Chimeric antigen receptors specific for B cell maturation antigen (BCMA) and / or transmembrane activator and CAML interactor (TACI)

Abstract

Genetically engineered immune cells (e.g., T cells or NK cells) that express anti-BCMA, anti-TACI, or anti-BCMA / anti-TACI chimeric antigen receptors and their use in cancer treatment. In some embodiments, the genetically engineered immune cells may be armed CAR-T or CAR-NK cells that further express arming polypeptides to enhance the characteristics of the CAR-T or CAR-NK cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 376,530, filed September 21, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been filed electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on September 20, 2023, is named 112139-0073-7007WO00_SEQ.XML, and is 197,654 bytes in size. [Background technology]

[0003] Chimeric antigen receptor (CAR-T) T cells are genetically engineered T cells that express an artificial T cell receptor for use in immunotherapy. The artificial T cell receptor (known as a chimeric antigen receptor) can specifically bind to disease cell antigens, such as cancer antigens. Upon binding to disease cells, the CAR-T cells are activated and eliminate the disease cells.

[0004] Although CAR-T cell therapy has demonstrated efficacy in the treatment of some blood cancers, the efficacy of treatment can be affected by various factors, such as tumor antigen escape, for example, when the expression level of the tumor antigen is reduced to a level at which CAR-T cells cannot participate in and mediate cytotoxic activity. In some cases, tumor cells can avoid killing by expressing an alternative form of the target antigen that lacks the binding epitope for the CAR. In other cases, tumor cells can avoid killing by switching to a genetically related but phenotypically distinct disease (a so-called lineage switch).

[0005] Therefore, it is of great interest to develop improved CAR-T approaches to address such challenges. Summary of the Invention

[0006] The present disclosure is based, at least in part, 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 for successful inhibition of tumor growth in animal models. Accordingly, provided herein are such CAR constructs, the expression of such immune cells, and therapeutic uses in cancer treatment.

[0007] In some aspects, the present disclosure provides a bispecific chimeric antigen receptor (CAR) specific for B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), the bispecific CAR comprising: (a) a first antigen binding portion specific for TACI; (b) a second antigen binding portion specific for BCMA; (c) a costimulatory signaling domain; and (d) a cytoplasmic signaling domain.

[0008] In some cases, the first antigen-binding portion specific for TACI of (a) comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) included. V H contains the same heavy chain CDRs as in the reference antibody, and V L comprises the same heavy chain CDRs as in a 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 of the first antigen-binding portion specific for TACI H and V L is the V of the reference antibody H and V L is the same as

[0009] In some examples, the first antigen-binding portion specific for TACI is a single-chain variable fragment (anti-TACI scFv). In particular examples, the anti-TACI scFv comprises the amino acid sequence of any one of SEQ ID NOs: 75, 84, 93, and 102 (e.g., SEQ ID NO: 75).

[0010] In some embodiments, the second antigen-binding portion specific for BCMA in (a) comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) included. V H teeth, (hi) a heavy chain complementarity determining region (CDR) 1 comprising X1YX2MH, wherein X1 is S or D and X2 is A or G; (hii) a heavy chain CDR2, (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) a heavy chain CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 43); and (hiii) a heavy chain CDR3, (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) a heavy chain CDR3 comprising VQX6PGAFDI (SEQ ID NO: 181), wherein X6 is P or S; Alternatively, or in addition, V L teeth, (li) a light chain CDR1, (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, and X 10 is G or S, and X11 is G or N, RX8X9X 10 ISSYLX 11 (SEQ ID NO: 3), and (lii) a light chain CDR2, (lii-a) WNDQRPS (SEQ ID NO: 59), (lii-b)DASNX 12 ET (SEQ ID NO: 4), wherein X 12 is L or V, DASNX 12 ET (SEQ ID NO: 4), 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, AX 13 SX 14 a light chain CDR2 comprising LQS (SEQ ID NO: 5); (liii) a light chain CDR3, (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, QQYDX 15 LPX 16 T (SEQ ID NO: 6), (liii-c) QHSYSTPHT (SEQ ID NO: 32), or (liii-d) a light chain CDR3 comprising QQLYS (sequence number 48).

[0011] In some cases, the V of the second antigen-binding moiety specific for BCMA His a V of a second antigen-binding portion that contains the same heavy chain CDRs as in the reference antibody and / or is specific for BCMA L comprises the same heavy chain CDRs as in the reference antibody, including BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, BC-08, or BC-09. In some examples, the reference antibody is BC-06. In some examples, the V of the first antigen-binding portion specific for BCMA H and V L is the V of the reference antibody H and V L is the same as

[0012] In some examples, the first antigen-binding portion specific for BCMA is a single chain variable fragment (anti-BCMA scFv). In particular examples, 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 (e.g., SEQ ID NO: 50).

[0013] Any of the bispecific CARs disclosed herein can include a costimulatory signaling domain. Examples include, but are not limited to, CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively, or in addition, the bispecific CAR can include a cytoplasmic signaling domain that can be derived from CD3ζ.

[0014] In some specific examples, a bispecific CAR comprises a fusion polypeptide comprising, from N-terminus to C-terminus, (i) a first antigen-binding moiety, (ii) a second antigen-binding moiety, (iii) a costimulatory signaling domain, and (iv) a cytoplasmic signaling domain. Alternatively, a bispecific CAR may comprise a fusion polypeptide comprising, from N-terminus to C-terminus, (i) a second antigen-binding moiety, (ii) the first antigen-binding moiety, (iii) a costimulatory signaling domain, and (iv) a cytoplasmic signaling domain. Any of the bispecific CARs disclosed herein may further comprise a hinge domain and a transmembrane domain located between (ii) and (iii). In some cases, a bispecific CAR may further comprise a peptide linker connecting the first antigen-binding moiety and the second antigen-binding moiety. Examples include GGGGS (SEQ ID NO: 104), GGGGSGGGGS (SEQ ID NO: 105), GGGGSGGGSGGGGGS (SEQ ID NO: 106), or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 107).

[0015] Any of the bispecific CARs disclosed herein may further comprise a signal peptide at the N-terminus.

[0016] In certain examples, the bispecific CAR disclosed herein may comprise an extracellular bispecific antigen-binding domain, which may comprise the amino acid sequence of SEQ ID NO: 163 or 164. In one example, the bispecific CAR may comprise the amino acid sequence of SEQ ID NO: 165 or 166.

[0017] In some aspects, provided herein is a nucleic acid or set of nucleic acids that collectively encode any of the bispecific CARs disclosed herein. In some cases, the nucleic acid comprises a first nucleotide sequence encoding the bispecific CAR. In some embodiments, the nucleic acid may further comprise a second nucleotide sequence encoding an armor polypeptide disclosed herein that enhances T cell functionality, and a third nucleotide sequence encoding a self-cleaving peptide located between the first and second nucleotide sequences. The nucleic acid or set of nucleic acids may be an expression vector, optionally a viral vector.

[0018] Exemplary arming polypeptides include, but are not limited to, IL-2, IL-5, IL-15, a costimulatory ligand, an anti-PDL1 antibody, or a fusion polypeptide comprising an anti-PDL1 antibody. In some cases, the arming polypeptide is a fusion protein comprising an anti-PDL1 antibody (e.g., a single-chain variable fragment (scFv)) and an IL-2 polypeptide. Exemplary arming polypeptides are provided in Table 11 below, each of which is within the scope of this disclosure.

[0019] Further provided herein are engineered immune cells that express the bispecific CAR disclosed herein and, optionally, further express the arming polypeptides disclosed herein. Such engineered immune cells may comprise any of the nucleic acids disclosed herein encoding the bispecific CAR and, optionally, the arming polypeptide. In some embodiments, the engineered immune cells may be T cells, NK cells, or macrophages. In one example, the immune cells are T cells. In particular examples, the engineered immune cells (e.g., T cells or NK cells) provided herein may express the bispecific CAR of SEQ ID NO: 165 or 166, as well as an arming polypeptide, e.g., 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.

[0020] In another aspect, the disclosure features an anti-TACI chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for TACI, a costimulatory signaling domain, and a cytoplasmic signaling domain, wherein the extracellular antigen-binding domain specific for BCMA is as disclosed herein. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively, or in addition, the cytoplasmic signaling domain is derived from CD3ζ. In some examples, the anti-TACI CAR may further comprise a hinge domain and / or a transmembrane domain between the extracellular antigen-binding domain specific for TACI and the costimulatory domain. In some examples, the anti-TACI CAR may further comprise both a hinge domain and a transmembrane domain. The anti-TACI CAR may further comprise a spacer between the hinge domain and the transmembrane domain. In particular examples, an 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).

[0021] In another aspect, the disclosure features an anti-BCMA chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specific for BCMA, a costimulatory signaling domain, and a cytoplasmic signaling domain, wherein the extracellular antigen-binding domain specific for BCMA is as disclosed herein. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. Alternatively, or in addition, the cytoplasmic signaling domain is derived from CD3ζ. In some examples, the anti-BCMA CAR may further comprise a hinge domain and / or a transmembrane domain between the BCMA-specific extracellular antigen-binding domain and the costimulatory domain. In some examples, the anti-BCMA CAR may comprise both a hinge domain and a transmembrane domain. The anti-BCMA CAR may further comprise a spacer between the hinge domain and the transmembrane domain. In particular examples, an 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).

[0022] Also provided herein is a nucleic acid comprising a first nucleotide sequence encoding an anti-TACI CAR described herein or an anti-BCMA CAR described herein. The nucleic acid may further comprise a second nucleotide sequence encoding an arming polypeptide disclosed herein that enhances T cell functionality, and a third nucleotide sequence encoding a self-cleaving peptide located between the first and second nucleotide sequences. In some cases, the nucleic acid is an expression vector, optionally a viral vector.

[0023] Further provided herein are engineered immune cells that express the anti-BCMA CAR and / or anti-TACI CAR disclosed herein, and optionally also express any of the arming polypeptides disclosed herein. In some embodiments, the engineered immune cells can be T cells, NK cells, or macrophages. In one example, the engineered immune cells are T cells.

[0024] Additionally, the present disclosure provides a method for eliminating unwanted cells in a subject, the method comprising administering to a subject in need thereof an effective amount of engineered immune cells disclosed herein that express any of the anti-BCMA CARs, anti-TACI CARs, and anti-BCMA / anti-TACI bispecific CARs disclosed herein, and a pharmaceutical composition comprising same. Such engineered immune cells may be armored T cells that further express one or more arming polypeptides, such as those disclosed herein.

[0025] In some embodiments, the unwanted cells are cancer cells. In some embodiments, the subject is a human cancer patient. For example, the human cancer patient may have BCMA + and / or TACI + The cancer cells may include 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.

[0026] Also within the scope of the present disclosure are genetically engineered immune cells or pharmaceutical compositions, including those disclosed herein, for use in cancer treatment. Further provided herein is the use of genetically engineered immune cells or pharmaceutical compositions comprising such for the manufacture of a medicament for use in cancer treatment.

[0027] In addition, the present disclosure provides a method for producing a heavy chain variable region (V H ) and the light chain variable region (V L ) H and V L The chains contain the same complementarity determining regions (CDRs, including CDR1, CDR2, and CDR3) as one of the reference antibodies (e.g., TC-01) provided in Table 2 below. In some cases, the anti-TACI antibody contains the same V chain as the reference antibody, such as TC-01. H and V L In some examples, the anti-TACI antibody is a single chain antibody, such as those provided in Table 2 (e.g., SEQ ID NO: 75).

