Antibodies targeting TACI and chimeric antigen receptors

Abstract

The present invention provides antibodies, antibody-drug conjugates, bispecific T cell engagers (BiTEs), and chimeric antigen receptors (CARs) that target transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Such antibodies, antibody-drug conjugates, BiTEs, and CARs can be used, for example, in methods of treating cancer (e.g., multiple myeloma), autoimmune disorders (e.g., characterized by high titers of antibodies that contribute to the disorder), or plasma cell dyscrasias (e.g., plasma cell proliferative disorders) in a subject in need thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 856,998, filed on June 4, 2019, entitled "ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS THAT TARGET TACI", U.S. Provisional Application No. 62 / 907,930, filed on September 30, 2019, entitled "ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS THAT TARGET TACI", and U.S. Provisional Application No. 63 / 012,735, filed on April 20, 2020, entitled "ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS THAT TARGET TACI", under 35 U.S.C. § 119(e), and the entire contents of each of these applications are incorporated herein by reference. Background of the Invention

[0003] Chimeric antigen receptors (CARs) direct the response of cytotoxic T cells against target cells expressing a selected target antigen, most commonly a tumor antigen or tumor-associated antigen. A CAR is a modified form of a T cell receptor in which the antigen-binding domain is replaced with an antigen-binding domain of an antibody that specifically binds a derivative target antigen. Engagement of the target antigen on the surface of the target cell by the CAR expressed, for example, on a T cell ("CAR-T cell" or "CAR-T") facilitates killing of the target cell. Summary of the Invention

[0004] Antibodies, antibody-drug conjugates, bispecific T cell engagers (BiTEs), and chimeric antigen receptors (CARs) that target transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) are described herein and can be used to treat subjects having a disease or disorder such as cancer, plasma cell disease or disorder, or autoimmune disease or disorder.

[0005] Some aspects of the present disclosure provide antibodies that specifically bind to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), wherein the antibody comprises:

[0006] (i) heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 1; and / or

[0007] (ii) The light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of the light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:2.

[0008] In some embodiments, the antibody comprises:

[0009] (i) CDR-H1 shown in SEQ ID NO:26, CDR-H2 shown in SEQ ID NO:27, and CDR-H3 shown in SEQ ID NO:28; CDR-L1 shown in SEQ ID NO:29, CDR-L2 shown in SEQ ID NO:30, and CDR-L3 shown in SEQ ID NO:31;

[0010] (ii) CDR-H1 shown in SEQ ID NO:32, CDR-H2 shown in SEQ ID NO:33, and CDR-H3 shown in SEQ ID NO:34; CDR-L1 shown in SEQ ID NO:35, CDR-L2 shown in SEQ ID NO:36, and CDR-L3 shown in SEQ ID NO:31; or

[0011] (iii) CDR-H1 shown in SEQ ID NO:37, CDR-H2 shown in SEQ ID NO:38, and CDR-H3 shown in SEQ ID NO:39; CDR-L1 shown in SEQ ID NO:40, CDR-L2 shown in SEQ ID NO:30, and CDR-L3 shown in SEQ ID NO:41.

[0012] In some embodiments, the antibody binds to TACI with a K of about 2 nM or lower, D optionally wherein the antibody binds to TACI with a K between about 500 pM and about 1 nM, D further optionally wherein the antibody binds to TACI with a K between about 700 pM and about 900 pM, D and binds to TACI. In some embodiments, the antibody binds to TACI with a K of about 861 pM. D

[0013] ​In some embodiments, the antibody comprises a heavy chain variable domain (VH) that contains an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1; and / or a light chain variable domain (VL) that contains an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, and the VL comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID NO:1 and a VL containing the amino acid sequence of SEQ ID NO:2.

[0014] In some embodiments, the antibody is a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment that specifically binds to TACI. In some embodiments, the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv and (Fab')2 fragments. In some embodiments, the antibody is an IgG antibody, optionally an IgG1 antibody. In some embodiments, the antibody is an scFv. In some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the scFv is fused to Fc.

[0015] Another aspect of the invention described herein relates to a composition comprising any one of the antibodies described herein.

[0016] Another aspect of the invention described herein relates to a polynucleotide encoding any one of the antibodies described herein.

[0017] Another aspect of the invention described herein relates to a vector comprising any one of the polynucleotides described herein.

[0018] Another aspect of the invention described herein relates to a host cell comprising any one of the vectors described herein.

[0019] In one embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0020] In another embodiment, the host cell is a prokaryotic cell. In one embodiment, the prokaryotic cell is Escherichia coli (E. coli).

[0021] Another aspect of the invention described herein relates to a method for generating an antibody that specifically binds to TACI, the method comprising culturing any of the host cells described herein in a medium. In one embodiment, the method further comprises recovering the antibody from the host cell or the medium.

[0022] Another aspect of the invention described herein relates to an antibody-drug conjugate comprising any of the antibodies described herein.

[0023] Another aspect of the invention described herein relates to a chimeric antigen receptor (CAR) polypeptide comprising an extracellular target binding domain, wherein the extracellular target binding domain comprises a TACI binding domain.

[0024] In one embodiment, the CAR polypeptide comprises a transmembrane domain and an intracellular signaling domain.

[0025] In one embodiment, the CAR polypeptide further comprises one or more co-stimulatory domains.

[0026] In one embodiment, the TACI binding domain does not comprise APRIL, BAFF, CAMLG or a portion thereof.

[0027] In one embodiment, the TACI binding domain binds to TACI with a K D of about 2 nM or lower. In one embodiment, the TACI binding domain binds to TACI with a K D between about 500 pM and about 1 nM. In one embodiment, the TACI binding domain binds to TACI with a K D between about 700 pM and about 900 pM. In one embodiment, the TACI binding domain binds to TACI with a K D of about 861 pM.

[0028] In one embodiment, the TACI binding domain comprises an antibody or an antigen-binding fragment thereof.

[0029] Another aspect of the invention described herein relates to a CAR polypeptide comprising an extracellular target binding domain, the extracellular target binding domain comprising any of the antibodies described herein or an antigen-binding fragment thereof.

[0030] In one embodiment of any aspect, the TACI binding domain comprises an anti-TACI single-chain variable fragment (scFv).

[0031] In one embodiment in any aspect, the anti-TACI scFv comprises a heavy chain variable domain (VH) containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0032] In one embodiment in any aspect, the VH comprises the amino acid sequence of SEQ ID NO: 1.

