin.

[0190] NK cells can be activated by any method known in the art, for example, in the presence of one or more agents selected from the group consisting of: CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, and K562 cell line. For a description of useful methods for expanding NK cells, see, for example, U.S. Patent Nos. 7,435,596 and 8,026,097. For example, the NK cells used in the methods of the present disclosure can be preferentially expanded by exposure to cells that lack or poorly express major histocompatibility complex I and / or II molecules, and the cells have been genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CD137L). Such cell lines include, but are not necessarily limited to, K562 [ATCC, CCL 243; Lozzio et al., Blood 45(3):321-334 (1975); Klein et al., Int. J. Cancer 18:421-431 (1976)], and Wilms' tumor cell line HFWT (Fehniger et al., Int Rev Immunol 20(3-4):503-534 (2001); Harada H et al., Exp Hematol 32(7):614-621 (2004)), endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoblastoma cell line HuH-6, small cell lung cancer cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB 19 and N1369, embryonal carcinoma cell line NEC 14 from testis, cervical cancer cell line TCO-2, and bone marrow metastatic neuroblastoma cell line TNB 1 [Harada et al., Jpn. J. Cancer Res 93:313-319 (2002)]. Preferably, the cell lines used lack or poorly express both MHC I and II molecules, such as the K562 and HFWT cell lines. A solid support can be used instead of the cell line. Such a support should preferably have attached to its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and / or a proliferative response, or capable of binding to a molecule having such an effect, thereby acting as a scaffold. The support can have CD137 ligand protein, CD137 antibody, IL-15 protein, or IL-15 receptor antibody attached to its surface. Preferably, the support will have an IL-15 receptor antibody and a CD137 antibody bound to its surface.

[0191] According to the present disclosure, a patient can be treated by infusing a therapeutically effective dose of immune cells (such as T lymphocytes or NK cells) expressing a CAR polypeptide (such as the anti-BCMA monospecific CAR, anti-TACI monospecific CAR, or anti-BCMA / anti-TACI bispecific CAR listed in Table 4 above) and optionally an armored polypeptide listed in Table 11 in the range of about 10 5 to 10 9 CAR+ cells. The infusion can be repeated at a frequency and number of times that the patient can tolerate until a desired response is achieved. Appropriate infusion doses and schedules will vary from patient to patient but can be determined by the treating physician for a particular patient. In some examples, an initial dose of about 10 6 cells / Kg can be infused and gradually increased to 10 8 or more cells / kg.

[0192] The specific dosing regimens used in the methods described herein, i.e., dose, timing, and repetition, will depend on the specific subject and the subject's medical history. The appropriate dose of the CAR-expressing immune cells (e.g., armored CAR-T cells) used will depend on the type of cancer to be treated, the severity and course of the disease, previous therapies, the patient's clinical history and response to immunotherapy, and the judgment of the attending physician.

[0193] In some embodiments, genetically engineered immune cells (e.g., armored CAR-T cells) expressing any of the CAR constructs disclosed herein (e.g., anti-BCMA CAR, anti-TACI CAR, or anti-BCMA / TACI bispecific CAR) can be used in combination with other types of therapies for cancer (such as chemotherapy, surgery, radiotherapy, gene therapy, etc.). Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent can be reduced due to additive or synergistic effects.

[0194] V. Kits for Therapeutic Applications

[0195] The present disclosure also provides kits for using genetically engineered immune cells (such as T lymphocytes, NK cells, or macrophages) expressing an anti-BCMA CAR, anti-TACI CAR, or anti-BCMA / anti-TACI bispecific CAR as described herein and optionally an armored polypeptide. See, for example, Table 11. Such kits can include one or more containers that include the genetically engineered immune cells, which can be formulated in a pharmaceutical composition further including a pharmaceutically acceptable carrier.

[0196] In some embodiments, the kits described herein include genetically engineered immune cells that can be expanded in vitro. The immune cells can express any of the CARs disclosed herein, e.g., any of the anti-BCMACAR, anti-TACICAR, and anti-BCMA / TACI bispecific CARs, such as those provided in Table 4 above. The immune cells can be armored CAR-T or CAR-NK cells that further express an armored polypeptide.

[0197] In some embodiments, the kit can additionally include instructions for any of the methods described herein. The included instructions can include a description of administering the genetically engineered immune cells disclosed herein to achieve a desired activity in a subject, e.g., eliminating target disease cells (such as cancer cells expressing BCMA, TACI, or both). The kit can further include a description of selecting a subject suitable for treatment based on identifying whether the subject requires treatment.

[0198] Instructions related to using the genetically engineered immune cells described herein generally contain information about the dosage, dosing regimen, and route of administration of the intended treatment. The container can be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the genetically engineered immune cells are for treating a disease or disorder associated with BCMA- and / or TACI-positive disease cells in a subject, delaying the onset of the disease or disorder, and / or alleviating the disease or disorder.

[0199] The kits provided herein employ suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packages, etc. Packaging in combination with a specific device (such as an inhaler, nasal administration device, or infusion device) is also contemplated. The kit can have a sterile inlet port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile inlet port.

[0200] The kit can optionally provide additional components such as buffers and explanatory information. Generally, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article comprising the contents of the above-described kit.

[0201] General technology

[0202] Unless otherwise indicated, the practice of the present disclosure will employ conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology techniques within the skill of the art. Such techniques are well explained in documents such as: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1989) Academic Press; Animal Cell Culture (edited by R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993 - 8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.P. Calos, 1987); Current Protocols in Molecular Biology (F.M.edited by Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); "Current Protocols in Immunology" (edited by J.E. Coligan et al., 1991); "Short Protocols in Molecular Biology" (John Wiley & Sons, 1999); "Immunobiology" (C.A. Janeway and P. Travers, 1997); "Antibodies" (P. Finch, 1997); "Antibodies: a practical approach" (edited by D. Catty., IRL Press, 1988 - 1989); "Monoclonal antibodies: a practical approach" (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); "Using antibodies: a laboratory manual" (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); "Antibodies" (edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995); "DNA Cloning: A practical Approach", Volumes I and II (edited by D.N. Glover, 1985); "Nucleic Acid Hybridization" (edited by B.D. Hames and S.J. Higgins, (1985)); "Transcription and Translation" (edited by B.D. Hames and S.J. Higgins, (1984)); "Animal Cell Culture" (edited by R.I. Freshney, (1986)); "Immobilized Cells and Enzymes" (IRL Press, 1986); and B.Perbal, A practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.).

[0203] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to the greatest extent based on the above description. Therefore, the following specific examples should be interpreted as merely illustrative and not limiting the remainder of the present disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter mentioned herein.

[0204] Example 1. Generation and characterization of anti-BCMA antibodies

[0205] This example illustrates the identification and characterization of exemplary anti-BCMA antibodies.

[0206] Anti-BCMA scFv antibody screening by mRNA display

[0207] mRNA display technology is used to 12-13 BCMA binders were identified in natural human scFv libraries. In brief, the scFv DNA library was first transcribed into an mRNA library, and then translated into an mRNA-scFv fusion library by covalent coupling of a puromycin linker, similar to the reported procedure (patent: US6258558B1, the relevant disclosure of which is incorporated by reference for the subject matter and purposes cited herein). The fusion library was first counter-selected multiple times with human IgG (negative protein) to remove non-specific binders, followed by selection for recombinant BCMA-Fc fusion protein and capture on protein G magnetic beads. The binder was washed off and then enriched with library-specific oligonucleotides by PCR amplification. In rounds 5-6, the scFv library was selected for the BCMA / HEK293 cell line. A total of 6 rounds of selection were performed to produce a highly enriched BCMA binding pool for screening.