[0028] Additionally, the present disclosure provides a method for the production of a heavy chain variable region (V H ) and the light chain variable region (V L ) H and V L The chains comprise the same complementarity determining regions (CDRs, including CDR1, CDR2, and CDR3) as one of the reference antibodies (e.g., BC-06) provided in Table 1 below. In some cases, the anti-BCMA antibody has the same V complementarity determining regions (V complementarity determining regions) as a reference antibody, such as BC-06. H and V L In some examples, the anti-BCMA antibody is a single chain antibody, such as those provided in Table 1 (e.g., SEQ ID NO: 50).

[0029] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief explanation of the drawings]

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0031] [Figure 1] 1 is a graph showing exemplary anti-BCMA antibodies (in scFv format) shown to specifically bind to BCMA-expressing CHOK1 cells but not to parental CHOK1 cells. [Figure 2] Figure 2A includes graphs showing binding of exemplary anti-BCMA antibodies (in scFv format) to cells expressing endogenous BCMA: H929 cell line, Figure 2B: MM1.S cell line, and Figure 2C: RPMI8226 cell line. [Figure 3] 1 is a graph showing the binding activity of exemplary anti-TACI antibodies (in scFv format) to various cells as indicated. [Figure 4]Figures showing the cytotoxic T lymphocyte (CTL) activity of exemplary monospecific anti-BCMA CAR-T cells against the various cells indicated. Figure 4A: CAR-T cells expressing the anti-BCMA construct EPLV102 against the various cell lines indicated. Figure 4B: CAR-T cells expressing the anti-BCMA construct EPLV103 against the various cell lines indicated. Figure 4C: CAR-T cells expressing the anti-BCMA construct EPLV104 against the various cell lines indicated. Figure 4D: CTL activity of the various anti-BCMA CAR constructs shown against K562-GFP and K562-BCMA-TACI-GFP cells. Figure 4E: IFNγ secretion by CAR-T cells expressing the various anti-BCMA CARs shown in the presence of target cells. [Figure 5] Figure 5A shows the CTL activity of CAR-T cells expressing EPLV101, EPLV102, and EPLV103 against the indicated target cells. Figure 5B shows the IFNγ secretion levels. [Figure 6] Figures showing CTL activity of CAR-T cells expressing EPLV102 against the indicated target cells are included. Figure 6A: E:T ratio of 10:1. Figure 6B: E:T ratio of 5:1. Figure 6C: Cell killing rate. Figure 6D: Percentage of CD4 / CD8 T cell subsets. Figure 6E: Percentage of effector T cell subsets. [Figure 7] Figures showing the CTL activity of CAR-T cells expressing EPLV102 under target cell re-challenge are included. Figure 7A: K562-GFP cells. Figure 7B: MM1R-GFP cells. [Figure 8] Figures 8A-8D show CTL activity of CAR-T cells expressing EPLV200, EPLV254, EPLV255, and EPLV256 against the indicated target cells. Figure 8A: Expression of anti-TACI CAR. Figures 8B-8D: CTL activity against K562-GFP, H929-GFP, and MM1R-GFP cells, respectively. Figure 8E: Cell killing rate. [Figure 9]Figures 9A-9C show the persistence of CAR-T cells expressing anti-TACI CAR constructs with different spacers under target cell re-challenge. Figure 9A: CAR expression levels. Figures 9B-9C: CTL activity against K562-GFP and MM1R-GFP cells, respectively. [Figure 10] Figures showing the expression of anti-BCMA / anti-TACI bispecific antibodies in immune cells are included. Figure 10A: Expression of the bispecific CAR construct EPLV217 in T cells from two donors. Figure 10B: Expression of the bispecific CAR construct EPLV302 in T cells from two donors. [Figure 11] Figures showing the CTL activity of CAR-T cells expressing the bispecific CAR EPLV217 against various target cells are included. 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. [Figure 12] Figures 12A-12B include figures showing the CTL activity of CAR-T cells expressing the bispecific CAR EPLV302 against various target cells. Figures 12A-12B: CTL activity of bispecific CAR-T cells from donors 994 and 995, respectively, upon re-challenge with K562-GFP cells at an E:T ratio of 1:1. Figures 12C-12D: CTL activity of bispecific CAR-T cells from donors 994 and 995, respectively, upon re-challenge with MM1R-GFP cells at an E:T ratio of 1:1. Figures 12E-12F: CTL activity of bispecific CAR-T cells from donors 994 and 995, respectively, upon re-challenge with H929-GFP cells at an E:T ratio of 1:1. Figures 12G-12H: Cell killing rates of CAR-T cells from donors 994 and 995, respectively. Figure 12I: IFNγ secretion. [Figure 13] Figures showing the in vivo activity of CAR-T cells expressing anti-BCMA monospecific CARs, anti-TACI monospecific CARs, and anti-BCMA / anti-TACI bispecific CARs in animal models are included. Figure 13A: Radiance levels. Figure 13B: Body weight. Figure 13C: Tumor growth. [Figure 14]Figures showing the bioactivity of engineered T cells expressing an anti-BCMA / TACI bispecific CAR alone (demilitarized CAR) or in combination with an arming polypeptide (armored CAR). Figure 14A: CAR expression levels 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. [Figure 15] Figures showing the anti-tumor activity of engineered T cells expressing anti-BCMA / TACI bispecific CARs alone (demilitarized CAR) or in combination with arming polypeptides (armored CAR). Figure 15A: Photographs showing tumor burden at various time points after CAR-T cell treatment. Figure 15B: Diagram showing the anti-tumor activity of armed CAR-T cells compared to disarmed CAR-T cells. [Figure 16] Figures showing the bioactivity of engineered NK cells expressing anti-BCMA / TACI bispecific CARs alone (demilitarized CAR) or in combination with arming polypeptides (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 various time points. DETAILED DESCRIPTION OF THE INVENTION

[0032] 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 (TNFRSF 17) and CD269. This receptor is expressed at high levels on hematopoietic cancer cells, such as multiple myeloma (MM) cells, but is not expressed on other normal tissues except for normal plasma cells. BCMA plays a role in regulating B-cell proliferation and survival, as well as maturation and differentiation into plasma cells.

[0033] 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 that is primarily expressed on the surface of B cells. TACI has three ligands: proliferation-inducing ligand (APRIL), B-cell-activating factor (BAFF), and calcium-modulating ligand (CAML). Binding of TACI to these ligands can trigger signaling pathways, leading to the regulation of cellular activity.

[0034] Provided herein are 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. In some embodiments, the CAR-T cells disclosed herein can further express an arming polypeptide that enhances CAR-T cell characteristics (e.g., proliferation and expansion, persistence, and / or cytotoxicity). The CAR-T cells disclosed herein can be used for the treatment of diseases involving BCMA and / or TACI (e.g., for use in cancer therapy). The bispecific CAR-T cells disclosed herein can activate CAR-T cells by bispecifically targeting one or both of two tumor antigens, BCMA and TACI. Such bispecific CAR-T cells are expected to be more effective in cancer therapy, for example, by preventing and / or treating target antigen escape. The armed CAR-T cells disclosed herein can further improve CAR-T cell efficiency, activate immune cells and / or mediate anti-tumor activity, overcome the suppressive tumor microenvironment, and thereby enhance cancer treatment.

[0035] I. Chimeric Antigen Receptor 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 unwanted cell, e.g., an antigen of interest (TAA) (here, BCMA and / or TACI). Generally, a CAR may comprise a fusion polypeptide comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment or scFv derived from an antibody specific for the target antigen), a costimulatory domain, and an intracellular signaling domain. In some cases, the fusion polypeptide may further comprise a hinge and transmembrane domain located C-terminal to the extracellular antigen-binding domain. In some embodiments, a CAR disclosed herein is a T cell receptor. In other embodiments, a CAR disclosed herein may be an NK cell receptor.

[0036] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located within the variable region of the immunoglobulin molecule. As used herein, the term "antibody," e.g., anti-BCMA antibody or anti-TACI antibody, refers to intact (e.g., full-length) polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., V), and the like. HThe term also encompasses any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies, for example, anti-galectin-9 antibodies, include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof); antibodies need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0037] A typical antibody molecule typically contains a heavy chain variable region (V H ) and the light chain variable region (V L ) included. V H Area and V L The regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). H and V Ltypically consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment 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.

[0038] In some embodiments, the antibody portions disclosed herein contain the same heavy and / or light chain complementarity determining regions (CDRs) or the same V as a reference antibody. H and / or V L They may share the same V H and / or V L Two antibodies that have CDRs mean that their CDRs are identical when determined by the same method (e.g., the Kabat, Chothia, AbM, Contact, or IMGT methods known in the art; see, e.g., bioinf.org.uk / abs / ). Such anti-BCMA or anti-TACI antibodies have the same V compared to the exemplary antibodies described herein. H , the same V L , or both.

[0039] In some embodiments, 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, modified as 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. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0040] In some embodiments, antibody portions disclosed herein may have one or more amino acid variations relative to a reference antibody. The amino acid residue variations disclosed in this disclosure (e.g., in framework regions and / or CDRs) may be conservative amino acid residue substitutions. As used herein, a "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 may be prepared by methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references summarizing such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.A.usubel, et al., eds., 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.

[0041] The anti-BCMA monospecific CARs, anti-TACI monospecific CARs, and anti-BCMA / anti-TACI bispecific CARs disclosed herein each comprise an anti-BCMA portion and / or an anti-TACI portion in the extracellular antigen-binding domain.

[0042] (a) Anti-BCMA binding part The anti-BCMA binding moiety in any of the CARs disclosed herein (e.g., any of the anti-BCMA monospecific CARs or anti-BMCA / anti-TACI bispecific CARs disclosed herein) comprises a heavy chain variable domain (V) of an anti-BCMA antibody connected by a peptide linker. H ) and the light chain variable domain (V LThe scFv fragment may be in the form of an scFv, which is a fusion polypeptide comprising a V H and V L The fragments can be in any orientation. In some cases, the scFv is arranged N-terminally to C-terminally in the V L fragment, a peptide linker, and V H Alternatively, the scFv may comprise, from N-terminus to C-terminus, H fragment, a peptide linker, and V L In some examples, the scFv may further comprise an N-terminal signal peptide to direct the CAR containing the scFv to the cell surface.

[0043] Exemplary anti-BCMA antibodies are provided below in Table 1, any of which are within the scope of the present disclosure. In one example, for example, an anti-BCMA antibody for use in constructing a monospecific or bispecific CAR provided herein can be clone BC-06. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0044] An anti-BCMA binding moiety (and anti-TACI binding moieties disclosed below) derived from a reference antibody refers to a binding moiety that has substantially similar structural and functional characteristics as the reference antibody. Structurally, the binding moiety may contain the same heavy and / or light chain complementary determining regions, or the same V H Chain and / or V LAlternatively, the binding moiety may have only a limited number of amino acid variations in one or more of the framework regions and / or one or more of the CDRs without significantly affecting binding affinity and specificity relative to the reference antibody. See discussion below.

[0045] In some embodiments, the anti-BCMA binding moiety may comprise one or more of the heavy and light chain complementarity determining region (CDR) motifs provided in Table 1 above, including, for example, all six CDR motifs provided in Table 1. In some examples, the anti-BCMA binding moiety may be derived from the anti-BCMA antibody BC-05. In some examples, the anti-BCMA binding moiety may be derived from the anti-BCMA antibody BC-06. In some examples, the anti-BCMA binding moiety may be derived from the anti-BCMA antibody BC-08.