[0033] In one embodiment in any aspect, the anti-TACI scFv comprises a light chain variable domain (VL) containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0034] In one embodiment in any aspect, the VL comprises the amino acid sequence of SEQ ID NO: 2.

[0035] In one embodiment in any aspect, the anti-TACI scFv comprises a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 2.

[0036] In one embodiment in any aspect, the VH is located at the N-terminus of the VL.

[0037] In another embodiment in any aspect, the VL is located at the N-terminus of the VH.

[0038] In one embodiment in any aspect, the VH and the VL are connected via a linker sequence.

[0039] In one embodiment in any aspect, the linker sequence comprises the amino acid sequence of SEQ ID NO: 3, 14, 15, 16 or 17.

[0040] In one embodiment in any aspect, the linker sequence comprises the amino acid sequence of SEQ ID NO: 3.

[0041] In one embodiment in any aspect, the TACI binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4 or 5.

[0042] In one embodiment in any aspect, the TACI binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0043] In one embodiment in any aspect, the transmembrane domain includes a hinge / transmembrane domain.

[0044] In one embodiment in any aspect, the hinge / transmembrane domain comprises the hinge / transmembrane domain of an immunoglobulin-like protein, CD28, CD8 or 4-1BB.

[0045] In one embodiment in any aspect, the hinge / transmembrane domain is the hinge / transmembrane domain of CD8, optionally wherein the hinge / transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO:7.

[0046] In one embodiment in any aspect, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, CD3ε or CD3θ.

[0047] In one embodiment in any aspect, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, optionally wherein the intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO:9.

[0048] In one embodiment in any aspect, the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, CD28, CD27, ICOS or OX40.

[0049] In one embodiment in any aspect, the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, optionally wherein the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO:8.

[0050] In one embodiment in any aspect, the CAR polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:10, 11, 12 or 13.

[0051] In one embodiment in any aspect, the extracellular target-binding domain further comprises a target-binding domain that binds to a second target other than TACI.

[0052] In one embodiment in any aspect, the second target is B cell maturation antigen (BCMA).

[0053] In one embodiment in any aspect, the target-binding domain comprises the ligand of the second target.

[0054] In one embodiment in any aspect, the target-binding domain comprises an antibody or an antigen-binding fragment thereof.

[0055] In one embodiment in any aspect, the antibody or an antigen-binding fragment thereof comprises a scFv.

[0056] In one embodiment in any aspect, the scFv is an anti-BCMA scFv.

[0057] In one embodiment in any aspect, the anti-BCMA scFv is located at the N-terminus of the anti-TACI scFv.

[0058] In one embodiment in any aspect, the anti-TACI scFv is located at the N-terminus of the anti-BCMA scFv.

[0059] In one embodiment in any aspect, the CAR polypeptide comprises an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 18-25.

[0060] Another aspect of the invention described herein relates to a CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 10.

[0061] Another aspect of the invention described herein relates to a CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0062] Another aspect of the invention described herein relates to a CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

[0063] Another aspect of the invention described herein relates to a CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 13.

[0064] Another aspect of the invention described herein relates to a CAR polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 18-25.

[0065] Another aspect of the invention described herein relates to a polynucleotide encoding any one of the CAR polypeptides described herein.

[0066] In one embodiment, the polynucleotide further comprises a suicide gene.

[0067] In one embodiment, the polynucleotide further comprises a sequence encoding a signal sequence.

[0068] Another aspect of the invention described herein relates to mammalian cells comprising any one of the CAR polypeptides described herein and / or any one of the polynucleotides described herein. In one embodiment, the mammalian cell is an induced pluripotent stem cell (iPSC). In one embodiment, the mammalian cell is an immune cell. In one embodiment, the immune cell is a T cell or a natural killer (NK) cell. In one embodiment, the mammalian cell is a human cell.

[0069] Another aspect of the invention described herein relates to a bispecific antibody that binds to TACI and CD3, wherein the bispecific antibody comprises a TACI-binding domain and a CD3-binding domain.

[0070] In one embodiment of any aspect, the TACI-binding domain binds to TACI with a K D of about 2 nM or lower. In one embodiment, the TACI-binding domain binds to TACI with a K D between about 500 pM and about 1 nM. In one embodiment, the TACI-binding domain binds to TACI with a K D between about 700 pM and about 900 pM. In one embodiment, the TACI-binding domain binds to TACI with a K D of about 861 pM.

[0071] In one embodiment of any aspect, the TACI-binding domain comprises a VH having an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and / or a VL having an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0072] Another aspect of the invention described herein relates to a bispecific antibody that specifically binds to TACI and CD3, wherein the bispecific antibody comprises a TACI-binding domain and a CD3-binding domain, and wherein the TACI-binding domain comprises a VH having the amino acid sequence of VH SEQ ID NO:1 and a VL having the amino acid sequence of SEQ ID NO:2.

[0073] In one embodiment of any aspect, the TACI-binding domain is located at the N-terminus of the CD3-binding domain.

[0074] In one embodiment of any aspect, the CD3-binding domain is located at the N-terminus of the TACI-binding domain.

[0075] In one embodiment of any aspect, the TACI-binding domain and the CD3-binding domain are linked by a linker sequence.

[0076] In one embodiment of any aspect, the linker sequence comprises the amino acid sequence of SEQ ID NO:3, 14, 15, 16 or 17.

[0077] In one embodiment of any aspect, the bispecific antibody is a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.

[0078] In one embodiment in any aspect, the bispecific antibody is a monoclonal antibody.

[0079] In one embodiment in any aspect, the bispecific antibody is a full-length antibody.

[0080] In another embodiment in any aspect, the bispecific antibody is an antibody fragment that specifically binds to TACI and CD3. In one embodiment, the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0081] In one embodiment in any aspect, the bispecific antibody is an IgG antibody. In one embodiment, the bispecific antibody is an IgG1 antibody.

[0082] Another aspect of the invention described herein relates to a composition comprising any of the bispecific antibodies described herein.

[0083] Another aspect of the invention described herein relates to a polynucleotide encoding any of the bispecific antibodies described herein.

[0084] Another aspect of the invention described herein relates to a vector comprising any of the polynucleotides encoding the bispecific antibodies described herein.

[0085] Another aspect of the invention described herein relates to a host cell comprising any of the said vectors (e.g., a vector comprising any of the polynucleotides encoding the bispecific antibodies described herein). In one embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell. In another embodiment, the host cell is a prokaryotic cell. In one embodiment, the prokaryotic cell is Escherichia coli (E. coli).