[0208] After 6 rounds of selection, the BCMA-enriched scFv library was cloned into the bacterial periplasmic expression vector pET22b and transformed into the top 10 competent cells. Each scFv molecule was engineered to have a C-terminal tag and a 6xHis tag for purification and assay detection. Clones from the top 10 cells were pooled, and a small amount of DNA was prepared and then transformed into the bacterial Rosetta II strain for expression. Single clones were picked, grown and induced with 0.1mM IPTG in 96-well plates for expression. Supernatants were collected after 16-24 hours of induction at 30°C for assays to identify anti-BCMA antibodies.

[0209] BCMA-binding screening by FACS was developed for the identification of individual anti-BCMA antibodies. Briefly, 100,000 BCMA / CHOK1 cells and CHOK1 cells were seeded into 96-well cell culture plates respectively. The cells and medium were cooled at 4°C and centrifuged at 1200 rpm for 6 minutes. The bacterial supernatant was diluted to a final 30% with cell medium and added to the BCMA / CHOK1 cells. The plates were incubated at 4°C with shaking for 1 hour. The cells were centrifuged as described above and the supernatant was removed. Then the plates were washed with 200 μl of complete medium at 4°C. 100 μl of 1:250 pre-diluted AF-647-conjugated anti-HIS tag antibody was added to the cells. The plates were incubated at 4°C in the dark with shaking for 30 minutes. The plates were washed twice as described and the cells were resuspended in 200 μl of complete medium. The cells were mixed well and read on an Attune NxT hematocytometer. Analysis was performed by using Attune NxT software to plot histograms of overlaying anti-BCMA scFv binding to both the negative cell line and the target cell line.

[0210] Selective anti-BCMA scFv clones were picked from glycerol stock plates and grown overnight in 5 mL cultures in Thomson 24-well plates with breathable membranes. Unless otherwise stated, this culture and all subsequent cultures described below were grown at 37°C and shaken at 225 RPM in Terrific Broth Complete supplemented with 1:5,000 dilution of Antifoam-204 plus 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol. Then the overnight starter culture was used to inoculate a larger culture, with the starter culture diluted 1:100 into the designated production culture and grown to an OD600 between 0.5 - 0.8. At this point, the culture was induced with IPTG at a final concentration of 0.1 mM and incubated overnight at 30°C. The next day, the culture was spun at 5,000 x g for 30 minutes to pellet the cells and then the supernatant was filter-sterilized through a 0.2 μm sterile PES membrane.

[0211] For purification, 3 uL of GE Ni Sepharose Excel resin per 1 mL of filtered supernatant was used. A disposable 10 mL or 20 mL BioRad Econo-Pac column was used. The resin was equilibrated with at least 20 column volumes (CV) of buffer A (1x PBS, pH 7.4, with additional NaCl added to 500 mM). The filtered and sterilized supernatant was purified by controlling the flow rate to 1 mL / min on the same packed resin bed or by pouring twice using gravity flow. Then, the column was washed with the following buffers: 10 CV of buffer A, 20 CV of buffer B (1x PBS, pH 7.4, with additional NaCl added to 500 mM, and 30 mM imidazole). If desired, two Detox buffers were used to remove endotoxin as an optional step. For the purification of 250 mL of expression culture, the antibody-bound column was sequentially washed with 20 CV of buffer C (1x PBS, pH 7.4, with additional NaCl added to 500 mM, 1% Tx114), 20 CV of buffer D (1x PBS, pH 7.4, with additional NaCl added to 500 mM, 1% Tx100 + 0.2% TNBP), and 40 CV of buffer E (1x PBS, pH 7.4, with additional NaCl added to 500 mM). The protein was eluted with elution buffer F (1x PBS, pH 7.4, with additional NaCl added to 500 mM, and 500 mM imidazole) in a total of six fractions (0.5 CV pre-elution, 5 x 1 CV elution). The fractions were run on a Bradford assay (100 ul of diluted Bradford solution + 10 ul of sample). The fractions with bright blue color were combined. The protein concentration was measured by the A280 extinction coefficient. SDS-PAGE gels were used to analyze the purity of the purified antibody.

[0212] Characterization of Exemplary Anti-BCMA scFv Antibodies

[0213] Exemplary anti-BCMA antibodies in scFv form isolated by mRNA library screening as disclosed above were analyzed to determine their antigen-binding affinities. The structural information of the exemplary anti-BCMA antibodies is provided in Table 1 above.

[0214] (a) BCMA Binding Activity Measured by ELISA

[0215] An ELISA assay was developed to determine the EC of the anti-BCMA antibody 50Briefly, a 384-well plate was immobilized with anti-human Fc antibody at a final concentration of 2 μg / mL in 1x PBS with a total volume of 25 μL / well. The plate was incubated overnight at 4 °C and then blocked with 80 μL of superblock / well for 1 hour. Human BCMA Fc was captured by the immobilized anti-hFc antibody. Serial 2-fold titrations of the purified anti-BCMA scFv were started from 200 nM. 25 μL of the diluted scFv was added to the wells immobilized with human BCMA and incubated for 1 hour with shaking. BCMA binding was detected by adding 25 μL of anti-Flag HRP diluted 1:5000 in 1x PBST. Between each step, the plate was washed 3 times with 1x PBST in a plate washer. Then, the plate was developed with 20 μL of TMB substrate for 5 minutes and the development was stopped by adding 20 μL of 2N sulfuric acid. The plate was read at OD450 nm on a Biotek plate reader and then graphed in Prism 8.1 software. The EC 50 value was calculated and shown in Table 5 below.

[0216] Table 5: BCMA binding activity of exemplary anti-BCMA antibodies determined by ELISA

[0217] Antibody (in scFv form) <![CDATA[EC 50 (nM)]]> BC - 01 0.938 BC - 02 1.916 BC - 03 2.319 BC - 04 1.964 BC - 05 0.5357 BC - 06 0.6201 BC - 07 0.8016 BC - 08 0.2989 BC - 09 0.5968

[0218] (b) BCMA Binding Activity Measured by Surface Plasmon Resonance (SPR)

[0219] The kinetic analysis of the anti-BCMA scFv has been evaluated by SPR technology using a Biacore T200. The assay was run using the Biacore T200 control software version 2.0. A Protein A sensor chip was used to capture the Fc fusion protein in the assay. For each cycle, 1 μg / mL of human BCMA-Fc protein was captured on flow cell 2 at a flow rate of 10 μL / min in 1x HBSP buffer on the Protein A sensor chip for 60 seconds. Serial 2-fold dilutions of HIS-tag purified anti-BCMA scFv were injected at a flow rate of 30 μL / min onto both the reference flow cell 1 and the BCMA-Fc capture flow cell 2 for 150 seconds, followed by a wash for 300 seconds. Then, the flow cells were regenerated with glycine pH 2 buffer (GE) at a flow rate of 30 μL / min for 30 seconds. Assays were performed on 8 concentration points of 300 - 0 nM for each anti-BCMA scFv in a 96-well plate. The kinetics of the binding of the scFv to the BCMA protein were analyzed using Biacore T200 evaluation software version 3.0. The specific binding response units were obtained by subtracting the binding to the reference flow cell 1 from the binding to the BCMA capture flow cell 2. The Kon, Koff, and KD values of the exemplary anti-BCMA antibodies (in scFv form) were calculated and shown in Table 6 below.