[0046] In some examples, an anti-BCMA binding moiety may comprise the same heavy chain CDRs as any of the reference antibodies (e.g., BC-05, BC-06, or BC-08) provided in Table 1 above. Alternatively, or in addition, an anti-BCMA binding moiety may have the same light chain CDRs as any of the reference antibodies (e.g., BC-05, BC-06, or BC-08) provided in Table 1 above. Such an anti-BCMA binding moiety may have the same V H Chain and / or V L Alternatively, the anti-BCMA binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework region in the reference antibody. For example, the anti-BCMA binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework region in the reference antibody.

[0047] In some embodiments, the anti-BCMA moiety may contain some level of variation in one or more CDRs relative to the CDRs of any of the reference antibodies provided in Table 1 above (e.g., BC-05, BC-06, or BC-08). For example, the anti-BCMA moiety may contain a V H Alternatively, or in addition, the anti-BCMA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of the reference antibody. L The antibody may comprise light chain CDRs that, individually or collectively, share at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity with the corresponding CDRs. As used herein, "individually" means that one CDR of the antibody shares the sequence identity shown with the corresponding CDR of a reference antibody (e.g., those in Table 1, such as BC-05, BC-06, or BC-08, or any of the anti-TACI reference antibodies disclosed below). "Collectively" means that the three Vs of the combined antibody share the sequence identity shown with the corresponding CDR of a reference antibody (e.g., those in Table 1, such as BC-05, BC-06, or BC-08, or any of the anti-TACI reference antibodies disclosed below). H or V L The CDRs are aligned with the corresponding three V H or V L It means that they share the sequence identity shown for the CDRs.

[0048] In some cases, the anti-BCMA portion may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of a reference antibody (e.g., BC-05, BC-06, or BC-08) provided in Table 1. In some cases, the anti-BCMA portion may comprise a heavy chain CDR3 that is the same as the heavy chain CDR3 of the reference antibody and may comprise one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0049] In some examples, the anti-BCMA moieties disclosed herein can be any of the anti-BCMA scFv fragments provided in Table 1, above. In particular examples, the anti-BCMA scFv can comprise the amino acid sequence of SEQ ID NO: 41, 50, or 62. Alternatively, the anti-BCMA moiety can comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to those provided in Table 1, e.g., SEQ ID NO: 41, 50, or 62. In other examples, the anti-BCMA moieties disclosed herein 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 those listed in Table 1, e.g., SEQ ID NO: 41, 50, or 62. H and V L It may contain sequences, but V H and V L It has the reverse orientation of the fragments.

[0050] Any of the anti-BCMA moieties disclosed herein (e.g., those provided in Table 1, such as SEQ ID NOs: 41, 50, or 62, or the reverse V H and V L or its counterpart with the same orientation) can be used to construct the anti-BCMA monospecific and / or anti-BCMA / anti-TACI bispecific CAR constructs disclosed herein.

[0051] (b) Anti-TACI binding part The anti-TACI binding moiety in any of the CARs disclosed herein (e.g., any of the anti-TACI monospecific CARs and / or anti-BCMA / anti-TACI bispecific CARs disclosed herein) comprises a heavy chain variable domain (V) of an anti-TACI antibody connected by a peptide linker. H ) and the light chain variable domain (V L The scFv fragment may be in the form of an scFv, which is a fusion polypeptide comprising a V H and V L The fragments can be in any orientation. In some cases, the scFv is arranged N-terminally to C-terminally in the V L fragment, a peptide linker, and V H Alternatively, the scFv may comprise, from N-terminus to C-terminus, Hfragment, a peptide linker, and V L In some examples, the scFv may further comprise an N-terminal signal peptide to direct the CAR containing the scFv to the cell surface.

[0052] In some embodiments, the anti-TACI binding moiety can be derived from any of those provided below in Table 2. The heavy and light chain complementarity determining regions provided in Table 2 (as well as Table 1) are based on the Chothia definition. [Table 2-1] [Table 2-2]

[0053] In some examples, an anti-TACI binding moiety can contain the same heavy chain CDRs as any of the reference antibodies listed in Table 2 above, e.g., TC-01. Alternatively, or in addition, an anti-TACI binding moiety can have the same light chain CDRs as those of the reference antibody, e.g., TC-01. Such an anti-TACI binding moiety can have the same V H Chain and / or V L Alternatively, the anti-TACI binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework region in the reference antibody. For example, the anti-TACI binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework region in the reference antibody.

[0054] In some embodiments, an anti-TACI binding moiety may contain some level of variation in one or more CDRs relative to that of a reference antibody (e.g., TC-01) provided in Table 2 above. For example, an anti-TACI binding moiety may contain a V HAlternatively, or in addition, the anti-TACI antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of the reference antibody. L It may comprise light chain CDRs that, individually or collectively, have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0055] In some cases, the anti-TACI binding portion may include up to 10 amino acid variations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively relative to those in the CDRs of a reference antibody (e.g., TC-01) provided in Table 2. In some cases, the anti-TACI binding portion may include a heavy chain CDR3 that is the same as the heavy chain CDR3 of the reference antibody and may include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0056] In some examples, an anti-TACI binding moiety disclosed herein can comprise the amino acid sequence of any of the anti-TACI scFv fragments provided in Table 2 above (e.g., SEQ ID NO: 75). Alternatively, an anti-TACI moiety 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 a reference antibody provided in Table 2 (e.g., SEQ ID NO: 75). In other examples, an anti-TACI binding moiety disclosed herein can comprise the V of the reference antibody. H and V L V identical to the sequence (e.g., SEQ ID NO: 75) H and V L sequence, but may contain the same V H and V L It has the reverse orientation of the fragments.

[0057] Any of the anti-TACI binding moieties disclosed herein (e.g., those provided in Table 2) can be used to construct the anti-TACI monospecific and / or anti-BCMA / anti-TACI bispecific CARs disclosed herein. In some examples, the anti-TACI moiety has the amino acid sequence of SEQ ID NO: 75, or the reverse V H and V L It may include its counterpart with orientation.

[0058] (c) Other components of the chimeric antigen receptor construct In addition to the extracellular antigen-binding domain disclosed herein, any of the CARs, including anti-BCMA CARs, anti-TACI CARs, or anti-BCMA / anti-TACI bispecific CARs, may further comprise one or more intracellular signaling domains (e.g., costimulatory and cytoplasmic signaling domains), 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 arming polypeptide within the host immune cell, which enhances the physical and / or biological characteristics of the host immune cell. See, for example, the disclosure herein. For example, the CAR coding sequence and suicide gene can be configured in a bicistronic expression cassette, and the CAR coding sequence and arming gene can be linked via a self-cleaving peptide (e.g., P2A or T2A) coding sequence. Examples are provided in Table 3 below. [Table 3]

[0059] Signaling domains Any of the CAR constructs disclosed herein, including anti-BCMA CARs, anti-TACI CARs, or anti-BCMA / anti-TACI bispecific CARs, typically comprise one or more intracellular signaling domains containing 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 signaling to induce an immune response, such as an effector function (secondary signaling). The cytoplasmic signaling domain can be any signaling domain involved in inducing cell signaling (primary signaling) that leads to immune cell proliferation and / or activation. The cytoplasmic signaling domains described herein are not costimulatory signaling domains known in the art that relay costimulatory or secondary signals to fully activate immune cells.

[0060] In some embodiments, the costimulatory signaling domain and the cytoplasmic signaling domain are for use in a CAR construct disclosed herein that is introduced into a T cell. In some cases, the costimulatory signaling domain can be derived from a costimulatory protein involved in T cell responses, such as a member of the B7 / CD28 family, a member of the TNF superfamily, a member of the SLAM family, or any other costimulatory 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 particular example, the costimulatory signaling domain is a 4-1BB signaling domain (e.g., SEQ ID NO: 116 in Table 3 above). In another particular example, the costimulatory signaling domain is a CD28 signaling domain (e.g., SEQ ID NO: 117 in Table 3 above).

[0061] The cytoplasmic signaling domain may contain an immunoreceptor tyrosine-based activation motif (ITAM) domain or may be ITAM-free. "ITAM," as used herein, generally refers to a conserved protein motif 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.

[0062] Hinge and transmembrane domains In some cases, a CAR construct disclosed herein (e.g., any of the anti-BCMA CARs, anti-TACI CARs, or anti-BCMA / anti-TACI bispecific CARs disclosed herein) may contain a transmembrane domain, which may be a hydrophobic alpha-helix that spans the membrane. A "transmembrane domain" may be a peptide fragment that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain may provide stability to a CAR containing it. An exemplary transmembrane domain may be the CD8 transmembrane domain or the CD28 transmembrane domain. In one example, the transmembrane domain may comprise SEQ ID NO: 109, 110, 111, or 112, as shown in Table 3 above.

[0063] Alternatively, or in addition, 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 function to provide flexibility to the CAR or a domain thereof or to prevent steric hindrance of the CAR or a domain thereof. The hinge domain may comprise 5 to 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 comprise SEQ ID NO: 106, as shown in Table 3 above.

[0064] (d) Anti-BCMA / anti-TACI bispecific CAR In some aspects, provided herein are anti-BCMA / anti-TACI bispecific CARs each comprising an anti-BCMA binding moiety (e.g., an anti-BCMA scFv such as those disclosed herein; see Table 1 above, such as SEQ ID NO: 50), an anti-TACI moiety (e.g., an anti-TACI scFv such as those disclosed; see Table 2 above, such as SEQ ID NO: 75), one or more intracellular signaling domains, such as a costimulatory 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 can be a single polypeptide comprising both the anti-BCMA and anti-TACI moieties. In other cases, the anti-BCMA / anti-TACI bispecific CAR can be a multi-chain (e.g., two-chain) molecule. The anti-BCMA and anti-TACI moieties can be located on separate polypeptides.

[0065] In some embodiments, an anti-BCMA / anti-TACI bispecific CAR disclosed herein may comprise an anti-BCMA binding moiety (e.g., scFv) derived from BC-06 and an anti-TACI binding moiety (e.g., scFv) derived from TC-01.

[0066] The anti-BCMA binding moiety (e.g., scFv) derived from BC-06 can be any of the anti-BCMA moieties for BC-06 disclosed above. In some cases, it can contain the same heavy and / or light chain CDRs as BC-06. In certain examples, the scFv contains the same V H and / or the same V L In some cases, the scFv may comprise, from the N-terminus to the C-terminus, L fragment (e.g., SEQ ID NO: 49), a peptide linker (e.g., SEQ ID NO: 104), and V H Alternatively, the scFv may comprise, from N-terminus to C-terminus, H fragment (e.g., SEQ ID NO: 45), a peptide linker (e.g., SEQ ID NO: 104), and V LIn one particular example, the anti-BCMA binding moiety may comprise SEQ ID NO: 50.

[0067] The anti-TACI binding moiety (e.g., scFv) derived from TC-01 can be any of the anti-TACI binding moieties related to TC-01 disclosed above. In some cases, the scFv contains the same heavy and / or light chain CDRs as TC-01. In certain examples, the scFv contains the same V H and / or the same V L In some cases, the scFv may comprise, from the N-terminus to the C-terminus, L fragment (e.g., SEQ ID NO: 74), a peptide linker (e.g., SEQ ID NO: 104), and V H Alternatively, the scFv may comprise, from N-terminus to C-terminus, H fragment (e.g., SEQ ID NO: 70), a peptide linker (e.g., SEQ ID NO: 104), and V L In one particular example, the anti-TACI portion may comprise SEQ ID NO:75.