[0086] Another aspect of the invention described herein relates to a method for producing a bispecific antibody that specifically binds to TACI and CD3, the method comprising culturing any of the host cells described herein (e.g., a host cell comprising any of the said vectors (e.g., a vector comprising any of the polynucleotides encoding the bispecific antibodies described herein)) in a culture medium.

[0087] In one embodiment, the method further comprises recovering the bispecific antibody from the host cell or the culture medium.

[0088] Another aspect of the invention described herein relates to methods of treating a disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject one or more of the following: (i) any CAR polypeptide described herein, any polynucleotide described herein, and / or any mammalian cell described herein; (ii) any antibody described herein; (iii) any antibody-drug conjugate described herein; and (iv) any bispecific antibody described herein.

[0089] In one embodiment, the disease or disorder is cancer, an autoimmune disorder, or a plasma cell disease or disorder.

[0090] In one embodiment, the disease or disorder is cancer.

[0091] In one embodiment, the cancer comprises cells that express TACI.

[0092] In one embodiment, the cancer is multiple myeloma.

[0093] In one embodiment, the subject is resistant to anti-BCMA therapy.

[0094] In one embodiment, the disease or disorder is an autoimmune disease or disorder.

[0095] In one embodiment, the autoimmune disease or disorder is characterized by a high titer of antibodies that contribute to the autoimmune disorder.

[0096] In one embodiment, the autoimmune disease or disorder is transplant rejection, graft-versus-host disease, or hemophilia with factor inhibitors.

[0097] In one embodiment, the disease or disorder is a plasma cell disease or disorder.

[0098] In one embodiment, the plasma cell disease or disorder is plasmacytosis, plasmacytoma, plasma cell leukemia, multiple myeloma, Waldenström macroglobulinemia, solitary plasmacytoma, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

[0099] Definitions

[0100] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to assist in describing particular embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs, unless otherwise defined. If there is a significant difference between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0101] Definitions of commonly used terms in immunology and molecular biology can be found in the following references: Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999 - 2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN - 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the entire contents of each of the foregoing documents are hereby incorporated by reference in their entirety.

[0102] In some embodiments, "activation" can refer to a T cell state that has been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation can refer to induced cytokine production. In other embodiments, activation can refer to detectable effector function. At a minimum, as used herein, an "activated T cell" is a proliferative T cell.

[0103] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0104] As used herein, "administering" refers to the giving of a dose of a compound (e.g., an anti-TACI antibody, an antibody-drug conjugate, a BiTE, and / or an anti-TACI CAR) or a composition (e.g., a pharmaceutical composition, such as a pharmaceutical composition comprising an anti-TACI antibody, an antibody-drug conjugate, a BiTE, and / or an anti-TACI CAR) to a subject. The compositions used in the methods described herein can be administered or formulated for administration by, for example, intramuscular, intravenous, intradermal, transdermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrathoracic, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, peritoneal, subcutaneous, subconjunctival, intravesical, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, local, by inhalation, by injection, by infusion, by continuous infusion, by direct local perfusion to bathe the target cells, by catheter, by lavage, in the form of a cream or in the form of a lipid composition. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). Preferably, the compound (e.g., the anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR of the present invention) or composition (e.g., a pharmaceutical composition comprising an anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR) is administered orally or formulated for oral administration.

[0105] The terms "anti-TACI antibody", "antibody that binds to TACI", and "antibody that specifically binds to TACI" refer to an antibody that is capable of binding to TACI with sufficient affinity such that the antibody can be used as a prophylactic, diagnostic, and / or therapeutic agent targeting TACI. In one embodiment, the anti-TACI antibody binds to an irrelevant non-TACI protein to an extent less than about 10% of the binding of the antibody to TACI, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to TACI has a dissociation constant (K -8 ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., from 10 -13 M to 10 -9 M, e.g., from 10 -13 M to 10 D M).

[0106] As used herein in the broadest sense, the term "antibody" encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody" can refer to, for example, a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) that are interconnected by disulfide bonds or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can consist of three domains, namely CH1, CH2, and / or CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of high variability called "complementary determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL can be composed of, for example, three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0107] An "antibody fragment" refers to a molecule that is different from a full-length antibody and that comprises a portion of the full-length antibody that specifically binds to an antigen (e.g., TACI) to which the full-length antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. These antibody fragments are obtained using conventional techniques, and the fragments are screened for utility in the same manner as full-length antibodies. Antibody fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of full-length immunoglobulins.

[0108] A "bispecific T cell engager", "BiTE antibody construct", or "BiTE" refers to a polypeptide that each includes single-chain variable fragments (scFvs) that are tandemly linked. Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). One scFv of a BiTE binds to a T cell receptor (TCR) (e.g., binds to the CD3ε subunit), and the other scFv binds to a target antigen (e.g., a tumor-associated antigen).

[0109] As used herein, the term "chimeric" refers to a fusion product of portions of at least two or more different polynucleotide molecules. In one embodiment, the term "chimeric" refers to a gene expression element that is generated by manipulating known elements or other polynucleotide molecules.

[0110] As used herein, the term "chimeric antigen receptor" or "CAR" or "multiple CARs" refers to an engineered T cell receptor that transplants ligand or antigen specificity onto a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). A CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immunoreceptor. As used herein, "CAR T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, a CAR has the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, for example, by utilizing the antigen-binding properties of a monoclonal antibody. Non-MHC-restricted antigen recognition confers on a CAR-expressing T cell the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape.

[0111] As used herein, the term "costimulatory ligand" includes molecules on an antigen-presenting cell (APC) that specifically bind to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands can include but are not limited to 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll-like receptor, and a ligand that specifically binds to B7-H3. Costimulatory ligands can also include but are not limited to an antibody that specifically binds to a costimulatory molecule present on a T cell, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Other costimulatory ligands known in the art can also be used according to standard methods.

[0112] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as but not limited to proliferation) of the T cell. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0113] The terms "reduce", "reduced", "reduction" or "inhibit" mean a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. Typically, such a reduction is statistically significant. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to the reference level. "Complete inhibition" is 100% inhibition compared to the reference level. Where appropriate, the reduction can preferably be reduced to an acceptable level within the normal range for an individual without a given disorder.

[0114] "Disease" is a state of health of an animal (e.g., a human) in which the animal is unable to maintain homeostasis, and in which the health of the animal will continue to deteriorate if the disease is not improved. In contrast, a "disorder" of an animal is a state of health in which the animal is able to maintain homeostasis, but the health state of the animal is less favorable compared to the situation in the absence of the disorder. If not treated in a timely manner, a disorder does not necessarily lead to a further decline in the health state of the animal.