[0220] Table 6: BCMA Binding Activity of Exemplary Anti-BCMA Antibodies Determined by SPR

[0221] Antibody ka (1 / Ms) kd (1 / s) KD (M) BC - 01 1.44E+05 4.31E-04 3.00E-09 BC - 02 9.16E+04 1.18E-03 1.29E-08 BC - 03 3.02E+05 3.59E-03 1.19E-08 BC - 04 1.14E+05 8.74E-04 7.68E-09 BC - 05 6.24E+04 6.46E-04 1.04E-08 BC - 06 3.19E+05 2.51E-03 7.87E-09 BC - 07 2.26E+05 5.92E-03 2.62E-08 BC - 08 1.78E+06 1.16E-03 6.53E-10 BC - 09 2.08E+05 4.07E-03 1.96E-08

[0222] (c) Binding of scFv Antibody to Cell - Surface BCMA Measured by FACS

[0223] To determine the binding selectivity and affinity of the anti-BCMA scFv for cells expressing BCMA, 200 nM of the purified anti-BCMA scFv antibody was diluted in complete medium and incubated with recombinant BCMA / CHOK1 and CHOK1 cells in a 96-well plate on ice for 1 hour. The cells were centrifuged at 1200 rpm for 6 minutes at 4 °C to remove the primary antibody. Then, the cells were washed once with 200 μL of complete medium / well. Samples were detected with pre-mixed anti-His streptavidin Alexa fluor 647 by adding 100 μL of diluted secondary antibody and incubated for 30 minutes at 4 °C in the dark. The samples were centrifuged at 1200 rpm for 5 minutes at 4 °C and washed twice with 200 μL of 1x PBS / well. The samples were reconstituted in 200 μL of 1x PBS and read on an Attune NxT hematology counter. Analysis was performed by plotting histograms of the overlay of anti-BCMA scFv binding to both the negative cell line and the target cell line using Attune NxT software. The anti-BCMA scFv showed selective binding to BCMA / CHOK1 cells rather than CHOK1 parental cells ( Figure 1 ). BCMA cell binding affinities for exemplary anti-BCMA antibodies (in scFv form) were also generated with serially diluted scFv as described above. EC 50 was calculated and shown in Table 7 below.

[0224] Table 7: BCMA Binding Activity of Exemplary Anti-BCMA Antibodies Determined by FACS

[0225]

[0226]

[0227] (d) Binding of Anti - BCMA Antibody to Endogenous BCMA - Expressing Cell Lines

[0228] To further characterize BCMA and TACI expression in recombinant and endogenous cell lines, a quantitative FACS assay was performed using Bunsen Laboratories Quantum Alexa Fluor 647 MESF microspheres for standard calibration according to the manufacturer's protocol. The parental CHOK1 cell line showed undetectable BCMA and TACI expression. TACI expression was absent in the H929 and U226B1 cell lines. BCMA expression was 4 - 6 fold higher than that of TACI in the MM1.R, MM1.S, and RPMI8226 multiple myeloma cell lines. The BCMA and TACI receptor copy numbers are summarized in Table 8.

[0229] Table 8: Copy numbers of BCMA and TACI in various cell lines

[0230] Cell Line BCMA Receptor TACI Receptor CHOK1 0 0 CHOK1 - BCMA 339834 0 H929 197350 0 MM1.S 29894 6409 RPMI8226 8532 1386 U266B1 42257 0 MM1.R 52419 12578 JVM - 2 18057 41088

[0231] Binding of anti - BCMA antibody (scFv) to endogenous BCMA was evaluated in the FACS binding assay as described above. 200 nM anti - BCMA scFv was tested on the negative cell line, H929, MM1.S, and RPMI8226. These exemplary antibodies exhibited similar binding patterns to different cell lines, and the binding intensity was correlated with the number of BCMA receptors on each cell line ( Figures 2A - 2C ).

[0232] The binding activity of the exemplary anti - BCMA clone BC - 06 (in scFv form; see Table 1 above) to multiple myeloma cell lines (BCMA+ or BCMA - ) was examined, and the EC 50 values are provided in Table 9 below.

[0233] Table 9: EC 50 values of clone BC - 06 against multiple myeloma and control cell lines

[0234] Cell Line MM1R H929 K562 <![CDATA[EC 50 (nM)]]> 0.74 2.85 Undetermined

[0235] Example 2. Generation and Characterization of Anti - TACI Antibodies

[0236] This example illustrates the identification and characterization of exemplary anti - TACI antibodies.

[0237] Screening of Anti - TACI scFv Antibodies by mRNA Display

[0238] Following the procedure described in Example 1 above, the mRNA display technique as described in Example 1 above was used to identify TACI binders from a 10 12 -13 natural human scFv library.

[0239] Characterization of Exemplary Anti-TACI scFv Antibodies

[0240] Exemplary anti-TACI antibodies in scFv form isolated by mRNA library screening as disclosed above were analyzed to determine their antigen-binding affinities. Structural information of the exemplary anti-TACI antibodies is provided in Table 2 above.

[0241] (a) TACI Binding Activity Measured by FACS

[0242] Anti-TACI scFv binders have been identified by anti-TACI supernatant FACS screening assay using JVM2, MM1.R, and RPMI8226 cell lines as described in the supernatant FACS screening assay above. 200 nM of purified scFv was subjected to FACS binding assay with TACI-negative cell lines K562, H929, and cancer cell lines expressing TACI, JVM2, MM1.R, RPMI8226. The assay procedure and data analysis are described in Example 1 above.

[0243] Anti-TACI scFv showed selective binding to the target cell lines. The binding level was correlated with the number of TACI receptors on the cell lines ( Figure 3 ). The EC 50 values of the exemplary anti-TACI antibodies (in scFv form) are summarized in Table 10 below.

[0244] Table 10: EC50 values of exemplary anti-TACI antibodies against various cell lines

[0245]

[0246] (b) TACI Binding Activity Measured by ELISA

[0247] ELISA binding assay was performed on the purified anti-TACI scFv. Recombinant TACI and negative proteins were immobilized on 384-well plates. Serial dilutions of 200 nM anti-TACI scFv clones were tested for binding in the above assay. Exemplary anti-TACI scFv generated from live cell selection (see above disclosure) did not bind to soluble recombinant TACI from multiple sources.

[0248] Example 3. Construction of Anti-BCMA Monospecific Chimeric Antigen Receptor (CAR)

[0249] This example illustrates the construction and characterization of an exemplary anti-BCMA monospecific chimeric antigen receptor (CAR).

[0250] Anti-BCMACAR Construct

[0251] Three selected anti-BCMA scFv candidates, BC-05, BC-06, and BC-08, were used to construct anti-BCMA monospecific CARs. These exemplary anti-BCMA monospecific CAR constructs contain, from the N-terminus to the C-terminus, an anti-BCMA scFv, a flag tag, an IgG4 hinge, a spacer, a CD28 transmembrane, a 4-1BB co-stimulatory signaling domain, and a CD3z intracellular signaling domain. The amino acid sequences of these exemplary anti-BCMA monospecific CARs are provided in Table 4 above.