[0068] In some embodiments, the bispecific anti-BCMA / anti-TACI binding moieties may be located on a single polypeptide. In some examples, the single polypeptide contains a spacer (peptide linker) between the anti-BCMA binding moiety and the anti-TACI binding moiety. Examples are provided in Table 3. See also the Examples below.

[0069] Any of the fusion polypeptides comprising anti-BCMA and anti-TACI portions may further comprise a costimulatory signaling domain and a cytoplasmic signaling domain such as those disclosed herein. Optionally, the fusion polypeptide may further comprise a hinge domain and a transmembrane domain, also disclosed herein. In some examples, the bispecific CAR may be included in a polycistronic expression cassette with arming genes (e.g., those listed in Table 11 below) via a self-cleaving peptide linker.

[0070] (e) anti-BCMA or anti-TACI monospecific CAR Also within the scope are anti-BCMA or anti-TACI monospecific CARs that comprise any of the anti-BCMA or anti-TACI binding moieties disclosed herein.

[0071] In some aspects, provided herein are anti-BCMA CARs, nucleic acids encoding such, and host cells expressing such. The anti-BCMA CAR may comprise (a) an extracellular binding domain, which may be any of the anti-BCMA binding moieties, 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 costimulatory signaling domain, such as those disclosed herein; and (c) a cytoplasmic signaling domain, such as those disclosed herein. The anti-BCMA CAR may further comprise a hinge domain and a transmembrane domain located C-terminal to the extracellular antigen-binding domain. In one example, the anti-BCMA CAR comprises the amino acid sequence of any one of SEQ ID NOs: 119-142 (e.g., SEQ ID NO: 127 or 128).

[0072] In some embodiments, provided herein are anti-TACI CARs, nucleic acids encoding such, and host cells expressing such. In some examples, the anti-TACI CAR may comprise (a) an extracellular binding domain, which may be any of the anti-TACI binding moieties, e.g., an anti-TACI scFv derived from any of the reference antibodies provided in Table 2 above; (b) a costimulatory signaling domain, such as those disclosed herein; and (c) a cytoplasmic signaling domain, such as those disclosed herein. The anti-TACI CAR may further comprise a hinge domain and a transmembrane domain located C-terminal to the extracellular antigen-binding domain. In one example, the anti-TACI CAR comprises any one of the amino acid sequences of SEQ ID NOs: 143-164 (e.g., SEQ ID NO: 143 or 144).

[0073] Exemplary anti-BCMA monospecific CARs, anti-TACI monospecific CARs, and anti-BCMA / anti-TACI bispecific CARs are provided below in Table 4, all of which are within the scope of the present disclosure. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9]

[0074] II. CAR-expressing immune cells In some aspects, provided herein are 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 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).

[0075] (a) Armed CAR-T cells Any of the CAR-expressing immune cells disclosed herein can be further engineered with additional mechanisms to reprogram the CAR-expressing cells to enhance their biological activity and / or persistence, thereby enhancing overall therapeutic efficacy. For example, CAR-expressing immune cells can be further engineered to express arming polypeptides to enhance the physical and / or biological characteristics of the CAR-T cells. Such CAR-T cells are known as armed CAR-T cells that co-express one or more CAR constructs and arming polypeptides that can enhance CAR-T cell characteristics, such as improved growth and / or persistence, increased efficacy, reduced toxicity, etc., or a combination thereof.

[0076] Exemplary arming polypeptides include, but are not limited to, suitable cytokines such as IL-2, IL-5, and / or IL-15, costimulatory 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 activating ligands (e.g., 4-1BBL). In some embodiments, the arming polypeptide can be a fusion polypeptide comprising, for example, a cytokine or fragment thereof (e.g., IL2 or IL15 or fragment thereof) and a checkpoint inhibitor (e.g., an anti-PDL1 fragment). Specific examples of arming polypeptides are provided below in Table 11, each of which is within the scope of the present disclosure. [Table 5-1] [Table 5-2]

[0077] Additional information on arming polypeptides can be found, for example, in WO2021 / 030633 and WO2022 / 159771, the relevant disclosures of each of which are incorporated by reference for the subject matter and purposes referenced herein.

[0078] In some cases, the coding sequences for the CAR construct and the arming polypeptide can be located within the same expression cassette. The two coding sequences can be separated by a ribosome entry site (IRES) or a coding sequence for a self-cleaving peptide (e.g., P2A or T2A) to produce two separate polypeptides (CAR and arming polypeptide). In other cases, two separate expression cassettes can be used to express the CAR construct and arming polypeptide in armed CAR-T cells.

[0079] In some instances, the arming polypeptide contains an N-terminal signal peptide so that the polypeptide can be secreted from the CAR-T cell. Alternatively, the arming polypeptide can be expressed as an intracellular or membrane-bound protein.

[0080] (a) Preparation of CAR-expressing immune cells 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), and optionally one or more arming polypeptides, such as those disclosed herein, into suitable immune cells, and collecting the resulting engineered immune cells that express the CAR on their cell surface.

[0081] The population of immune cells as starting parent cells can be obtained from any source, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as spleen, lymph nodes, thymus, stem cells, or tumor tissue. Suitable sources for obtaining host cells of the desired type will be apparent to those of skill in the art. In some embodiments, the population of immune cells is derived from PBMCs. Host cells of the desired type (e.g., T cells, NK cells, macrophages, or a combination thereof) can be expanded within the resulting population of cells by co-incubating the cells with stimulatory molecules. As a non-limiting example, anti-CD3 and anti-CD28 antibodies can be used to expand T cells. In some embodiments, cells of a specific type (e.g., T cells, NK cells, or macrophages) can be enriched from the immune cell population. Such enriched cell subpopulations can be expanded and / or activated in vitro before being genetically engineered for the introduction of a CAR-encoding expression cassette and / or an arming polypeptide-encoding expression cassette (which can be the same expression cassette).

[0082] A CAR polypeptide described 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 arming polypeptides, such as those disclosed herein (e.g., those provided in Table 11), and an expression vector for stable or transient expression of the CAR polypeptide and optionally arming polypeptide can be generated via conventional methods and introduced into immunized host cells. For example, a nucleic acid encoding a CAR polypeptide and optionally arming polypeptides can be cloned into one or more suitable expression vectors, e.g., a viral vector operably linked to a suitable promoter. Non-limiting examples of useful vectors of the present disclosure include viral vectors, such as, for example, retroviral vectors, including gammaretroviral vectors, adeno-associated viral vectors (AAV vectors), and lentiviral vectors. The nucleic acid and vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and join with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the end of the nucleic acid encoding the CAR polypeptide and, optionally, the arming 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 depends on the type of host cell for expression of the CAR polypeptide and, optionally, the arming polypeptide, but must be suitable for integration and replication in eukaryotic cells. Any of such nucleic acids encoding a CAR and, optionally, the arming polypeptide and expression vectors containing them are also within the scope of the present disclosure.

[0083] For the expression of the CAR polypeptide described herein, and optionally, the arming polypeptide, various promoters can be used, including, but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs such as HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, or herpes simplex tk virus promoter. Additional promoters for the expression of the CAR polypeptide, and optionally, the arming polypeptide, include any constitutively active promoter in immune cells. Alternatively, any regulatable promoter can be used so that its expression can be regulated in immune cells. In some embodiments, the promoter can be the pEF1α promoter.

[0084] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene such as a neomycin gene or a kanamycin gene for selecting stable or transient transfectants in host cells; an enhancer / promoter sequence derived from the immediate early gene of human CMV for high-level transcription; a transcription termination and RNA processing signal derived from SV40 for mRNA stability; an SV40 polyomavirus origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" (e.g., an HSV thymidine kinase or an inducible caspase such as iCasp9) for inducing lethality in cells carrying the vector, and a reporter gene for assessing expression of the CAR polypeptide.

[0085] In a specific embodiment, such a vector may also contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cells expressing the suicide gene to die. A suicide gene may be a gene that confers sensitivity to a drug, e.g., a cell in which the gene is expressed, causing the cell to die when contacted with or exposed to the drug. Suicide genes are known in the art (see, e.g., 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, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

[0086] The nucleic acids disclosed herein can include two coding sequences, one 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 the other for an arming polypeptide. The two coding sequences can be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically distinct) polypeptides. To this end, the nucleic acids described herein can contain a third nucleotide sequence located between the first and second coding sequences. 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 encode a self-cleaving peptide, such as, for example, a P2A, T2A, or F2A peptide (see, e.g., Kim et al., PLoS One. 2011;6(4):e18556). See also Figure 3.

[0087] Any of the vectors comprising nucleic acid sequences encoding the ACTR polypeptides described herein, and optionally, arming polypeptides, are also within the scope of this disclosure.

[0088] Such vectors, or sequences encoding the CAR polypeptide and, optionally, the arming polypeptide contained therein, can be delivered into host cells, such as host immune cells (e.g., T cells, NK cells, or macrophages), by any suitable method. Methods of 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).

[0089] After introducing into the host cells a vector encoding a CAR polypeptide provided herein (e.g., an anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR construct disclosed herein, such as those provided in Table 4), and optionally any of the arming polypeptides provided herein, the cells can be cultured under conditions that allow for expression of the CAR polypeptide and optionally the arming polypeptide. If expression of the CAR polypeptide and / or arming polypeptide is regulated by a regulatable promoter, the host cells can be cultured under conditions in which the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter, and the immune cells are cultured in the presence of an inducing molecule or under conditions in which the inducing molecule is produced. Determining whether a CAR polypeptide and / or arming polypeptide is expressed will be apparent to one of skill in the art and can be assessed by any known method, for example, detection of mRNA encoding the CAR polypeptide and / or encoding the arming polypeptide by quantitative reverse transcriptase PCR (qRT-PCR), or detection of the CAR or arming 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.

[0090] The methods for preparing host cells expressing any of the CAR polypeptides and optionally arming polypeptides described herein can also include activating the host cells ex vivo. Activating a host cell refers to stimulating the host cell to an activated state in which it can perform effector functions. The method of activating a host cell will depend on the type of host cell used to express the CAR polypeptide and optionally arming 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 phytohemoagglutinin. In another example, 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 (CAR-expressing cells) expressing any of the CAR polypeptides and optionally arming polypeptides (armed CAR cells) described herein are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be apparent to one of skill in the art and may include assessing the expression of one or more cell surface markers associated with cellular activation, cytokine expression or secretion, and cell morphology.

[0091] Methods for preparing host cells expressing any of the CAR polypeptides and, optionally, arming polypeptides described herein can include expanding the host cells ex vivo. Expanding the host cells can include any method that results in an increase in the number of cells expressing the CAR polypeptide and, optionally, arming polypeptide, e.g., allowing the host cells to grow or stimulating the host cells to grow. Methods for stimulating host cell growth will depend on the type of host cell used to express the CAR polypeptide and, optionally, arming polypeptide, and will be apparent to one of skill in the art. In some embodiments, host cells expressing any of the CAR polypeptides and, optionally, arming polypeptides described herein are expanded ex vivo prior to administration to a subject.

[0092] In some embodiments, host cells expressing a CAR polypeptide, and optionally an arming polypeptide, are expanded and activated ex vivo before administering the cells to a subject. Activation and expansion of the host cells can be used to allow for integration of the viral vector into the genome and expression of the gene encoding the CAR polypeptide, and optionally an arming polypeptide, described herein. When mRNA electroporation is used, electroporation may be more effective when performed on activated cells, but may not require activation and / or amplification.