[0115] The term "EC50" as used herein refers to the concentration of an antibody or an antigen-binding portion thereof that induces a response that is 50% of the maximum response (i.e., the midpoint between the maximum response and the baseline) in an in vivo or in vitro assay.

[0116] As used herein, the terms "effective amount", "effective dose", and "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" or "therapeutically effective amount" is defined as an amount sufficient to prevent, cure, or at least partially arrest the disease (e.g., diarrhea) and its complications in a patient who has the disease or is at risk of developing the disease. The amount effective for such use will depend on the severity of the disorder being treated and the general condition of the patient's own immune system.

[0117] In some embodiments, the term "engineered" and its grammatical equivalents as used herein can refer to one or more man-made designed alterations of nucleic acids (e.g., nucleic acids within the genome of an organism). In another embodiment, engineering can refer to the alteration, addition, and / or deletion of genes. An "engineered cell" can refer to a cell having added, deleted, and / or altered genes. The terms "cell" or "engineered cell" and their grammatical equivalents as used herein can refer to cells of human or non-human animal origin.

[0118] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. Epitopes can be formed by both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by contiguous amino acids typically remain upon exposure to a denaturing solvent, while epitopes formed by tertiary folding typically are lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996). Epitopes can also be defined by point mutations in a target protein (e.g., TACI) that affect the binding of the antibody (e.g., monoclonal antibody).

[0119] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain that contains an Fc region as defined herein.

[0120] As used herein, the term "host cell" is intended to mean a cell into which an expression vector has been introduced. It is understood that such term is intended to refer not only to a particular subject cell but also to the progeny of such cell. Because certain modifications may occur in progeny as a result of mutation or environmental influences, such progeny may in fact be different from the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0121] The term "human antibody" includes antibodies having variable and constant regions (if present) with human germline immunoglobulin sequences. A human antibody can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by somatic mutation in vivo) (see Lonberg, N. et al. (1994) Nature 368(6474):856-859); Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93 and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (i.e., humanized antibodies).

[0122] An "isolated antibody" is an antibody that has been identified and separated and / or recovered from components of its natural environment and / or is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to TACI is substantially free of antibodies that specifically bind to antigens other than TACI). Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified to: (1) greater than 95% by weight of antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues as measured by a spinning cup sequenator, or (3) homogeneity by SDS-PAGE using Coomassie TM Blue or preferably silver staining. Isolated antibodies include recombinant intracellular antibodies in situ because at least one component of the natural environment of the antibody will be absent. Similarly, isolated antibodies include antibodies surrounding recombinant cells in culture medium. However, isolated antibodies are generally prepared by at least one purification step.

[0123] As used herein, the term "K D " is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, when using recombinant TACI as an analyte and an antibody as a ligand in a BIACORE 3000 instrument and measuring by surface plasmon resonance (SPR) technology, the antibody binds to TACI with a dissociation equilibrium constant (K -6 less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, 10 -10 M or even lower. D )

[0124] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" or "HuMab" refers to an antibody that exhibits a single binding specificity and has variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is produced by a hybridoma that comprises B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome that contains a human heavy chain transgene and a light chain transgene and that are fused to immortalized cells.

[0125] As used herein, the term "operably linked" refers to a first polynucleotide molecule (such as a promoter) that is linked to a second transcribable polynucleotide molecule (such as a gene of interest), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule and may or may not be adjacent. For example, if a promoter regulates or mediates the transcription of a gene of interest in a cell, the promoter is operably linked to the gene of interest.

[0126] In some embodiments, the polypeptides (or nucleic acids encoding such polypeptides) described herein can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide as determined by assays known in the art or described hereinafter. Functional fragments can contain conservative substitutions of the sequences disclosed herein.

[0127] In some embodiments, the polypeptides described herein can be variants of the polypeptides or molecules described herein. In some embodiments, the variants are conservatively modified variants. For example, conservatively substituted variants can be obtained by mutation of the native nucleotide sequence. As used herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide, but whose amino acid sequence differs from that of the native or reference polypeptide by one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide encompasses a sequence that contains one or more additions, deletions, or substitutions of nucleotides when compared to the native or reference DNA sequence, provided that the sequence encodes a variant protein or a fragment thereof that retains the activity of the non-variant polypeptide. A variety of PCR-based site-directed mutagenesis methods are known in the art and can be applied by one of ordinary skill in the art. For example, the variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence.

[0128] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences for maximum percent sequence identity and introducing gaps (if necessary) and not considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0129] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used interchangeably herein with "nucleic acid" to denote a polymer of nucleotides. Generally, a polynucleotide is composed of nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) that are naturally found in DNA or RNA, linked by phosphodiester bonds. However, the term encompasses molecules containing nucleotides or nucleotide analogs that contain chemically or biologically modified bases, modified backbones, etc., whether or not present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it should be understood that both DNA and RNA are provided, and in each case both single-stranded and double-stranded forms are provided (as well as the complement of each single-stranded molecule). The "polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or the sequence information that biochemically characterizes a particular nucleic acid (i.e., a string of letters used as abbreviations for bases). Unless otherwise specified, the polynucleotide sequences presented herein are presented in the 5' to 3' direction.

[0130] The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically having a length between about 2 and 60 amino acids. The polypeptides used herein generally contain amino acids such as the 20 most common L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, for example, by the addition of chemical entities (such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functionalization, etc.). A polypeptide having a non-polypeptide moiety covalently or non-covalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA techniques, or synthesized by chemical means (such as conventional solid-phase peptide synthesis, etc.). The term "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and / or the sequence information that biochemically characterizes the polypeptide (i.e., a string of letters or three-letter codes used as abbreviations for amino acid names). Unless otherwise indicated, the polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction.

[0131] In some embodiments, the nucleic acid encoding a polypeptide as described herein (e.g., a CAR polypeptide) is contained within a vector, such as an expression vector. In some aspects described herein, the nucleic acid sequence encoding a given polypeptide as described herein is operably linked to the vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, the vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replicating when combined with appropriate control elements and can transfer a gene sequence to a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like.

[0132] As used herein, the term "expression vector" refers to a vector that directs the expression of, for example, RNA or a polypeptide from a sequence linked to transcriptional regulatory sequences on the vector. The expressed sequence will typically but not necessarily be heterologous to the cell. The expression vector can contain additional elements. For example, the expression vector can have two replication systems, such that it can be maintained in two organisms, e.g., for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, where appropriate, the secretion of the protein, including, where applicable, but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, as well as polypeptides obtained by translation of the mRNA transcribed from the gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene can or can not include regions before and after the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0133] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein, in place of a non-essential viral gene. The vector and / or particle can be used for the purpose of transferring nucleic acid into cells in vitro or in vivo. Many forms of viral vectors are known in the art.