[0252] Generation of lentiviral vectors for producing anti-BCMA CARs

[0253] According to standard molecular biology methods, the coding sequences of the above exemplary anti-BCMA CARs were cloned into lentiviral vectors. Using polyethyleneimine (PEI) transfection reagent, the resulting lentiviral vectors were co-transfected with an LV-MAX packaging mixture into Expi HEK293 according to the manufacturer's protocol. The transfected cells were grown with shaking at 37°C at an 8% CO2 level for 72 hours. The supernatant was harvested by centrifugation at 3200 rpm for 10 minutes at room temperature and vacuum filtration using a 0.45 µm PES membrane. The virus was concentrated by ultracentrifugation at 8000 rpm at 4°C for 1 hour (Beckman Coulter). The pellet was then resuspended in lentiviral stabilizer, aliquoted immediately, and stored at -80°C.

[0254] Transduction of virus vectors encoding BCMA CARs into immune cells and their expansion

[0255] PBMCs were isolated from the LRS compartment of fresh healthy donors using density gradient centrifugation with Lymphoprep and a SepMate 50 kit from Stemcell Technology. Then, CD3+ Pan T cells were isolated from PBMCs using an EasySep human T cell isolation kit according to the Stemcell Technology protocol. The Pan T cells were activated with human T-activator CD3 / CD28 dynabeads at a bead-to-cell ratio of 1:1 for 24 hours and then transduced with lentivirus in the presence of dynabeads and 1 mg / mL protamine sulfate. Spinoculation was performed at 300 g at 25°C for 2 hours. The cells and virus were incubated at 37°C for 24 hours. The next day, the cells were removed from the beads and virus. The cells were grown in X-vivo 15 (Lonza) medium containing 5% human serum with recombinant human IL15 and IL7 (Peprotech) for 2 weeks. The medium was changed every 2 - 3 days with the addition of fresh cytokines.

[0256] Characterization of CAR-T Cells Expressing Anti-BCMACAR

[0257] (i) Anti - BCMACAR Expression

[0258] CAR surface expression was evaluated by surface staining with an anti-flag tag antibody directly conjugated to Mix-n-stain AF647. Briefly, 100,000 lentivirally transduced T cells were incubated with 0.1 ul anti-flag-AF647 together in the dark with shaking at 4°C for 1 hour. The cells were centrifuged at 1,300 rpm for 5 minutes, the supernatant was removed, and the cells were washed with 200 uL 1x PBS. The resulting sample was resuspended in 200 uL 1x PBS. The percentage of surface expression was quantified by reading the fluorescence-stained cells on an Attune NxT flow cytometer. The results of this study showed that the CAR expression levels of different CAR constructs (see Table 4 above) were in the range of 50%-85%.

[0259] (ii) Cytotoxic T Lymphocyte (CTL) Activity

[0260] Human PBMC and Pan T cell isolation, viral transduction, and T cell expansion were described above. To screen for different CAR activities, a real-time image-based CTL activity assay was performed using GFP-engineered target cells. Briefly, 10,000 transduced T cells were incubated with 10,000 K562-GFP, BCMA / TACI / K562-GFP in RPMI1640 medium with 10% FBS at effector (CAR-T) to target cell ratios of 10:1, 5:1, and 1:1, respectively. No cytokines were added. The assay ran for 64 hours, and the GFP of the target cells was imaged and quantified by a Cytation 5 scanner. After the CTL assay, IFNγ was detected using a human IFNγ Duoset ELISA kit (R&D System). Briefly, the supernatant was collected after the CTL assay was terminated at 64 hours. Recombinant IFNγ was serially diluted and included in the assay to generate a standard curve. The supernatant IFNγ and recombinant IFNγ were assayed according to the provided manufacturer's protocol. The data were analyzed using Prism 8.0 software.

[0261] Figures 4A - 4C The CTL activities with different target cells and the range of effector to target cell ratios are shown. As Figure 4D shown, the endpoint CTL activities of different CARs were calculated. Figure 4EIFNγ levels are shown. BCMA monospecific CARs exhibit dose-dependent target-specific CTL activity and are associated with IFNγ secretion. Multiple donors have been screened with different CARs and similar CAR CTL activity has been shown ( Figures 5A - 5B ).

[0262] (iii) CTL Assay of CAR - T Cells Expressing Anti - BCMA Construct EPLV102

[0263] To further evaluate the CTL activity of EPLV102-expressing CAR-T cells, PBMCs from multiple donors were transduced with lentivirus and CAR-Ts were expanded as described above. In the donors shown here, transduced or untransduced T cells were co-cultured with K562, BCMA / K562, H929, and MM1R GFP cells at effector-to-target cell ratios of 10:1 and 5:1 for 66 hours. Target-specific CTL activity was observed in both E / T ratios ( Figures 6A - 6C ). Similar CTL activity was observed with multiple donors.

[0264] The CAR-T cell phenotype is associated with T cell persistence. After the CTL assay, the CAR-T cell phenotype was analyzed using a panel of antibodies detecting T cell differentiation markers by FACS assay. Briefly, as described above, transduced T cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, and anti-CD62L. Analysis was performed by Attune NxT software. Gating was performed on CD3, CD4, CD8 positive CAR-T cells and Tn, Tscm, Tcm, and Tem cells, and the results showed comparable CD8 and CD4 CAR+ T cells ( Figure 6D ) and a high percentage of Tcm ( Figure 6E ).

[0265] (iv) EPLV102 CAR - T Persistence Evaluated by Multiple Rounds of Target Cell Attack

[0266] To further test CAR-T persistence, multiple rounds of target cell attack experiments were performed. EPLV102 was transduced into Pan T cells and expanded. 10,000 transduced T cells were attacked with 10,000 K562 and MM1R GFP cells for 48 hours, and then the transduced T cells were attacked with fresh 10,000 target cells for an additional 48 hours in RPMI1640 medium without additional cytokines. CTL activity was quantitatively imaged every 2 hours with Cytation 5 and analyzed by Prism 8.0 software. EPLV102 exhibited sustained CTL activity in 2 rounds of target attack and re-attack experiments ( Figures 7A - 7B ).

[0267] Example 4. Construction of anti-TACI monospecific chimeric antigen receptor (CAR)

[0268] This example illustrates the construction and characterization of an exemplary anti-TACI monospecific chimeric antigen receptor (CAR).

[0269] Generation of anti-TACI CAR constructs and CAR-T cells expressing the anti-TACI CAR constructs

[0270] Four selected anti-TACI scFv candidates, TC-01, TC02, TC-03, and TC04, were used to construct anti-TACI monospecific CARs. These CAR constructs contained, from the N-terminus to the C-terminus, anti-TACI scFv, a flag tag, a CD8a spacer, and transmembrane, 41BB, and CD3z intracellular signaling domains. The amino acid sequences of these anti-TACI CAR constructs are provided in Table 4 above.

[0271] According to standard molecular biology methods, the coding sequences of the anti-TACI CAR constructs were cloned into lentiviral vectors. As per routine practice, the resulting lentiviral vectors were used to transduce PBMCs for anti-TACI CAR expression. See the above disclosure.