[0093] In some cases, the CAR polypeptide and / or arming polypeptide are transiently expressed (e.g., for 3-5 days) in a suitable host cell. Transient expression can be advantageous when there is potential toxicity and should be useful in the early stages of clinical trials for possible side effects.

[0094] (b) Pharmaceutical Composition Any of the engineered immune cells expressing a CAR and optionally an arming polypeptide 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 mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0095] The phrase "pharmaceutically acceptable," when used in connection with compositions of the present disclosure, refers to molecular entities and other components of such compositions that are physiologically tolerated and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in mammals, particularly humans. "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions used in the present methods may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.

[0096] 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 non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0097] For examples of additional useful medications, see also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0098] IV. Therapeutic uses Any of the engineered immune cells (e.g., T cells, NK cells, or macrophages) expressing a CAR 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 arming polypeptide also disclosed herein (e.g., those provided in Table 11), can be used for therapeutic purposes, e.g., to eliminate unwanted cells that express BCMA and / or TACI. In some examples, the engineered immune cells are armed CAR-T cells that express any of the anti-BCMA, anti-TACI, or anti-BCMA / TACI bispecific CAR constructs, such as those provided in Table 4 above, together with an arming polypeptide, such as those provided in Table 11 above.

[0099] To practice the methods described herein, an effective amount of immune cells (NK cells, T lymphocytes, or macrophages) expressing any of the CARs described 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), and optionally, an arming polypeptide (e.g., those provided in Table 11 above), or a pharmaceutical composition thereof, can be administered to a subject in need of treatment via a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of each agent (e.g., NK cells, T lymphocytes, or macrophages expressing a CAR, and optionally, an arming polypeptide) that confers a therapeutic effect on a subject upon administration. Determining whether an amount of cells or compositions described herein achieves a therapeutic effect will be apparent to one of skill in the art. An effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, gender, and weight, the duration of treatment, the nature of any concurrent therapy, if any, the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorate, improves, reduces, or slows the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient.

[0100] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to that amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. Within the context of the present disclosure, the term "therapeutically effective" refers to that amount of a compound or pharmaceutical composition sufficient to delay the onset of, halt the progression of, reduce, or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0101] In some embodiments, the methods of the present disclosure can be used to eliminate or inhibit disease cells that express BCMA and / or TACI. Thus, any of the immune cells disclosed herein can be used to eliminate or inhibit disease cells that express BCMA and / or TACI. + and / or TACI + BCMA in cancer cells + and / or TACI + The methods disclosed herein can be used to treat diseases associated with diseased cells. + and / or TACI + It may be used to treat cancers involving cancer cells, such as multiple myeloma, lung cancer, stomach cancer, breast cancer, testicular cancer, and the like.

[0102] In some embodiments, an effective amount of any of the engineered immune cells expressing a CAR 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 arming polypeptide (e.g., those provided in Table 11), may be provided to a subject in need of treatment via a suitable route, e.g., intravenous infusion. + and / or TACI + BMCA in cancer cells + and / or TACI + The human patient may have a disease associated with disease cells. In some cases, the human patient may have a disease associated with BCMA. + and / or TACI+ In some cases, the human patient may have multiple myeloma, lung cancer, stomach cancer, breast cancer, or testicular cancer.

[0103] In some embodiments, immune cells (e.g., NK and / or T cells) for use in the treatments disclosed herein can be autologous to the subject, i.e., immune cells can be obtained from a subject in need of treatment, genetically engineered for expression of a CAR polypeptide, and then administered to the same subject. In one particular embodiment, prior to reintroduction into the subject, autologous immune cells (e.g., T lymphocytes, NK cells, or macrophages) are activated and / or expanded ex vivo. Administration of autologous cells to a subject can result in reduced host cell rejection compared to administration of non-autologous cells.

[0104] 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 for expression of a CAR polypeptide, and administered to a second subject of the same species as the first subject. For example, the allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor. In certain embodiments, the T lymphocytes are allogeneic T lymphocytes in which expression of endogenous T cell receptors has been inhibited or eliminated. In one particular embodiment, the allogeneic T lymphocytes are activated and / or expanded ex vivo before being introduced into the subject. The T lymphocytes can be activated by any method known in the art, for example, in the presence of anti-CD3 / CD28, IL-2, and / or phytohemoagglutinin.

[0105] 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 the K562 cell line. See, e.g., U.S. Patent Nos. 7,435,596 and 8,026,097 for a description of useful methods for expanding NK cells. For example, NK cells used in the methods of the present disclosure lack or poorly express major histocompatibility complex I and / or II molecules and can be preferentially expanded by exposure to cells genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CDI37L). Such cell lines include K562 [ATCC, CCL243, 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 carcinoma cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB19 and N1369, testicular embryonic carcinoma cell line NEC14, cervical carcinoma cell line TCO-2, and bone marrow metastatic neuroblastoma cell line TNB1 [Harada, et al., Jpn. J. Cancer Res 93:313-319(2002)]. Preferably, the cell line used lacks or expresses insufficiently both MHCI and II molecules, such as K562 and HFWT cell lines. A solid support may be used instead of a 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 a molecule with such an affect, thereby functioning as a scaffold.The support may have a CD137 ligand protein, a CD137 antibody, an IL-15 protein, or an IL-15 receptor antibody bound to its surface. Preferably, the support has an IL-15 receptor antibody and a CD137 antibody bound to its surface.

[0106] According to the present disclosure, a patient is 5 ~10 9 Patients may be treated by infusing a therapeutically effective amount of immune cells, such as T lymphocytes or NK cells, expressing a CAR polypeptide, such as an anti-BCMA monospecific CAR, anti-TACI monospecific CAR, or anti-BCMA / anti-TACI bispecific CAR listed in Table 4 above, in the range of CAR+ cells, and optionally an arming polypeptide listed in Table 11. Infusions can be repeated as frequently and as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient and can be determined by the treating physician for a particular patient. In some examples, infusions of about 10 6 Inject an initial dose of cells / kg, 10 8 The dose may be gradually increased to more than 100 cells / kg.

[0107] The particular dosage regimen used in the methods described herein, i.e., dose, timing, and repetition, will depend on the particular subject and the subject's medical history. The appropriate dosage of the CAR-expressing immune cells (e.g., armed CAR-T cells) used will depend on the type of cancer being treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to immune cell therapy, and the discretion of the attending physician.

[0108] In some embodiments, engineered immune cells (e.g., armed 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) may be utilized in combination with other types of cancer therapy, such as chemotherapy, surgery, radiation therapy, or gene therapy. Such therapy may be administered simultaneously or sequentially (in any order) with immunotherapy according to the present disclosure. When co-administered with additional therapeutic agents, the suitable therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0109] V. Kits for Therapeutic Use The present disclosure also provides kits for the use of engineered immune cells (e.g., T lymphocytes, NK cells, or macrophages) expressing an anti-BCMA CAR, an anti-TACI CAR, or an anti-BCMA / anti-TACI bispecific CAR, and optionally, an arming polypeptide, as described herein. See, e.g., Table 11. Such kits may include one or more containers containing the engineered immune cells, which may be formulated into a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0110] In some embodiments, the kits described herein comprise genetically engineered immune cells that can be expanded in vitro. The immune cells can express any of the CARs disclosed herein, for example, any of the anti-BCMA CARs, anti-TACI CARs, and anti-BCMA / TACI bispecific CARs, such as those provided in Table 4 above. The immune cells can be armed CAR-T or CAR-NK cells that further express an arming polypeptide.

[0111] In some embodiments, the kit may additionally include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the engineered immune cells disclosed herein to achieve an intended activity in a subject, e.g., eliminating target disease cells, such as cancer cells that express BCMA, TACI, or both. The kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment.

[0112] Instructions for use of the genetically engineered immune cells described herein generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided with kits of the present disclosure are typically instructions on a label or package insert. The label or package insert indicates that the genetically engineered immune cells are used to treat, delay the onset of, and / or alleviate a disease or disorder associated with BCMA and / or TACI-positive disease cells in a subject.

[0113] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices, or injection devices, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The container may have a sterile access port.

[0114] Kits may optionally provide additional components such as buffers and interpretive information. Typically, kits include a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0115] general technologyThe practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds., (1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985>>; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984>>, Animal Cell Culture (R.I. Freshney, ed. (1986>>, Immobilized Cells and Enzymes (lRL Press, (1986>>, and are fully described in the literature such as B. Perbal, A practical Guide To Molecular Cloning (1984), F.M. Ausubel et al. (eds.).

[0116] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced therein.

[0117] Example 1. Generation and characterization of anti-BCMA antibodies This example demonstrates the identification and characterization of exemplary anti-BCMA antibodies.

[0118] Anti-BCMA scFv antibody screening by mRNA display mRNA display technology, 12~13 This method was used to identify BCMA binders from a natural human scFv library. Briefly, the scFv DNA library was first transcribed into an mRNA library and then translated into an mRNA-scFv fusion library by covalent conjugation via a puromycin linker, similar to a reported procedure (Patent: US6258558B1, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referenced herein). The fusion library was first selected multiple times with human IgG (a negative protein) to remove nonspecific binders, followed by selection against recombinant BCMA-Fc fusion protein and capture on protein G magnetic beads. Binders were eluted and then enriched by PCR amplification using library-specific oligos. In rounds 5-6, the scFv library was selected against the BCMA / HEK293 cell line. A total of six rounds of selection were performed to generate a highly enriched BCMA-binding pool for screening.

[0119] After six 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 Flag and 6xHis tag for purification and assay detection. Clones from the top 10 cells were pooled, miniprep DNA was prepared, and then transformed into the bacterial Rosetta II strain for expression. Single clones were picked, expanded, and induced with 0.1 mM IPTG in 96-well plates for expression. Supernatants were collected after 16–24 h of induction at 30°C for assay to identify anti-BCMA antibodies.

[0120] A BCMA binding screening 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 culture medium were cooled to 4°C and spun down at 1200 rpm for 6 minutes. The bacterial supernatant was diluted to a final concentration of 30% with cell culture medium, and the BCMA / CHOK1 cells were added. The plate was incubated with shaking at 4°C for 1 hour. The cells were spun down and the supernatant removed as described above. The plate was then washed with 200 μl of complete medium at 4°C. 100 μl of 1:250 prediluted AF-647-conjugated anti-HIS tag antibody was added to the cells. The plate was incubated in the dark at 4°C for 30 minutes with shaking. The plate was washed twice as described, and the cells were reconstituted in 200 μl of complete medium. Cells were mixed thoroughly and read on an Attune NxT cytometer. Analysis was performed using Attune NxT software, which plots overlaying histograms of anti-BCMA scFv binding to both negative and target cell lines.

[0121] Selective anti-BCMA scFv clones were selected from glycerol stock plates and grown overnight in 5 mL cultures in Thomson 24-well plates with breathable membranes. This culture, and all subsequent cultures described below, were grown at 37°C in Terrific Broth Complete supplemented with 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol, shaken at 225 RPM unless otherwise specified, and a 1:5,000 dilution of antifoam-204 was also added. This overnight starter culture was then used to inoculate larger cultures, 1:100 dilutions of the starter culture, designated production cultures, and grown to an OD600 of 0.5-0.8. At this point, the cultures were induced with a final concentration of 0.1 mM IPTG and incubated overnight at 30°C. The next day, the culture was spun at 5,000 xg for 30 minutes to pellet the cells, and the supernatant was then filter sterilized through a 0.2 um sterile PES membrane.