[0134] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence or "transgene" capable of being expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in extrachromosomal DNA at a high copy number in a subject, thereby eliminating the potential effects of chromosomal integration.

[0135] The term "pharmaceutical composition" refers to a preparation in a form that renders the bioactivity of the active ingredient(s) contained therein effective and that contains no other components that are unacceptably toxic to the subject to which the formulation is administered.

[0136] "Pharmaceutically acceptable carrier" refers to the components in a pharmaceutical formulation other than the active ingredient(s) that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0137] As used herein, the terms "specifically bind" and "specifically binds" refer to a physical interaction between two molecules, compounds, cells, and / or particles, wherein the specificity and affinity of the first entity for binding to the second target entity are greater than its specificity and affinity for binding to a third entity that serves as a non-target. In some embodiments, specifically binding can refer to the affinity of the first entity for the second target entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more than its affinity for a third non-target entity under the same conditions. A reagent that is specific for a given target is a reagent that exhibits specific binding to the target under the assay conditions used. Non-limiting examples include antibodies or ligands that recognize and bind to a cognate binding partner (e.g., a stimulatory and / or co-stimulatory molecule present on a T cell) protein. For example, when measured by surface plasmon resonance (SPR) technology, such as using a recombinant TACI as the analyte and the antibody as the ligand in a BIACORE 3000 instrument, a specific antibody can bind to its target with an affinity of approximately less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M, or 10 -10 M, or even lower (K D ). In some embodiments, the affinity of the antibody for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

[0138] As used herein, "signal peptide" or "signal sequence" refers to a peptide at the N-terminus of a newly synthesized protein that serves to direct the nascent protein into the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 signal peptide.

[0139] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (APC) (e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule" or "costimulatory molecule") on a T cell, thereby mediating a primary response of the T cell, including but not limited to proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and particularly encompass MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0140] The term "stimulatory molecule" as used herein means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0141] "Subject" or "individual" is a mammal. Mammals include but are not limited to domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, deer, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0142] A subject can be a subject who has previously been diagnosed or identified as having or suffering from a condition (e.g., the diseases and disorders described herein) or one or more complications associated with such a condition, and optionally, has undergone treatment for the condition or one or more complications associated with the condition. Alternatively, a subject can also be a subject who has not previously been diagnosed as having such a condition or related complications. For example, a subject can be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or a subject who does not exhibit risk factors.

[0143] A "subject in need of treatment" for a particular condition can be a subject who has the condition, has been diagnosed as having the condition, or is at risk of developing the condition.

[0144] The term "transmembrane activator and calcium modulator and cyclophilin ligand interactor" or "TACI" is also known as tumor necrosis factor receptor (TNFR) superfamily member 13B (TNFRSF13B) and is a lymphocyte-specific member of the TNFR superfamily. TACI interacts with two other members of the tumor necrosis factor (TNF) family, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), and controls T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0145] As used herein, the terms "treat", "treatment", "treating" or "ameliorate" refer to a therapeutic treatment, where the goal is to reverse, alleviate, improve, inhibit, slow down or prevent the progression or severity of a condition associated with a disease or disorder, e.g., glioblastoma, glioma, acute lymphoblastic leukemia or other cancers, diseases or disorders. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only an improvement in symptoms or markers, but also a halt or at least a slowing of the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, remission (whether partial or total) and / or decreased mortality, whether detectable or not. The "treatment" of a disease also includes providing relief of the symptoms or side effects of the disease (including palliative treatment).

[0146] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably and refer to antigens that are differentially expressed by cancer cells and can thus be exploited to target cancer cells. Cancer antigens are antigens that may potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells but are not necessarily expressed. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed transiently, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., the activated ras oncogene), tumor suppressor genes (e.g., mutant p53), and fusion proteins generated by internal deletions or chromosomal translocations. Other cancer antigens can also be encoded by viral genes, such as genes carried on RNA and DNA tumor viruses.

[0147] Other terms are defined within the description of the various aspects and embodiments of the present technology, as described below.

[0148] The constructs and methods described herein offer several advantages. For example, one advantage provided by the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and anti-TACI CARs described herein is that targeting TACI allows for mitigation of the BCMA antigen escape problem. Although anti-BCMA therapies have yielded promising results in multiple myeloma to date, it has also been shown that targeting BCMA results in relapse and / or BCMA antigen loss following BCMA CAR T cell therapy. Although the frequency of such events has not been determined, an overall objective response rate of 85% has been observed in recent studies, but the median progression-free survival was 11.8 months (Raje et al., N Engl J Med. 2019;380(18):1726-1737), indicating that monospecific targeting of BCMA with CAR T cells may not be a curative therapy for most patients. Reports of disease resistance due to BCMA antigen loss under selective pressure from anti-BCMA CAR T cell therapy have also emerged. Thus, targeting TACI provides a unique advantage in treating diseases affected by BCMA antigen escape.

[0149] The foregoing summary is intended to illustrate some embodiments, advantages, features, and uses of the technology disclosed herein in a non-limiting manner. Other embodiments, advantages, features, and uses of the technology disclosed herein will become apparent from the detailed description, drawings, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] The drawings are not intended to be drawn to scale. This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with one or more color drawings will be provided by the Office upon request and payment of the necessary fee. In the drawings:

[0151] Figure 1 is a diagram showing a general method for generating anti-transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) antibodies and their use in anti-TACI chimeric antigen receptor (CAR) T cells described herein.

[0152] Figure 2 is a diagram showing the results of a surface plasmon resonance assay using an anti-TACI antibody.

[0153] Figure 3 is a diagram showing the design of anti-TACI CARs referred to herein as "anti-TACI (H / L)" and "anti-TACI (L / H)".

[0154] Figure 4 is a series of diagrams showing BCMA and TACI expression on the MM1S, MM1S BCMA knockout, K562, K562-TACI, and K562-BCMA cell lines.

[0155] Figure 5 is a series of graphs showing TACI expression in T cells.

[0156] Figure 6 is a graph showing the transfection efficiency of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells as measured by mCherry expression. Multiplicity of infection (MOI) = 10.

[0157] Figure 7 is a graph showing the killing activity of untransduced (UTD), anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells using MM1S cells as target cells.