[0272] Characterization of CAR-T cells expressing anti-TACI CAR

[0273] (i) CTL Activity of Anti - TACI CAR T Cells

[0274] Human PBMC and Pan T cell isolation, viral transduction, and T cell expansion were described above. To screen for different CAR activities, a real-time image-based CTL activity assay was performed using GFP-engineered target cells. Pan T cells transduced with CAR constructs EPLV200, EPLV254, EPLV255, and EPLV256 were incubated with 100,000 K562, H929 TACI-negative, and MM1R TACI-positive GFP cells at effector-to-target cell ratios of 5:1 and 1:1 as described for 72 hours and imaged by Cytation 5.

[0275] The TACI monospecific CAR showed 50%-90% surface expression ( Figure 8A ). Figures 8B - 8D The real-time CTL activities of different target cells with an E / T ratio of 1:1 are shown in Figure 8E . As shown in

[0276] (ii) CAR - T Persistence Evaluation

[0277] To further test the effect of different spacers on CAR-T persistence, multi-round target cell re-challenge experiments were performed on CAR-T cells expressing EPLV300 (short spacer) and EPLV301 (medium spacer containing IgG4 hinge and CD28 transmembrane). See the structural information provided in Table 4 above. Nucleic acids encoding the CAR construct were transduced into Pan T cells and amplified in 2 donors. 10,000 transduced T cells were challenged with 10,000 K562 and MM1R GFP cells for 48 hours, and then the transduced T cells were re-challenged with fresh 20,000 target cells for an additional 68 hours in RPMI medium without additional cytokines. Both short spacer CAR surface and medium spacer CAR surface were expressed at 70%-90%( Figure 9A ). CTL activity was quantified by imaging target-GFP cells every 2 hours with Cytation 5 and analyzed by Prism 8.0 software. EPLV300 showed consistently better CTL activity in 2 rounds of target attack and re-challenge experiments in both donors( Figures 9B - 9C ).

[0278] Example 5. Construction and Characterization of Anti-BCMA and Anti-TACI Bispecific CARs

[0279] This example illustrates the construction and characterization of an exemplary anti-BCMA / anti-TACI bispecific (CAR).

[0280] Anti-BCMA / anti-TACI bispecific CAR construct

[0281] The same anti-BCMA scFv and anti-TACI scFv used in the monospecific CAR constructs disclosed above were used to construct an anti-BCMA / anti-TACI bispecific CR construct, where the anti-BCMA and anti-TACI scFv fragments are in tandem orientation in the orientation of anti-BCMA scFv to anti-TACI scFv. The bispecific CAR construct further includes a flag tag, a CD8a spacer, and a transmembrane (EPLV217) or an IgG4 hinge, a CD28 transmembrane (EPLV302), a 4-1BB co-stimulatory signaling domain, and a CD3z intracellular signaling domain. The amino acid sequence of an exemplary bispecific CAR construct is shown in Table 4 above.

[0282] According to standard molecular biology methods, the coding sequence of the bispecific CAR construct was cloned into a lentiviral vector. According to routine practice, the resulting lentiviral vector was used to transduce PBMCs for bispecific CAR expression. See the above disclosure.

[0283] Characterization of CAR-T Cells Expressing Anti-BCMA / Anti-TACI Bispecific CAR

[0284] Human PBMC and Pan T cell isolation, viral transduction, and T cell expansion were described above. A real-time image-based CTL activity assay was performed using GFP-engineered target cells. The CAR constructs EPLV217 with CD8a TM and EPLV302 with CD28 TM-transduced pan T cells were incubated with 100,000 K562 BCMA - / TACI - double-negative, H929 BCMA + / TACI - and MM1R BCMA + / TACI + double-positive GFP cells at an effector-to-target cell ratio of 1:1 as described and incubated for 72 hours, and imaged in multiple donors by Cytation 5. A target cell re-challenge assay was also performed with EPLV302 to examine the persistence of the bispecific CAR.

[0285] The BCMA / TACI bispecific CAR showed 60%-80% surface expression in multiple donors, as Figures 10A - 10B shown respectively.

[0286] Figures 11A - 11C CTL activities of CAR-T cells expressing EPLV217 against different target cells are shown in. EPLV217-expressing CAR-T cells exhibited specific and effective CTL activities against different target cell lines ( Figure 11D ) as well as robust IFNγ release ( Figure 11E ).

[0287] In the re-challenge assay, the CTL activities of EPLV302-expressing CAR-T cells also showed effective and persistent target-specific CTL activities against the target cell lines ( Figures 12A - 12H ). IFNγ levels were correlated with CTL activities in different target cells ( Figure 12I ). Multiple donors were screened with two forms of CAR and similar results were shown.

[0288] Example 6. In Vivo Efficacy Study in Disseminated MM1R-Luciferase Model

[0289] The in vivo antitumor efficacy of anti-BCMA monospecific, anti-TACI monospecific, and anti-BCMA / anti-TACI bispecific CAR-T cells was evaluated in a disseminated MM1R-luciferase model in NCG mice. Three CAR constructs (EPLV200, EPLV257, and EPLV217) were transduced into Pan T cells and amplified in vitro for 4 days. 5 x 10 5 MM1R-luciferase cells were inoculated into NCG mice. On day 8, mice were administered PBS control and 1e6 of each CAR-T cell. Mice were imaged every 3 - 4 days and body weight was measured.

[0290] As Figure 13A shown, on day 38, all treatment groups showed tumor growth inhibition and all CAR-Ts effectively eradicated tumor cells in all mice. There was no weight loss in the treatment groups ( Figure 13B ). In this study, all mice survived.

[0291] On day 53, the BCMA monospecific CAR-T treated group was re-challenged with 5 x 10 5 MM1R-luciferase cells. No additional CAR-T cells were injected. No tumor growth was detected in BCMA CAR-T (EPLV257) treated mice for up to 87 days. All mice survived. Control mice treated with PBS showed robust tumor growth ( Figure 13C ).

[0292] Example 7. Characterization of Engineered T Cells Expressing Anti-BCMA / TACI Bispecific CAR and Armored Polypeptides

[0293] This example evaluated the biological activity of engineered T cells expressing an anti-BCMA / TACI bispecific CAR construct and an armored polypeptide comprising an anti-PDL1 fragment and a mutant IL2 fragment.

[0294] An anti-BCMA / TACI bispecific CAR (EPLV302) was constructed with the following components (from the N-terminus to the C-terminus): a signal peptide, an anti-BCMA scFv, a peptide linker, an anti-TACI scFv, a flag tag, a CD8a spacer, and a transmembrane (or IgG4 hinge), a CD28 transmembrane, a 41BB co-stimulatory domain, and a CD3z intracellular signaling domain. To co-express the armored polypeptide, the coding sequence of the anti-BCMA / TACI bispecific CAR was ligated (at its 3'-end) with the coding sequences of a T2A peptide, a second signal peptide, an anti-PDL1 fragment, and a mutant IL-2 from 5' to 3'. See the EPLV302 (with armored polypeptide) and EPLV327 (without armored polypeptide) structural information provided in Table 4 above. The coding sequences were cloned into a lentiviral vector according to standard molecular biology methods.