[0122] Purification involved the use of 3 μL of GE Ni Sepharose Excel resin per mL of filtered supernatant. Disposable 10 mL or 20 mL BioRad Econo-Pac columns were used. The resin was equilibrated with at least 20 column volumes (CV) of Buffer A (1× PBS, pH 7.4, containing additional NaCl added to 500 mM). The filter-sterilized supernatant was purified by gravity flow, either at a controlled flow rate of 1 mL / min or by pouring twice over the same packed resin bed. The column was then washed with the following buffers: 10 CV Buffer A, 20 CV Buffer B (1× PBS, pH 7.4, containing additional NaCl to 500 mM and 30 mM imidazole). Two Detox buffers were used to remove endotoxins as an optional step, if needed. For purification of 250 mL of expression culture, the antibody binding column was washed sequentially with 20 CV of buffer C (1x PBS pH 7.4, containing additional NaCl to 500 mM, 1% Tx114), 20 CV of buffer D (1x PBS pH 7.4, containing additional NaCl to 500 mM, 1% Tx100 + 0.2% TNBP), and 40 CV of buffer E (1x PBS pH 7.4, containing additional NaCl to 500 mM). Protein was eluted with elution buffer F (1x PBS pH 7.4, containing additional NaCl to 500 mM, and 500 mM imidazole) in a total of six fractions (0.5 CV pre-elution, 5x 1 CV elution). Fractions were subjected to a Bradford assay (100 μl of diluted Bradford solution + 10 μl of sample). Bright blue fractions were pooled. Protein concentration was measured by A280 elongation index. SDS-PAGE gel to analyze the purity of the purified antibody.

[0123] Characterization of Exemplary Anti-BCMA scFv Antibodies As disclosed above, exemplary anti-BCMA antibodies in scFv format isolated by mRNA library screening were analyzed to determine their antigen binding affinity. Structural information for exemplary anti-BCMA antibodies is provided in Table 1 above.

[0124] (a) BCMA binding activity by ELISA EC of anti-BCMA antibodies 50 An ELISA assay was developed to determine the binding of BCMA to the antibody. Briefly, 384-well plates were immobilized with anti-human Fc antibody at a final concentration of 2 μg / mL in 1x PBS in a total volume of 25 μL per well. The plates were incubated overnight at 4°C and subsequently blocked with 80 μL of Superblock per well for 1 hour. Human BCMA-Fc was captured via the immobilized anti-hFc antibody. Purified anti-BCMA scFv was serially titrated 2-fold from 200 nM. 25 μL of diluted scFv was added to the human BCMA-immobilized wells and incubated with shaking for 1 hour. 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 three times with 1x PBST in a plate washer. The plate was then developed with 20 μL of TMB substrate for 5 minutes and stopped by adding 20 μL of 2N sulfuric acid. Plates were read on a Biotek plate reader at OD 450 nm and then plotted using Prism 8.1 software. 50 The values ​​were calculated and are shown in Table 5 below. [Table 6]

[0125] (b) BCMA binding activity measured by surface plasmon resonance (SPR) Kinetic analysis of anti-BCMA scFv was evaluated using SPR technology with a Biacore T200. Assays were performed using 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 in 1x HBSP buffer on the Protein A sensor chip at a flow rate of 10 μl / min for 60 seconds. Two-fold serially diluted HIS-tagged purified anti-BCMA scFv was injected into both reference flow cell 1 and BCMA-Fc capture flow cell 2 at a flow rate of 30 μl / min for 150 seconds, followed by a 300-second wash. The flow cells were then regenerated with glycine pH 2 buffer (GE) at a flow rate of 30 μl / min for 30 seconds. Eight concentration points ranging from 300 to 0 nM per anti-BCMA scFv were assayed in a 96-well plate. The kinetics of scFv binding to BCMA protein were analyzed using Biacore T200 evaluation software version 3.0. Specific binding response units were derived from subtraction of binding from BCMA capture flow cell 2 to reference flow cell 1. K, K, and K values ​​were calculated for exemplary anti-BCMA antibodies (in scFv format) shown in Table 6 below. [Table 7]

[0126] (c) scFv antibody binding to cell surface BCMA via FACS To determine the binding selectivity and affinity of anti-BCMA scFv binding to BCMA-expressing cells, 200 nM purified anti-BCMA scFv antibody was diluted in complete medium and incubated with recombinant BCMA / CHOK1 cells and CHOK1 cells in a 96-well plate on ice for 1 hour. Cells were spun down at 1200 rpm for 6 minutes at 4°C to remove the primary antibody. Cells were then washed once with 200 μL of complete medium per well. Samples were detected with premixed anti-His biotin streptavidin Alexa fluor 647 by adding 100 μL of diluted secondary antibody and incubated in the dark for 30 minutes at 4°C. Samples were spun down at 1200 rpm for 5 minutes at 4°C and washed twice with 200 μL of 1x PBS per well. Samples were reconstituted in 200 μL of 1x PBS and read on an Attune NxT cytometer. Analysis was performed using Attune NxT software, which plots overlaid histograms of anti-BCMA scFv binding to both the negative and target cell lines. The anti-BCMA scFvs demonstrated selective binding to BCMA / CHOK1 cells and not to parental CHOK1 cells (Figure 1). BCMA cell binding affinities (in scFv format) for exemplary anti-BCMA antibodies were also generated using the serially diluted scFvs described above. EC 50 was calculated and is shown in Table 7 below. [Table 8]

[0127] (d) Anti-BCMA antibody binding to endogenous BCMA-expressing cell lines To further characterize BCMA and TACI expression in recombinant and endogenous cell lines, quantitative FACS assays were performed using Bangs Laboratories Inc. Quantum Alexa Fluor 647 MESF microsphere beads for standard calibration according to the manufacturer's protocol. The parental CHOK1 cell line showed undetectable BCMA and TACI expression. There was no TACI expression in the H929 and U226B1 cell lines. BCMA showed 4- to 6-fold higher expression than TACI in the MM1.R, MM1.S, and RPMI8226 multiple myeloma cell lines. BCMA and TACI receptor copy numbers are summarized in Table 8. [Table 9]

[0128] Binding of anti-BCMA antibodies (scFv) to endogenous BCMA was assessed using the FACS binding assay described above. 200 nM anti-BCMA scFv was tested on the negative cell lines H929, MM1.S, and RPMI8226. These exemplary antibodies showed similar binding patterns across different cell lines, with binding strength correlating with the number of BCMA receptors on each cell line (Figures 2A-2C).

[0129] The binding activity of the exemplary anti-BCMA clone BC-06 (in scFv format, see Table 1 above) against multiple myeloma cell lines (BCMA+ or BCMA-) was examined and EC 50 The values ​​are provided in Table 9 below. [Table 10]

[0130] Example 2. Generation and characterization of anti-TACI antibodies This example demonstrates the identification and characterization of exemplary anti-TACI antibodies.

[0131] Anti-TACI scFv antibody screening by mRNA display The mRNA display technology described in Example 1 above was used to generate 10 12~13 was used for the identification of TACI binders from a naive human scFv library.

[0132] Characterization of Exemplary Anti-TACI scFv Antibodies As disclosed above, exemplary anti-TACI antibodies in scFv format isolated by mRNA library screening were analyzed to determine their antigen binding affinity. Structural information for exemplary anti-TACI antibodies is provided in Table 2 above.

[0133] (a) TACI binding activity by FACS Anti-TACI scFv binders were identified by anti-TACI supernatant FACS screening assays 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 assays using the TACI-negative cell lines JVM2, MM1.R, and RPMI8226, K562, and H929, as well as TACI-expressing cancer cell lines. Assay procedures and data analysis were described in Example 1 above.

[0134] The anti-TACI scFv demonstrated selective binding to the target cell lines. The level of binding correlates with the TACI receptor number on the cell line (Figure 3). EC values ​​of exemplary anti-TACI antibodies (in scFv format) 50 The values ​​are summarized in Table 10 below. [Table 11]

[0135] (b) TACI binding activity by ELISA Purified anti-TACI scFvs were subjected to ELISA binding assays. Recombinant TACI and negative proteins were immobilized on 384-well plates. 200 nM anti-TACI scFv clones were serially diluted and tested for binding in the above assay. An exemplary anti-TACI scFv generated from live cell selection (see disclosure above) did not bind to soluble recombinant TACI from multiple sources.

[0136] Example 3. Construction of anti-BCMA monospecific chimeric antigen receptor (CAR) This example demonstrates the construction and characterization of an exemplary anti-BCMA monospecific chimeric antigen receptor (CAR).

[0137] Anti-BCMA CAR construct 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 N- to C-terminus, an anti-BCMA scFv, a flag tag, an IgG4 hinge, a spacer, a CD28 transmembrane, a 4-1BB costimulatory 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.

[0138] Production of lentiviral vectors to produce anti-BCMA CARs The coding sequence of the exemplary anti-BCMA CAR described above was cloned into a lentiviral vector according to standard molecular biology methods. The resulting lentiviral vector was co-transfected with LV-MAX packaging mix into Expi HEK293 cells using polyethyleneimine (PEI) transfection reagent according to the manufacturer's protocol. The transfected cells were grown at 37°C with shaking at 8% CO2 for 72 hours. The supernatant was centrifuged at 3200 rpm for 10 minutes at room temperature and collected by vacuum filtration using a 0.45 μm PES membrane. The virus was concentrated by ultracentrifugation (Beckman Coultier) at 8000 rpm for 1 hour at 4°C. The pellet was then resuspended in lentiviral stabilizer, immediately aliquoted, and stored at -80°C.

[0139] Transduction and propagation of immune cells with viral vectors encoding BCMA CAR PBMCs were isolated from the LRS chamber of fresh healthy donors using density gradient centrifugation lymphoprep and a SepMate 50 kit from Stemcell Technology. CD3+ Pan T cells were then isolated from the PBMCs using the EasySep Human T Cell Isolation Kit according to the Stemcell Technology protocol. Pan T cells were activated with human T activator CD3 / CD28 Dynabeads at a 1:1 bead-to-cell ratio for 24 hours and then transduced with lentivirus in the presence of Dynabeads and 1 mg / mL protamine sulfate. Spinoculation was performed at 300 x g for 2 hours at 25°C. 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 for 2 weeks in 5% human serum containing recombinant human IL-15 and IL-7 (Peprotech) in X-vivo 15 (Lonza) medium. The medium was changed every 2–3 days, and fresh cytokines were added.

[0140] Characterization of CAR-T cells expressing anti-BCMA CAR (i) Anti-BCMA CAR expression CAR surface expression was assessed by surface staining with anti-Flag tag antibody directly conjugated with Mix-n-stain AF647. Briefly, 100,000 lentiviral-transduced T cells were incubated with 0.1 μl of anti-Flag AF647 for 1 hour in the dark with shaking at 4°C. The cells were spun down at 1,300 rpm for 5 minutes, the supernatant removed, and washed with 200 μL of 1x PBS. The resulting sample was reconstituted in 200 μL of 1x PBS. The surface expression percentage was quantified by reading the fluorescently stained cells on an Attune NxT flow cytometer. Results from this study indicate that CAR expression levels for different CAR constructs (see Table 4 above) ranged from 50 to 85%.

[0141] (ii) Cytotoxic T lymphocyte (CTL) activity Isolation, viral transduction, and T cell expansion of human PBMCs and Pan T cells were performed as described above. To screen for various CAR activity, 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 or BCMA / TACI / K562-GFP cells at effector (CAR-T) to target cell ratios of 10:1, 5:1, and 1:1 in RPMI 1640 medium containing 10% FBS. No cytokines were added. The assay was performed for 64 hours, and GFP in target cells was imaged and quantified using a Cytation 5 scanner. IFNγ was detected after the CTL assay using the Human IFNγ Duoset ELISA kit (R&D System). Briefly, the supernatant was collected after the CTL assay was completed at 64 hours. Recombinant IFNγ was serially diluted and included in the assay to generate a standard curve. Supernatant IFNγ and recombinant IFNγ were assayed according to the manufacturer's protocol provided. Data were analyzed using Prism 8.0 software.