[0158] Figure 10 is a graph showing the killing activity of UTD, anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells using MM1S BCMA knockout cells as target cells.

[0159] Figure 9 is a series of graphs showing CD45RO and CCR7 expression on T cells to determine the T cell immunophenotype (e.g., naive T cells, central memory (CM) T cells, effector memory (EM) T cells, and late effector T cells (EMRA)). Enrichment of the CMT cell population was observed.

[0160] Figure 10 is a series of graphs showing CD95 and CD127 expression on the naive T cell population (i.e., CD45-CCR7+ T cells) identified in Figure 9. CD95 and CD127 are biomarkers for the T stem cell memory (TSCM) population.

[0161] Figure 11 is a graph showing CD107a expression of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells. CD107a is a marker for cytotoxic degranulation.

[0162] Figures 12A - 12C are a series of graphs showing the binding of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells to soluble TACI (sTACI) (Figure 12A), soluble BCMA (sBCMA) (Figure 12B), and soluble APRIL (sAPRIL) (Figure 12C).

[0163] Figures 13A - 13E are a series of graphs showing cytokine production of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells incubated with MM1S target cells (Figure 13A), MM1S BCMA knockout cells (Figure 13B), K562-BCMA cells (Figure 13C), K562-TACI cells (Figure 13D), and U266 cells (Figure 13E).

[0164] Figure 14 is a graph showing the population doublings of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells in response to stimulation with target cells expressing K562.

[0165] Figure 15 is a graph showing the experimental design for testing UTD, anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells in vivo. Mice were injected intravenously (i.v.) with 1 x 10 6 MM1S cells. Two weeks later, the mice were injected with 2 x 10 6 CART or UTD cells. Bioluminescence images were taken twice a week.

[0166] Figures 16A and 16B are graphs taken by bioluminescence imaging, which show the tumor burden in mice.

[0167] Figure 17 is a graph showing the quantification (photons / second) of the tumor burden in mice measured by bioluminescence.

[0168] Figure 18 is a graph showing the tumor killing of UTD, anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells.

[0169] Figure 19 is a graph showing the survival proportions of mice after treatment with UTD, anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells.

[0170] Figures 20A - 20E show the functionality of anti-TACI CAR. Figure 20A shows the construct designs of anti-TACI and control anti-BCMA CAR. Figure 20B shows BCMA and TACI staining. Figure 20C shows the luciferase-based killing assays of anti-TACI CAR and anti-BCMA CAR (control) against multiple myeloma cell lines MMlS, RPMl-8266, and U266. Figure 20D shows the in vivo luciferase imaging of MMlS after CAR treatment. Figure 20E shows the binding of soluble TACI to BCMA and TACI CAR.

[0171] Figures 21A - 21E show a comparison of tandem bispecific BCMA - TACI CAR with TriPRIL. Figure 21A shows the dual - targeting CAR T - cell construct design. Figure 21B shows the transduction efficiency of the construct. Figure 21C shows a luciferase - based killing assay of the dual - targeting CAR against multiple myeloma cell lines MMlS, RPMl - 8226, and U266. Figure 21D shows the expression of BCMA and TACI on MMlS cells expressing non - specific CRISPR / Cas9, CRISPR / Cas9 targeting BCMA, and CRISPR / Cas9 targeting TACI. Figure 21E shows a luciferase - based killing assay against the MMlS knockout cell line from Figure 21D in flat - bottom plates.

[0172] Figures 22A - 22C show a luciferase - based killing assay using CARs targeting three different myeloma lines. T cells were incubated with tumor cell lines expressing CBG - GFP for 18 hours, then lysed and analyzed for luciferase expression.

[0173] Figure 23 shows the staining of peripheral blood mononuclear cells (PBMCs) with TACI antibody measured by flow cytometry.

[0174] Figure 24 shows the transduction efficiency of normal donor T cells with anti - TACI H - L, anti - TACI L - H, or anti - BCMA measured as the percentage of mCherry - positive cells in flow cytometry analysis.

[0175] Figures 25A - 25B show the measurement of the binding of soluble TACI protein to untransduced T cells and to T cells expressing anti - TACI H - L, anti - TACI L - H, or anti - BCMA. Cells were incubated with fluorescent soluble TACI protein at different concentrations, and then the fluorescence of the cells was measured using flow cytometry, expressed as the percentage of TACI - positive cells (Figure 25A) or the median fluorescence intensity (MFI) relative to the signal measured in untransduced cells (Figure 25B).

[0176] Figures 26A - 26H show the effect of CAR T cells on RPMI8226 subcutaneous tumors in mice. Figure 26A shows the timeline of the experiment. Figures 26B - 26F show the individual growth curves of RPMI8226 tumors in mice treated with anti - TACI H - L CAR (Figure 26B), anti - TACI L - H CAR (Figure 26C), anti - BCMA CAR (Figure 26D), or untransduced T cells (Figure 26E, negative control) or untreated (Figure 26F, negative control) mice. Figure 26G shows the survival of mice over time. Figure 26H shows the intravenous administration of 2x10 6The number of CAR T cells per microliter in the blood of mice 21 days after adoptive transfer of CAR T cells.

[0177] Figures 27A - 27B show that anti-TACI CAR T cells specifically act on tumor cells expressing TACI. Figure 27A shows flow cytometry analysis of MM1S Cas9 cancer cells with or without Cas9-mediated disruption of TACI, and stained for TACI and BCMA. Figure 27B shows the cytotoxicity against MM1S Cas9 and MM1S Cas9 TACI knockout cells resulting from co-incubation with anti-TACI H-L CAR, anti-TACI L-H CAR, anti-BCMA CAR, or untransduced T cells at different T cell-to-cancer cell ratios.

[0178] Figure 28 shows the binding affinity of bispecific and monospecific CAR T cells to their respective soluble antigens measured by flow cytometry. The top two panels show the binding of fluorescent BCMA to: anti-TACI H-L CAR, anti-TACI L-H CAR, anti-BCMA, anti-TACI / anti-BCMA bispecific CAR, anti-BCMA / anti-TACI bispecific CAR, or untransduced T cells. The left two panels show the percentage of BCMA- or TACI-positive cells. The right two panels show the median fluorescence intensity of each cell population relative to untransduced cells.