[0295] Human PBMCs and Pan T cells were isolated and transduced with the viral vectors disclosed above and amplified as disclosed in the examples above. The expression of the bispecific CAR in T cells transduced with the bispecific CAR coding sequence or in combination with the coding sequence of the armored polypeptide was examined. Engineered T cells showed approximately 25%-50% surface expression of the bispecific CAR with or without co-expressed armored polypeptide. Figure 14A 。

[0296] In addition, a real-time image-based CTL activity assay was performed using target cells engineered to express GFP. Briefly, pan T cells engineered with the CAR construct EPLV302 (non-armored) or construct EPLV327 (armored) were incubated with 100,000 K562 BCMA- / TACI-double-negative or MM1R BCMA+ / TACI+ double-positive GFP cells at an effector-to-target cell ratio of 1:1 as described for 114 hours and then imaged in multiple donors by Cytation 5. As Figure 14B shown, T cells expressing the bispecific CAR showed high cytotoxic activity against MM1R-GFP cells but not K562-GFP cells with or without the armored polypeptide.

[0297] Next, a target cell re-challenge assay was performed using pan T cells engineered to be EPLV302 or EPLV327 at an effector-to-target cell ratio of 0.5:1 for 168 hours to study the persistence of T cells expressing the bispecific non-armored CAR and armored CAR. In the cancer cell re-challenge assay, both non-armored bispecific CAR-T cells and armored bispecific CAR-T cells showed sustained CTL activity for up to 168 hours. In addition, the release of IFNγ was measured at 48 hours, 120 hours, and 168 hours. AsFigures 14C - 14E As shown, the IFNγ levels measured at 48 hours and 120 hours using different target cells were correlated with CTL activity, and the IFNγ level of T cells expressing armored CAR was higher than that of T cells expressing non-armored CAR at 168 hours. Figure 14E Multiple donors were screened with two forms of CAR and similar results were shown.

[0298] Example 8. In vivo anti-tumor activity of engineered T cells expressing anti-BCMA / TACI bispecific CAR and armored polypeptide

[0299] To evaluate the anti-tumor activity of engineered T cells expressing bispecific armored CAR and armored polypeptide (EPLV327; see Table 4 above), 6-8 week-old female NCG mice (Charles River Laboratories, Wilmington, MA) were intravenously inoculated with 5×10 5 MM1R-luc multiple myeloma tumor cells suspended in serum-free medium. Seven days after tumor cell inoculation, the mice were randomly divided into the following 3 treatment groups:

[0300] (1) Vehicle (n = 5 mice),

[0301] (2) Non-armored CAR (1E6 CAR-T cell dose; n = 18 mice), and

[0302] (3) EPC-004 (EPLV327) (1E6 CAR-T cell dose; n = 18 mice).

[0303] Seven days after tumor cell inoculation, CAR-T cells were administered by intravenous injection. After intraperitoneal injection of the luciferin substrate, the tumor burden was determined weekly by bioluminescence imaging. Mice in the vehicle group showed a high tumor burden, while the tumor burden in mice of groups (2) and (3) was significantly reduced. Figure 15A Animals treated with non-armored CAR and EPC-004 showed significant tumor growth inhibition over time compared to the vehicle control group.

[0304] In addition, compared to non-armored CAR-T cells, EPC-004 CAR-T cells showed significantly greater anti-tumor activity, as Figure 15B shown. Adverse clinical events related to tumor burden were monitored in the animals over time. By day 28 after treatment began, all vehicle control animals were euthanized due to tumor burden, while animals in the non-armored CAR and EPC-004 treatment groups survived through day 35.

[0305] Example 9. Development of CAR-NK cells

[0306] Following conventional methods, BCMA single-specific (EPLV310) and TACI single-specific (EPLV300) CAR candidates (see Table 4 above) were constructed, containing anti-BCMA scFv or anti-TACI scFv, flag tag, IgG4 hinge, CD28 transmembrane, 41BB and CD3z intracellular signaling domains. A bispecific CAR (EPLV302) candidate in tandem form was also constructed. The structural information of this bispecific CAR is also provided in Table 4 above. According to standard molecular biology methods, the coding sequences of the single-specific and bispecific CAR constructs were cloned into a lentiviral vector.

[0307] As described previously, lentiviruses carrying the coding sequences of the single-specific or bispecific CAR were generated. PBMC were isolated from the LRS compartment of fresh healthy donors using density gradient centrifugation with lymphoprep and the SepMate 50 kit from StemCell Technologies. Then, NK cells were isolated from PBMC using the NK cell isolation kit from Miltenyi according to the manufacturer's protocol. NK cells were activated with CTS X-pander medium (ThermoFisher) and a cytokine mixture at a concentration of 0.5 - 1 x 106 cells / mL for 6 - 7 days. After activation, 1 mg / mL protamine sulfate and 1 uM BX795 were added. Spinoculation was performed at 1000 g at 32 °C for 45 minutes. The NK cells and virus were incubated at 37 °C for 24 hours. The next day, the virus was removed, and the transduced NK cells were replenished with fresh medium and cytokine mixture. The medium was changed every 2 - 3 days with the addition of fresh cytokines.

[0308] CAR surface expression in the transduced NK cells was evaluated by surface staining using CD56-PE and AF647-conjugated anti-flag tag antibodies. Briefly, 100,000 lentivirally transduced NK cells were incubated with 0.5 μL CD56-PE and 0.1 μL anti-flag-AF647 together in the dark with shaking at 4 °C for 1 hour. The cells were centrifuged at 1,300 rpm for 5 minutes, the supernatant was removed, and the cells were washed with 200 μL 1x PBS. The resulting sample was reconstituted in 200 μL 1x PBS. The surface expression percentage was quantified by reading the fluorescence-stained cells on an Attune NxT flow cytometer. Figure 16AShows the levels of NK cells expressing CD56, which is an NK cell marker. NK cells with a purity of approximately 90%-95% were observed during expansion. CAR expression of different CAR constructs gated on the NK cell population was in the range of 40%-90%. Figure 16B 。

[0309] Human PBMC and NK cell isolation, viral transduction, and T cell expansion were described above. Both BCMA, TACI monospecific, and BCMA / TACI bispecific CARs showed robust surface expression in multiple donors, as shown above. A real-time image-based CTL activity assay was performed using MM1R target cells engineered with GFP. NK cells transduced with the CAR constructs EPLV302 (bispecific), EPLV310 (BCMA monospecific), and EPV300 (TACI monospecific) were incubated with 100,000 MM1R BCMA+ / TACI+ double-positive GFP cells at an effector-to-target cell ratio of 5:1 as described for 212 hours and imaged in multiple donors by Cytation 5. A target cell re-challenge assay was also performed. All CAR-NK constructs exhibited cancer cell killing activity. Bispecific CAR-NK showed more persistent activity than monospecific CAR-NK in the 3rd and 4th re-challenges ( Figure 16C ). A robust release of IFNγ levels was associated with persistent CTL activity ( Figure 16D ).

[0310] Other embodiments

[0311] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature for the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0312] From the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0313] Equivalent solutions

[0314] Although several embodiments of the present invention have been described and shown herein, those of ordinary skill in the art will readily conceive of various other devices and / or structures for performing the functions described herein and / or obtaining these results and / or one or more of these advantages, and each of such variations and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that the embodiments of the present invention may be practiced otherwise than as specifically described and claimed within the scope of the appended claims and their equivalents. Embodiments of the present disclosure relate to each and every separate feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods that do not mutually contradict are included within the scope of the present invention as disclosed herein.

[0315] All definitions defined and used herein are to be understood as controlling dictionary definitions, definitions in incorporated documents by reference, and / or ordinary meanings of the defined terms.