[0142] CTL activity using different target cells and a range of effector-to-target cell ratios is shown in Figures 4A-4C. Endpoint CTL activity for different CARs was calculated as shown in Figure 4D. IFNγ levels are shown in Figure 4E. BCMA monospecific CARs exhibited dose-dependent target-specific CTL activity, which correlated with IFNγ secretion. Multiple donors were screened with different CARs and showed similar CAR CTL activity (Figures 5A-5B).

[0143] (iii) CTL assay of CAR-T cells expressing the anti-BCMA construct EPLV102 To further evaluate the CTL activity of EPLV102-expressing CAR-T cells, PBMCs from multiple donors were transduced with lentivirus and CAR-T cells propagated as described above. For the donor shown here, transduced or non-transduced T cells were cocultured 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 at both effector-to-target ratios (Figures 6A-6C). Similar CTL activity was observed across multiple donors.

[0144] CAR-T cell phenotype correlates with T cell persistence. Following the CTL assay, CAR-T cell phenotype was analyzed using a FACS assay with a panel of antibodies detecting T cell differentiation markers. Briefly, transduced T cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, and anti-CD62L as described above. Analysis was performed using Attune NxT software. Gating was performed on CD3-, CD4-, and CD8-positive CAR-T cells and Tn, Tscm, Tcm, and Tem cells. Results showed comparable CD8 and CD4 CAR+ T cells (Figure 6D) and a high percentage of Tcm (Figure 6E).

[0145] (iv) The persistence of EPLV102 CAR-T was assessed by multiple rounds of target cell challenge. To further test the persistence of CAR-T, we performed multiple rounds of target cell challenge experiments. EPLV102 was transduced into Pan T cells and expanded. 10,000 transduced T cells were challenged with 10,000 K562 and MM1R GFP cells for 48 hours. Subsequently, the transduced T cells were rechallenged with 10,000 fresh target cells for another 48 hours in RPMI 1640 medium without additional cytokines. CTL activity was quantified by imaging every 2 hours using Cytation 5 and analyzed using Prism 8.0 software. EPLV102 demonstrated sustained CTL activity in two rounds of target challenge and rechallenge experiments (Figures 7A-7B).

[0146] Example 4. Construction of anti-TACI monospecific chimeric antigen receptor (CAR) This example demonstrates the construction and characterization of an exemplary anti-TACI monospecific chimeric antigen receptor (CAR).

[0147] Generation of anti-TACI CAR constructs and CAR-T cells expressing such Four selected anti-TACI scFv candidates, TC-01, TC02, TC-03, and TC04, were used to construct anti-TACI monospecific CARs. These CAR constructs contain, from N- to C-terminus, an anti-TACI scFv, a flag tag, a CD8a spacer and transmembrane domain, 41BB, and a CD3z intracellular signaling domain. The amino acid sequences of these anti-TACI CAR constructs are provided in Table 4 above.

[0148] The coding sequence of anti-TACI CAR construct was cloned into lentiviral vector according to standard molecular biology method.The obtained lentiviral vector was used to transduce PBMC for anti-TACI CAR expression according to conventional method.See above disclosure.

[0149] Characterization of CAR-T cells expressing anti-TACI CAR (i) CTL activity of anti-TACI CAR T cells Isolation, viral transduction, and T cell expansion of human PBMCs and Pan T cells were performed as described above. To screen for various CAR activity, a real-time image-based CTL activity assay was performed using GFP-engineered target cells. Pan T cells transduced with the CAR constructs EPLV200, EPLV254, EPLV255, and EPLV256 were incubated with 100,000 K562, H929 TACI-negative, and MM1R TACI-positive GFP cells at effector / target cell ratios of 5:1 and 1:1, as described, for 72 hours and imaged using Cytation 5.

[0150] The TACI monospecific CARs showed 50-90% surface expression (Figure 8A). Real-time CTL activity using different target cells at a 1:1 E / T ratio is shown in Figures 8B-8D. The endpoint CTL activity of different CARs was calculated as shown in Figure 8E. Multiple donors were screened with different TACI CARs and showed similar results.

[0151] (ii) Evaluation of CAR-T durability To further test the effect of different spacers on CAR-T persistence, multiple rounds of target cell rechallenge experiments were performed on CAR-T cells expressing EPLV300 (short spacer) and EPLV301 (medium spacer containing an IgG4 hinge and CD28 transmembrane). See the structural information provided in Table 4 above. Pan T cells were transduced with nucleic acids encoding the CAR constructs and expanded in two donors. 10,000 transduced T cells were challenged with 10,000 K562 and MM1R GFP cells for 48 hours, followed by rechallenge with 20,000 fresh target cells in RPMI medium without additional cytokines for an additional 68 hours. Both short-spacer and medium-spacer CAR surfaces were expressed at 70–90% (Figure 9A). CTL activity was quantified by imaging target GFP cells every 2 hours using Cytation 5 and analyzed using Prism 8.0 software. EPLV300 demonstrated persistently better CTL activity in two rounds of target challenge and rechallenge experiments in two donors (FIGS. 9B-9C).

[0152] Example 5: Construction and characterization of anti-BCMA and anti-TACI bispecific CARs This example demonstrates the construction and characterization of an exemplary anti-BCMA / anti-TACI bispecific (CAR).

[0153] Anti-BCMA / anti-TACI bispecific CAR construct Anti-BCMA / anti-TACI bispecific CAR constructs were constructed using the same anti-BCMA and anti-TACI scFvs used in the monospecific CAR constructs disclosed above, with the anti-BCMA and anti-TACI scFv fragments in tandem in the anti-BCMA to anti-TACI scFv orientation. The bispecific CAR constructs further comprise a flag tag, a CD8a spacer and transmembrane (EPLV217), or an IgG4 hinge, a CD28 transmembrane (EPLV302), a 4-1BB costimulatory signaling domain, and a CD3z intracellular signaling domain. The amino acid sequences of exemplary bispecific CAR constructs are provided in Table 4 above.

[0154] The coding sequence of the bispecific CAR construct is cloned into a lentiviral vector according to standard molecular biology methods.The resulting lentiviral vector is used to transduce PBMCs for bispecific CAR expression according to conventional methods.See above disclosure.

[0155] Characterization of CAR-T cells expressing anti-BCMA / anti-TACI bispecific CARs Isolation, viral transduction, and T cell expansion of human PBMCs and Pan T cells were performed as described above. A real-time image-based CTL activity assay was performed using GFP-engineered target cells. The CAR construct EPLV217 bearing the CD8a TM and EPLV302 bearing CD28 TM-transduced Pan T cells were transduced with 100,000 K562 BCMA T cells at a 1:1 effector-to-target cell ratio. - / TACI - Double negative, H929 BCMA + / TACI - , and MM1R BCMA + / TACI - Incubation with double-positive GFP cells for 72 hours and imaged in multiple donors by Cytation 5. Target cell re-challenge assays are also performed with EPLV302 to confirm persistence of the bispecific CAR.

[0156] The BCMA / TACI bispecific CARs showed 60-80% surface expression in multiple donors, as shown in Figures 10A-10B, respectively.

[0157] The CTL activity of EPLV217-expressing CAR-T cells against different target cells is shown in Figures 11A-11C. EPLV217-expressing CAR-T cells demonstrated specific and potent CTL activity (Figure 11D) and robust IFNγ release (Figure 11E) against different target cell lines.

[0158] The CTL activity of EPLV302-expressing CAR-T cells also demonstrated potent and sustained target-specific CTL activity against target cell lines in rechallenge assays (Figures 12A-12H). IFNγ levels correlated with CTL activity in different target cells (Figure 12I). Multiple donors were screened with both CAR formats and showed similar results.

[0159] Example 6: In vivo efficacy studies in a disseminated MM1R-luciferase model The in vivo anti-tumor 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 expanded in vitro for 4 days. 5x10 5 NCG mice were inoculated with MM1R-luciferase cells. On day 8, mice were administered PBS control and 1e6 of each CAR-T cell. Mice were imaged and weighed every 3-4 days.

[0160] As shown in Figure 13A, all treatment groups demonstrated tumor growth inhibition, and all CAR-Ts effectively eradicated tumor cells in all mice by day 38. There was no weight loss in any treatment group (Figure 13B). All mice survived throughout the study.

[0161] The BCMA monospecific CAR-T treatment group also received 5 × 10 5 The mice were re-challenged with 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).

[0162] Example 7: Characterization of engineered T cells expressing anti-BCMA / TACI bispecific CAR and arming polypeptides This example evaluates the bioactivity of engineered T cells expressing an anti-BCMA / TACI bispecific CAR construct and arming polypeptides comprising an anti-PDL1 fragment and a variant IL2 fragment.

[0163] An anti-BCMA / TACI bispecific CAR (EPLV302) was constructed containing the following components (from N- to C-terminus): signal peptide, anti-BCMA scFv, peptide linker, anti-TACI scFv, flag tag, CD8a spacer and transmembrane (or IgG4 hinge), CD28 transmembrane, 41BB costimulatory domain, and CD3z intracellular signaling domain. To coexpress the arming polypeptides, the coding sequence of the anti-BCMA / TACI bispecific CAR was linked (to its 3' end) to the 5' to 3' coding sequence, T2A peptide, second signal peptide, anti-PDL1 fragment, and mutant IL-2. See the structural information for EPLV302 (without arming polypeptide) and EPLV327 (with arming polypeptide) provided in Table 4 above. The coding sequences were cloned into lentiviral vectors according to standard molecular biology methods.

[0164] Human PBMCs and Pan T cells were isolated, transduced with the viral vectors described above, and expanded as described in the previous examples. Expression of the bispecific CAR in T cells transduced with the bispecific CAR coding sequence or in combination with the coding sequence for an arming polypeptide was examined. The engineered T cells showed approximately 25-50% surface expression of the bispecific CAR, with or without co-expressed arming polypeptide. Figure 14A.

[0165] Furthermore, we performed a real-time, image-based CTL activity assay using target cells engineered to express GFP. Briefly, pan T cells engineered with the CAR construct EPLV302 (disarmed) or the construct EPLV327 (armed) were incubated with 100,000 K562 BCMA / TACI double-negative or MM1R BCMA / TACI double-positive GFP cells at a 1:1 effector / target cell ratio for 114 hours and then imaged by Cytation 5 in multiple donors. As shown in Figure 14B, T cells expressing bispecific CARs, regardless of the presence or absence of arming polypeptides, exhibited high cell-killing activity against MM1R-GFP cells but not against K562-GFP cells.

[0166] To investigate the persistence of T cells expressing bispecific disarmed and armed CARs, we performed target cell rechallenge assays for 168 hours using pan T cells engineered with either EPLV302 or EPLV327 at an effector-to-target cell ratio of 0.5:1. Both disarmed and armed bispecific CAR T cells demonstrated sustained CTL activity up to 168 hours in the cancer cell rechallenge assay. Furthermore, IFNγ release was measured at 48, 120, and 168 hours. As shown in Figures 14C-14E, IFNγ levels measured at 48 and 120 hours correlated with CTL activity using different target cells, and IFNγ levels of T cells expressing armed CARs at 168 hours were higher than those of T cells expressing disarmed CARs (Figure 14E). Multiple donors were screened with both CAR formats and showed similar results.