[0179] Figure 29 shows the measurement of cytokine concentrations in the supernatants of cultures of MM1S (top panel) or RPMI8226 (bottom panel) cancer cells cultured alone or co-cultured with anti-TACI / anti-BCMA bispecific CAR, anti-BCMA / anti-TACI bispecific CAR, or untransduced T cells. The left panel shows the interleukin-2 (IL-2) concentration, the middle panel shows the interferon-γ (IFNγ) concentration, and the right panel shows the tumor necrosis factor α (TNFα) concentration.

[0180] Figures 30A - 30E show the results from the MM1S challenge model using bispecific CAR T cells. Figure 30A shows the timeline of the experiment, where 1 x 10 6 MM1S cells were intravenously injected into mice on day -14, and then 2 x 10 6CAR T cells and bioluminescence imaging measurements were performed on days 0, 4, 7, 11, and 14. Figures 30B, 30C, and 30D show the bioluminescence fluxes from MM1S cells measured in mice untreated or treated with T cells from three different donors (transduced with anti-TACI / anti-BCMA bispecific CAR or anti-BCMA / anti-TACI bispecific CAR, or untransduced). Figure 30E shows a representative bioluminescence image from a mouse treated with cells from donor 1.

[0181] Figures 31A - 31F show the results from the RPMI8226 subcutaneous tumor model. Figure 31A shows the timeline of the experiment, in which 5 x 10 6 RPMI8226 cells were implanted subcutaneously into mice on day - 14, and then 2 x 10 6 CAR T cells were injected intravenously on day 0, and caliper measurements of the tumors were taken on days 0, 4, 7, 10, and 14. Figure 31B shows the number of CAR T cells per microliter in the blood of mice on day 14 (left) and day 21 (right). Figures 31C, 31D, 31E, and 31F show the tumor volume curves measured in mice treated with anti-TACI / anti-BCMA bispecific CAR (Figure 31C), anti-BCMA / anti-TACI bispecific CAR (Figure 31D), or untransduced (Figure 31E) T cells or untreated mice (Figure 31F).

[0182] Figures 32A - 32B show the experimental results of monitoring T cell exhaustion after repeated antigen stimulation. Figure 32A shows the timeline of the experiment, in which 1 x 10 6 T cells (untransduced or transduced with anti-BCMA CAR, anti-TACI / anti-BCMA bispecific CAR, or anti-BCMA / anti-TACI bispecific CAR) were stimulated with irradiated K562 cells overexpressing BCMA on day 0, and flow cytometry analysis was performed on days 0, 7, 14, 21, and 28 to monitor exhaustion and memory phenotypes. T cells were counted at each time point, renormalized to 1 x 10 6 cells, and restimulated. Figure 32B shows the flow cytometry measurement results of Tim-3 and Lag3 on T cells over time.

[0183] Figures 33A - 33C show the analysis results of T cell phenotypes in repeatedly stimulated CAR T cells. Figure 33A shows the timeline of the experiment, in which 1 x 10 6CAR T cells, and were counted and analyzed by flow cytometry at days 0, 7, 14, and 21. The CAR T cells were restimulated at days 7 and 14. Figure 33B shows, according to the definitions shown on the left (central memory (CM): CCR7 + CD45RA – ; effector memory (EM): CCR7 – CD45RA – ; naive: CCR7 + CD45RA + ; terminally differentiated effector (TDE): CCR7 – CD45RA + ), the percentages of each type of CAR T cell (anti-BCMA, anti-TACI / anti-BCMA bispecific, or anti-BCMA / anti-TACI bispecific) classified into each effector phenotype by flow cytometry. Figure 33C shows the growth of CAR T cells over time, counted as cells / 10 6 . DETAILED DESCRIPTION

[0184] The present disclosure provides anti-transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) antibodies, antibody-drug conjugates thereof, bispecific T cell engagers (BiTEs) targeting TACI, and anti-TACI chimeric antigen receptors (CARs). Such antibodies, antibody-drug conjugates, BiTEs, and CARs can be used, for example, in methods of treating cancer, autoimmune disorders, or plasma cell diseases or disorders in a subject in need thereof.

[0185] Anti-TACI Antibodies

[0186] The present invention provides isolated antibodies that specifically bind to TACI.

[0187] In some aspects, the present disclosure provides the heavy and light chain variable domain sequences and the heavy and light chain CDR sequences of the anti-TACI antibodies described herein. When different definition systems (e.g., IMGT definition, Kabat definition, or Chothia definition) are used, the CDRs of an antibody may have different amino acid sequences. A definition system annotates each amino acid in a given antibody sequence (e.g., a VH or VL sequence) with a number, and the numbers corresponding to the heavy and light chain CDRs are provided in Table 3. CDR sequences of examples of anti-TACI antibodies according to different definition systems are provided in Table 4.

[0188] In some embodiments, the anti-TACI antibodies of the present disclosure comprise one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any of the anti-TACI antibodies selected from Table 4. In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 provided in Table 4 for each numbering system. In some embodiments, the anti-TACI antibodies of the present disclosure comprise one or more of the CDR-L (e.g., CDR-L1, CDR-L2, and CDR-L3) amino acid sequences from any of the anti-TACI antibodies selected from Table 4. In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 provided in Table 4 for each numbering system.

[0189] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided in Table 4 for each numbering system. In some embodiments, the CDR3 domains of the heavy and light chains of the antibody can play a particularly important role in the binding specificity / affinity of the antibody for the antigen. Thus, the anti-TACI antibodies of the present disclosure can comprise at least the heavy chain and / or light chain CDR3 of any of the anti-TACI antibodies provided in Table 4.

[0190] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable domain having the amino acid sequence of SEQ ID NO:1. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 of the light chain variable domain having the amino acid sequence of SEQ ID NO:2.

[0191] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1 having the amino acid sequence of SEQ ID NO:26 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO:27 (according to the IMGT definition system), CDR-H3 having the amino acid sequence of SEQ ID NO:28 (according to the IMGT definition system), CDR-L1 having the amino acid sequence of SEQ ID NO:29 (according to the IMGT definition system), CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the IMGT definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the IMGT definition system).