[0316] All references, patents, and patent applications disclosed herein are incorporated by reference relative to the subject matter to which each is cited, and in some cases, may cover the entire document.

[0317] Unless the context clearly dictates the contrary, the indefinite articles "a" and "an" as used herein in the specification and claims shall be understood to mean "at least one".

[0318] As used herein in the specification and claims, the phrase "and / or" shall be understood to mean "any one or both" of the elements so combined, i.e., elements that coexist in some cases and separate in other cases. Multiple elements listed with "and / or" shall be construed in the same manner, i.e., "one or more" of the elements so combined. Other elements may optionally exist in addition to the elements specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" may in one embodiment refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0319] As used herein in this specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted inclusively, i.e., including at least one of many elements or elements in a list, but also including more than one element and optionally additional unlisted items. Only expressly stated contrary terms such as "only one" or "exactly one" or when used in a claim, "consisting of" refer to including exactly one element of many elements or elements in a list. In general, when preceded by exclusive terms such as "any one", "one of...", "only one of...", or "exactly one of...", the term "or" as used herein shall be interpreted only as indicating exclusive alternatives (i.e., "one or the other but not both"). When used in a claim, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0320] As used in this specification and the claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one element of each specifically listed element in the list of elements, and does not exclude any combination of the elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to in the phrase "at least one", whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can mean at least one that optionally includes more than one A, no B (and optionally includes elements other than B); in another embodiment, it can mean at least one that optionally includes more than one B, no A (and optionally includes elements other than A); in yet another embodiment, it can mean at least one that optionally includes more than one A, and at least one that optionally includes more than one B (and optionally includes other elements); and so on.

[0321] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order of the steps or acts recited in the method.

Claims

1. A bispecific chimeric antigen receptor (CAR) that is specific for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), said bispecific CAR comprising: (a) A first antigen-binding portion that is specific for TACI; (b) A second antigen-binding portion that is specific for BCMA; (c) A co-stimulatory signaling domain; and (d) A cytoplasmic signaling domain.

2. The bispecific CAR according to claim 1, wherein the first antigen-binding portion specific for TACI in (a) comprises a heavy-chain variable region (V H ) and a light-chain variable region (V L ); wherein the V H comprises heavy-chain CDRs identical to the heavy-chain CDRs in a reference antibody, and the V L comprises heavy-chain CDRs identical to the heavy-chain CDRs in the reference antibody; and wherein the reference antibody is TC-01, TC-02, TC-03 or TC-04; optionally wherein the reference antibody is TC-01.

3. The bispecific CAR according to claim 2, wherein the V of the first antigen-binding portion specific for TACI H and the V L are the same as the V H and V L of the reference antibody.

4. The bispecific CAR according to any one of claims 1 to 3, wherein the first antigen-binding portion that is specific for TACI is a single-chain variable fragment (anti-TACI scFv).

5. The bispecific CAR according to claim 9, wherein the anti-TACI scFv comprises the amino acid sequence of any one of SEQ ID NO: 75, 84, 93, and 102; optionally wherein the anti-TACI scFv comprises the amino acid sequence of SEQ ID NO:

75.

6. The bispecific CAR according to any one of claims 1 to 5, wherein the second antigen-binding portion specific for BCMA in (a) comprises a heavy chain variable region (V H ) and a light chain variable region (V L ); Wherein The said V H comprises: (hi) Heavy chain complementarity-determining region (CDR) 1, said heavy chain CDR1 comprising X1YX2MH, wherein X1 is S or D, and X2 is A or G; (hii) Heavy chain CDR2, said heavy chain CDR2 comprising (hii-a) X3IX4YDGSX5KYYADSVKG (SEQ ID NO: 1), wherein X3 is V or F, X4 is S or R, and X5 is D or N; (hii-b) FIRSKAYGGTTEYAASVKG (SEQ ID NO: 27); or (hii-c) GISWNSGSIGYADSVKG (SEQ ID NO: 43); and (hiii) Heavy chain CDR3, said heavy chain CDR3 comprising (hiii-a) DEHQVVPNYRFDF (SEQ ID NO: 56), (hiii-b) DWEDPLYYYDTPF (SEQ ID NO: 35), (hiii-c) DWDYYDSSGYYPDALGI (SEQ ID NO: 16), (hiii-d) DLWDGIVGAPAGY (SEQ ID NO: 9), (hiii-e) DLTTITPGY (SEQ ID NO: 22), (hiii-f) DLWEFGGDYADY (SEQ ID NO: 63), (hiii-g) GPHYDILTSNWFDP (SEQ ID NO: 28), or (hiii-h) VQX6PGAFDI (SEQ ID NO: 181), wherein X6 is P or S; and Wherein the V L includes: (li) Light chain CDR1, said light chain CDR1 comprising (li-a) SGSGSNIGSNDVS (SEQ ID NO: 58), (li-b) QASQDIX7NYLN (SEQ ID NO: 2), wherein X7 is N or S, or (Li-C)RX8X9X 10 ISSYLX 11 (SEQ ID NO:3), wherein X8 is A or S, X9 is S or T, X 10 is G or S, and X11 is G or N; (lii) Light chain CDR2, said light chain CDR2 comprising (lii-a)WNDQRPS (SEQ ID NO:59), (lii-b)DASNX 12 ET (SEQ ID NO:4), wherein X 12 is L or V, or (lii-c)AX 13 SX 14 LQS (SEQ ID NO:5), wherein X 13 is A or T, and X 14 is S or T; and (liii) light chain CDR3, wherein the light chain CDR3 comprises (liii-a) AAWDDSLNGWV (SEQ ID NO:60), (liii-b)QQYDX 15 LPX 16 T (SEQ ID NO:6), wherein X 15 is K or N, and X 16 is F, L or Y, (liii-c) QHSYSTPHT (SEQ ID NO:32), or (liii-d) QQLYS (SEQ ID NO:48).

7. The bispecific CAR according to claim 6, wherein the V of the second antigen-binding portion specific for BCMA H comprises a heavy chain CDR identical to the heavy chain CDR in a reference antibody, and / or wherein the V of the second antigen-binding portion specific for BCMA L comprises a heavy chain CDR identical to the heavy chain CDR in the reference antibody; the reference antibody is BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, BC-08 or BC-09; optionally wherein the reference antibody is BC-06.

8. The bispecific CAR according to claim 7, wherein the V of the second antigen-binding portion specific for BCMA H and the V L are the same as the V H and V L of the reference antibody.

9. The bispecific CAR according to any one of claims 1 to 8, wherein the second antigen-binding portion specific for BCMA is a single-chain variable fragment (anti-BCMA scFv).

10. The bispecific CAR according to claim 9, wherein the anti-BCMA scFv comprises the amino acid sequence of any one of SEQ ID NOs: 15, 20, 25, 34, 41, 50, 53, 62 and 66; optionally wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO:

50.

11. The bispecific CAR according to any one of claims 1 to 10, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from: CD28, 4-1BB, OX40, ICOS, CD27, CD40 or CD40L.

12. The bispecific CAR according to claim 11, wherein the cytoplasmic signaling domain is derived from CD3ζ.

13. The bispecific CAR according to any one of claims 1 to 12, wherein the bispecific CAR comprises: (a) a fusion polypeptide, the fusion polypeptide comprising from N-terminus to C-terminus (i) the first antigen-binding portion, (ii) the second antigen-binding portion, (iii) the co-stimulatory signaling domain and (iv) the cytoplasmic signaling domain; or (b) a fusion polypeptide, the fusion polypeptide comprising from N-terminus to C-terminus (i) the second antigen-binding portion, (ii) the first antigen-binding portion, (iii) the co-stimulatory signaling domain and (iv) the cytoplasmic signaling domain.