[0167] Example 8: In vivo anti-tumor activity of engineered T cells expressing anti-BCMA / TACI bispecific CAR and arming polypeptides To assess the antitumor activity of engineered T cells expressing a bispecific armed CAR and arming polypeptide (EPLV327; see Table 4 above), 6- to 8-week-old female NCG mice (Charles River Laboratories, Wilmington, MA) were injected with 5 × 10 T cells suspended in serum-free medium. 5 Mice were inoculated intravenously with MM1R-luc multiple myeloma tumor cells. Seven days after tumor cell inoculation, mice were randomized into three treatment groups as follows: (1) Vehicle (n = 5 mice); (2) demilitarized CAR (1E6 CAR-T cell dose; n = 18 mice), and (3) EPC-004 (EPLV327) (1E6 CAR-T cell dose; n = 18 mice).

[0168] CAR-T cells were administered by intravenous injection 7 days after tumor cell inoculation. Tumor burden was determined weekly by bioluminescence imaging after intraperitoneal injection of luciferin substrate. Mice in the vehicle group exhibited high tumor burden, while the tumor burden of mice in groups (2) and (3) was significantly reduced. Figure 15A. Animals treated with disarmed CAR and EPC-004 showed significant tumor growth inhibition over time compared to the vehicle control group.

[0169] Furthermore, EPC-004 CAR-T cells exhibited significantly greater antitumor activity compared to disarmed CAR-T cells, as shown in Figure 15B. Animals were monitored over time for adverse clinical events related to tumor burden. By day 28 after treatment initiation, all vehicle control animals were sacrificed due to tumor burden, while animals in both the disarmed CAR and EPC-004 treatment groups survived beyond day 35.

[0170] Example 9: Development of CAR-NK cells BCMA monospecific (EPLV310) and TACI monospecific (EPLV300) CAR candidates (see Table 4 above) containing anti-BCMA or anti-TACI scFv, flag tag, IgG4 hinge, CD28 transmembrane, 41BB, and CD3z intracellular signaling domains were constructed according to routine methods. A bispecific CAR (EPLV302) candidate was also constructed in tandem format. Structural information for this bispecific CAR is also provided in Table 4 above. The coding sequences for the monospecific and bispecific CAR constructs were cloned into lentiviral vectors according to standard molecular biology methods.

[0171] Lentivirus carrying the coding sequences for monospecific or bispecific CARs was produced as previously described. PBMCs were isolated from the LRS chamber of fresh healthy donors using density gradient centrifugation with lymphoprep and a SepMate 50 kit from Stemcell Technology. NK cells were then isolated from the PBMCs using a Miltenyi NK cell isolation kit according to the manufacturer's protocol. NK cells were activated for 6–7 days at a concentration of 0.5–1 x 106 cells / mL using CTS X-pander medium (ThermoFisher) and a cytokine cocktail. After activation, 1 mg / mL protamine sulfate and 1 μM BX795 were added. Spinoculation was performed at 1000 g for 45 minutes at 32°C. NK cells and virus were incubated for 24 hours at 37°C. The next day, virus was removed, and transduced NK cells were replenished with fresh medium and cytokine cocktail. Medium was changed and fresh cytokines were added every 2–3 days.

[0172] CAR surface expression on transduced NK cells was assessed by surface staining using CD56-PE and AF647-conjugated anti-flag tag antibodies, respectively. Briefly, 100,000 lentiviral-transduced NK cells were incubated with 0.5 μl of CD56-PE and 0.1 μl of anti-flag-AF647 at 4°C for 1 hour in the dark with shaking. Cells were spun down at 1,300 rpm for 5 minutes, the supernatant removed, and washed with 200 μl of 1x PBS. The resulting sample was reconstituted in 200 μl of 1x PBS. The surface expression percentage was quantified by reading fluorescently stained cells on an Attune NxT flow cytometer. Figure 16A shows the level of NK cells expressing the NK cell marker CD56. Approximately 90-95% purity of NK cells was observed during expansion. CAR expression of different CAR constructs gated on the NK cell population ranged from 40-90%. Figure 16B.

[0173] Isolation, viral transduction, and T cell expansion of human PBMCs and NK cells were described above. Both BCMA, TACI monospecific, and BCMA / TACI bispecific CARs demonstrated robust surface expression in multiple donors, as shown above. A real-time image-based CTL activity assay was performed using GFP-engineered MM1R target cells. 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 a 5:1 effector-to-target cell ratio for 212 hours and imaged by Cytation 5 in multiple donors. A target cell rechallenge assay was also performed. All CAR-NK constructs demonstrated cancer cell killing activity. The bispecific CAR-NK showed more sustained activity than the monospecific CAR-NK at the third and fourth rechallenges (Figure 16C). The release of robust IFNγ levels correlated with sustained CTL activity (FIG. 16D).

[0174] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0175] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present invention to adapt it to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the following claims.

[0176] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0177] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0178] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0179] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0180] The phrase "and / or," as used herein in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," may refer in one embodiment to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0181] As used herein in the specification and claims, "or" should 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 construed as inclusive, i.e., including at least one, but also more than one, of a number or list of elements and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "any of," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0182] As used herein in the specification and claims herein, the phrase "at least one" in connection with 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 of the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements of the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to the specifically identified elements or not, may optionally be present. Thus, as a non-limiting example, "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") may refer in one embodiment to at least one A, optionally including more than one, with no B (and optionally including elements other than B); in another embodiment to at least one B, optionally including more than one, with no A (and optionally including elements other than A); in yet another embodiment to at least one A, optionally including more than one, and at least one B, optionally including more than one, with other elements; etc.

[0183] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

1. A bispecific chimeric antigen receptor (CAR) specific for B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), (a) a first antigen-binding moiety specific for TACI; (b) a second antigen-binding moiety specific for BCMA; and (c) a costimulatory signaling domain; and (d) a cytoplasmic signaling domain.

2. (a) the first antigen-binding portion specific for TACI is a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and V Ｈ contains the same heavy chain CDRs as in the reference antibody, and said V Ｌ comprises the same heavy chain CDRs as in the reference antibody, wherein the reference antibody is TC-01, TC-02, TC-03, or TC-04, and optionally the reference antibody is TC-01.

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

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

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

75.

6. (a) the second antigen-binding portion specific for BCMA comprises a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ), The V Ｈ but, (hi) heavy chain complementarity determining region (CDR) 1, １ YX ２ MH, wherein X １ is S or D, and X ２ is A or G; and (hii) a heavy chain CDR2 comprising: (hii-a)X ３ IX ４ YDGSX ５ KYYADSVKG (SEQ ID NO: 1), wherein X ３ is V or F, and X ４ is S or R, and X ５ is D or N, X ３ IX ４ YDGSX ５ KYYADSVKG (SEQ ID NO: 1), (hii-b) FIRSKAYGGTTEYAASVKG (SEQ ID NO: 27), or (hii-c) a heavy chain CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 43); and (hiii) a 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)VQX ６ PGAFDI (SEQ ID NO: 181), wherein X ６ is P or S, VQX ６ and a heavy chain CDR3 comprising PGAFDI (SEQ ID NO: 181); The V Ｌ but, (li) a light chain CDR1 comprising: (li-a) SGSGSNIGSNDVS (SEQ ID NO: 58), (li-b)QASQDIX ７ NYLN (SEQ ID NO: 2), wherein X ７ is N or S, QASQDIX ７ NYLN (SEQ ID NO: 2), or (li-c)RX ８ X ９ X １０ ISSYLX １１ (SEQ ID NO: 3), wherein X ８ is A or S, and X ９ is S or T, and X １０ is G or S, and X11 is G or N, RX ８ X ９ X １０ ISSYLX １１ (SEQ ID NO: 3); and (lii) a 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, DASNX １２ ET (SEQ ID NO: 4), 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, AX １３ SX １４ a light chain CDR2 comprising LQS (SEQ ID NO: 5); (liii) a light chain CDR3 comprising: (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, QQYDX １５ LPX １６ T (SEQ ID NO: 6), (liii-c) QHSYSTPHT (SEQ ID NO: 32), or (liii-d) a light chain CDR3 comprising QQLYS (SEQ ID NO: 48).

7. the V of the second antigen-binding portion specific for BCMA Ｈ the V of the second antigen-binding portion comprises the same heavy chain CDRs as in the reference antibody and / or is specific for BCMA Ｌ comprises the same heavy chain CDRs as in the reference antibody, wherein the reference antibody is BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, BC-08, or BC-09, and optionally the reference antibody is BC-06.

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

9. The bispecific CAR of 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. 10. The bispecific CAR of 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, and optionally the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO:

50.

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

12. The bispecific CAR of claim 11, wherein said cytoplasmic signaling domain is derived from CD3ζ.

13. the bispecific CAR (a) a fusion polypeptide comprising, from N-terminus to C-terminus, (i) the first antigen-binding portion, (ii) the second antigen-binding portion, (iii) the costimulatory signaling domain, and (iv) the cytoplasmic signaling domain; or (b) a fusion polypeptide comprising, from N-terminus to C-terminus, (i) the second antigen-binding portion, (ii) the first antigen-binding portion, (iii) the costimulatory signaling domain, and (iv) the cytoplasmic signaling domain.

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

15. The bispecific CAR of claim 13 or 14, further comprising a peptide linker connecting the first antigen-binding moiety and the second antigen-binding moiety.

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

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

18. The bispecific CAR of claim 1, comprising the amino acid sequence of SEQ ID NO:

182.

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

20. A nucleic acid or a set of nucleic acids collectively encoding the bispecific CAR of any one of claims 1 to 19.

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

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

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

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

178.

25. The nucleic acid or 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. 25. A genetically engineered immune cell expressing a bispecific CAR according to any one of claims 1 to 19, and optionally further expressing an arming polypeptide according to any one of claims 22 to 24.

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

28. A genetically engineered immune cell according to claim 26 or 27, comprising a nucleic acid according to any one of claims 20 to 25.

29. 29. The genetically engineered immune cell of any one of claims 26 to 28, which is a T cell, a 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 costimulatory signaling domain, and a cytoplasmic signaling domain, wherein the extracellular antigen-binding domain specific for TACI is the anti-TACI chimeric antigen receptor (CAR) described in any one of claims 2 to 5.

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

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

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

34. The anti-TACI CAR of claim 30, comprising the amino acid sequence of any one of SEQ ID NOs: 143 to 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 costimulatory signaling domain, and a cytoplasmic signaling domain, wherein the extracellular antigen-binding domain specific for BCMA is the anti-BCMA chimeric antigen receptor (CAR) described in any one of claims 6 to 10.

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

37. The anti-BCMA CAR of claim 35 or 36, further comprising a hinge domain and / or a transmembrane domain between the extracellular antigen-binding domain specific for BCMA and the costimulatory domain.

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

39. The anti-BCMA CAR of claim 35, comprising the amino acid sequence of any one of SEQ ID NOs: 119 to 142, and optionally, 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 the anti-TACI CAR of any one of claims 30 to 34 or the anti-BCMA CAR of any one of claims 35 to 39.

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

42. 41. The nucleic acid of claim 40, wherein the arming polypeptide is as defined in claim 23 or 24.

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

44. 43. 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 an arming polypeptide according to claim 41 or 42.

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

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

47. 47. The method of claim 46, wherein the undesired cells are cancer cells.

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

49. The human cancer patient is ＋ and / or TACI ＋ 49. The method of claim 48, comprising cancer cells.

50. 50. The method of 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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