14. The bispecific CAR according to claim 13, further comprising a hinge domain and a transmembrane domain, the hinge domain and the transmembrane domain being located between (ii) and (iii).

15. The bispecific CAR according to claim 13 or claim 14, further comprising a peptide linker that links the first antigen-binding portion and the second antigen-binding portion.

16. The bispecific CAR according to claim 15, wherein the peptide linker comprises the amino acid sequence of GGGGS (SEQ ID NO:104), GGGGSGGGGS (SEQ ID NO:105), GGGGSGGGGSGGGGS (SEQ ID NO:106) or GSTSGSGKPGSGEGSTKG (SEQ ID NO:107).

17. The bispecific CAR according to any one of claims 1 to 16, which further comprises a signal peptide at the N-terminus.

18. The bispecific CAR according to claim 1, which comprises the amino acid sequence of SEQ ID NO:

182.

19. The bispecific CAR according to claim 18, which comprises the amino acid sequence of SEQ ID NO: 165 or 166.

20. A nucleic acid or a set of nucleic acids that jointly encode the bispecific CAR according to any one of claims 1 to 19.

21. The nucleic acid or a set of nucleic acids according to claim 16, wherein the nucleic acid comprises a first nucleotide sequence encoding the bispecific CAR according to any one of claims 13 to 19.

22. The nucleic acid or a set of nucleic acids according to claim 21, wherein the nucleic acid further comprises a second nucleotide sequence encoding an armored polypeptide that enhances T cell functionality; and a third nucleotide sequence encoding a self-cleaving peptide, and the third nucleotide sequence is located between the first nucleotide sequence and the second nucleotide sequence.

23. The nucleic acid or a set of nucleic acids according to claim 22, wherein the armored polypeptide is IL-2, IL-5, IL-15, a co-stimulatory ligand, an anti-PDL1 antibody, or a fusion polypeptide comprising the anti-PDL1 antibody; optionally wherein the anti-PDL1 antibody is a single-chain variable fragment (scFv) that is fused to an IL-2 polypeptide.

24. The nucleic acid or a set of nucleic acids according to claim 23, wherein the armored polypeptide comprises the amino acid sequence of SEQ ID NO: 168, 170, 172, 174, 178, or 180; optionally wherein the armored polypeptide comprises the amino acid sequence of SEQ ID NO:

178.

25. The nucleic acid or a set of nucleic acids according to any one of claims 20 to 24, wherein the nucleic acid is an expression vector, optionally a viral vector.

26. A genetically engineered immune cell that expresses the bispecific CAR according to any one of claims 1 to 19, and optionally further expresses the armored polypeptide set forth in any one of claims 22 to 24.

27. The genetically engineered immune cell according to claim 26, which expresses (a) the bispecific CAR comprising the amino acid sequence of SEQ ID NO: 165 or 166; and (b) the armored polypeptide comprising the amino acid sequence of SEQ ID NO: 177 or 178 or the armored polypeptide comprising the amino acid sequence of SEQ ID NO: 173 or 174.

28. The genetically engineered immune cell according to claim 26 or 27, which comprises the nucleic acid according to any one of claims 20 to 25.

29. The genetically engineered immune cell according to any one of claims 26 to 28, which is a T cell, an NK cell, or a macrophage, optionally wherein the immune cell is a T cell.

30. An anti-TACI chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for TACI, a co-stimulatory signaling domain, and a cytoplasmic signaling domain; wherein the extracellular antigen-binding domain specific for TACI is set forth in any one of claims 2 to 5.

31. The anti-TACI CAR according to claim 33, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from: CD28, 4-1BB, OX40, ICOS, CD27, CD40 or CD40L; and / or wherein the cytoplasmic signaling domain is derived from CD3ζ.

32. The anti-TACI CAR according to claim 30 or claim 31, further comprising a hinge domain and / or a transmembrane domain located between the extracellular antigen-binding domain specific for TACI and the co-stimulatory domain.

33. The anti-TACI CAR according to claim 32, comprising both the hinge domain and the transmembrane domain, and wherein the anti-TACI CAR further comprises a spacer located between the hinge domain and the transmembrane domain.

34. The anti-TACI CAR according to claim 30, comprising an amino acid sequence of any one of SEQ ID NOs: 143-162; optionally wherein the anti-TACI CAR comprises the amino acid sequence of SEQ ID NO: 143 or 144.

35. An anti-BCMA chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for BCMA, a co-stimulatory signaling domain, and a cytoplasmic signaling domain; wherein the extracellular antigen-binding domain specific for BCMA is set forth in any one of claims 6 to 10.

36. The anti-BCMA CAR according to claim 35, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from: CD28, 4-1BB, OX40, ICOS, CD27, CD40 or CD40L; and / or wherein the cytoplasmic signaling domain is derived from CD3ζ.

37. The anti-BCMA CAR according to claim 35 or claim 36, further comprising a hinge domain and / or a transmembrane domain located between the extracellular antigen-binding domain specific for BCMA and the co-stimulatory domain.

38. The anti-BCMA CAR according to claim 37, comprising both the hinge domain and the transmembrane domain, and wherein the anti-BCMA CAR further comprises a spacer located between the hinge domain and the transmembrane domain.

39. The anti-BCMA CAR according to claim 35, comprising an amino acid sequence of any one of SEQ ID NOs: 119-142; optionally wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 127 or 128.

40. A nucleic acid comprising a first nucleotide sequence encoding an anti-TACI CAR according to any one of claims 30 to 34 or an anti-BCMA CAR according to any one of claims 35 to 39.

41. The nucleic acid according to claim 40, further comprising a second nucleotide sequence encoding an armored polypeptide that enhances T cell functionality; and a third nucleotide sequence encoding a self-cleaving peptide, the third nucleotide sequence being located between the first nucleotide sequence and the second nucleotide sequence.

42. The nucleic acid according to claim 40, wherein the armored polypeptide is set forth in any one of claims 23 or 24.

43. The nucleic acid according to any one of claims 40 to 42, wherein the nucleic acid is an expression vector, optionally a viral vector.

44. A genetically engineered immune cell expressing an anti-TACI CAR according to any one of claims 30 to 34 and / or an anti-BCMA CAR according to any one of claims 35 to 39, and optionally further expressing the armored polypeptide set forth in claim 41 or claim 42.

45. The genetically engineered immune cell according to claim 44, which is a T cell, NK cell or macrophage, optionally wherein the immune cell is a T cell.

46. A method for eliminating unwanted cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a genetically engineered immune cell according to any one of claims 26 to 29 and 44 to 45, or a pharmaceutical composition comprising the genetically engineered immune cell.

47. The method according to claim 46, wherein the unwanted cells are cancer cells.

48. The method according to claim 46 or claim 47, wherein the subject is a human cancer patient.

49. The method according to claim 48, wherein the human cancer patient comprises BCMA + and / or TACI + cancer cells.

50. The method according to claim 49, wherein the cancer cells are multiple myeloma cells, lung cancer cells, gastric cancer cells, breast cancer cells or testicular cancer cells.

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