[0192] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-H1 having the amino acid sequence of SEQ ID NO:26, CDR-H2 having the amino acid sequence of SEQ ID NO:27, and CDR-H3 having the amino acid sequence of SEQ ID NO:28. "Together" means the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-L1 having the amino acid sequence of SEQ ID NO:29, CDR-L2 having the amino acid sequence of SEQ ID NO:30, and CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0193] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that are at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical in total compared to CDR-H1 having the amino acid sequence of SEQ ID NO:26, CDR-H2 having the amino acid sequence of SEQ ID NO:27, and CDR-H3 having the amino acid sequence of SEQ ID NO:28. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical in total compared to CDR-L1 having the amino acid sequence of SEQ ID NO:29, CDR-L2 having the amino acid sequence of SEQ ID NO:30, and CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0194] In some embodiments, the anti-TACI antibodies of the present disclosure comprise: a CDR-H1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H1 having the amino acid sequence of SEQ ID NO:26; a CDR-H2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H2 having the amino acid sequence of SEQ ID NO:27; and / or a CDR-H3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H3 having the amino acid sequence of SEQ ID NO:28. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise: a CDR-L1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L1 having the amino acid sequence of SEQ ID NO:29; a CDR-L2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L2 having the amino acid sequence of SEQ ID NO:30; and / or a CDR-L3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0195] In some embodiments, the anti-TACI antibodies of the present disclosure comprise a CDR-H1 having the amino acid sequence of SEQ ID NO:32 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:33 (according to the Kabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO:34 (according to the Kabat definition system), a CDR-L1 having the amino acid sequence of SEQ IDNO:35 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO:36 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the Kabat definition system).

[0196] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-H1 having the amino acid sequence of SEQ ID NO:32, CDR-H2 having the amino acid sequence of SEQ ID NO:33, and CDR-H3 having the amino acid sequence of SEQ ID NO:34. "Together" means the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-L1 having the amino acid sequence of SEQ ID NO:35, CDR-L2 having the amino acid sequence of SEQ ID NO:36, and CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0197] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that are together at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to CDR-H1 having the amino acid sequence of SEQ ID NO:32, CDR-H2 having the amino acid sequence of SEQ ID NO:33, and CDR-H3 having the amino acid sequence of SEQ ID NO:34. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are together at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to CDR-L1 having the amino acid sequence of SEQ ID NO:35, CDR-L2 having the amino acid sequence of SEQ ID NO:36, and CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0198] In some embodiments, the anti-TACI antibodies of the present disclosure comprise: a CDR-H1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H1 having the amino acid sequence of SEQ ID NO:32; a CDR-H2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H2 having the amino acid sequence of SEQ ID NO:33; and / or a CDR-H3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H3 having the amino acid sequence of SEQ ID NO:34. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise: a CDR-L1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L1 having the amino acid sequence of SEQ ID NO:35; a CDR-L2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L2 having the amino acid sequence of SEQ ID NO:36; and / or a CDR-L3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L3 having the amino acid sequence of SEQ ID NO:31.

[0199] In some embodiments, the anti-TACI antibodies of the present disclosure comprise a CDR-H1 having the amino acid sequence of SEQ ID NO:37 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:38 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO:39 (according to the Chothia definition system), a CDR-L1 having the amino acid sequence of SEQ ID NO:40 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO:41 (according to the Chothia definition system).

[0200] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-H1 having the amino acid sequence of SEQ ID NO:37, CDR-H2 having the amino acid sequence of SEQ ID NO:38, and CDR-H3 having the amino acid sequence of SEQ ID NO:39. "Together" means the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that together contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-L1 having the amino acid sequence of SEQ ID NO:40, CDR-L2 having the amino acid sequence of SEQ ID NO:30, and CDR-L3 having the amino acid sequence of SEQ ID NO:41.

[0201] In some embodiments, the anti-TACI antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that are together at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to CDR-H1 having the amino acid sequence of SEQ ID NO:37, CDR-H2 having the amino acid sequence of SEQ ID NO:38, and CDR-H3 having the amino acid sequence of SEQ ID NO:39. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are together at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to CDR-L1 having the amino acid sequence of SEQ ID NO:40, CDR-L2 having the amino acid sequence of SEQ ID NO:30, and CDR-L3 having the amino acid sequence of SEQ ID NO:41.

[0202] In some embodiments, an anti-TACI antibody of the present disclosure comprises: a CDR-H1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H1 having the amino acid sequence of SEQ ID NO:37; a CDR-H2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H2 having the amino acid sequence of SEQ ID NO:38; and / or a CDR-H3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-H3 having the amino acid sequence of SEQ ID NO:39. Alternatively or additionally, an anti-TACI antibody of the present disclosure comprises: a CDR-L1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L1 having the amino acid sequence of SEQ ID NO:40; a CDR-L2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L2 having the amino acid sequence of SEQ ID NO:30; and / or a CDR-L3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the CDR-L3 having the amino acid sequence of SEQ ID NO:41.

[0203] In some embodiments, an anti-TACI antibody of the present disclosure comprises a VH containing the amino acid sequence of SEQ ID NO:1. Alternatively or additionally, an anti-TACI antibody of the present disclosure comprises a VL containing the amino acid sequence of SEQ ID NO:2.

[0204] In some embodiments, an anti-TACI antibody of the present disclosure comprises a VH having no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH shown in SEQ ID NO:1. Alternatively or additionally, an anti-TACI antibody of the present disclosure comprises a VL having no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VL shown in SEQ ID NO:2.

[0205] In some embodiments, the anti-TACI antibodies of the present disclosure comprise a VH that contains an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the VH shown in SEQ ID NO:1. Alternatively or additionally, the anti-TACI antibodies of the present disclosure comprise a VL that contains an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the VL shown in SEQ ID NO:2.

[0206] In some embodiments, the anti-TACI antibodies of the present disclosure are humanized antibodies. In some embodiments, the humanized anti-TACI antibody comprises a humanized VH that contains CDR-H1 having the amino acid sequence of SEQ ID NO:26 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO:27 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO:28 (according to the IMGT definition system); and a humanized VL that contains CDR-L1 having the amino acid sequence of SEQ ID NO:29 (according to the IMGT definition system), CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the IMGT definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the IMGT definition system).

[0207] In some embodiments, the humanized anti-TACI antibody comprises a humanized VH that contains CDR-H1 having the amino acid sequence of SEQ ID NO:32 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO:33 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO:34 (according to the Kabat definition system); CDR-L1 having the amino acid sequence of SEQ ID NO:35 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO:36 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the Kabat definition system).

[0208] In some embodiments, the humanized anti-TACI antibody comprises a humanized VH that contains CDR-H1 having the amino acid sequence of SEQ ID NO:37 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO:38 (according to the Chothia definition system), CDR-H3 having the amino acid sequence of SEQ ID NO:39 (according to the Chothia definition system); CDR-L1 having the amino acid sequence of SEQ ID NO:40 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO:41 (according to the Chothia definition system).

[0209] In some embodiments, the anti-TACI antibody is an IgG, Fab fragment, F(ab') fragment, F(ab')2 fragment, scFv, or an scFv fused to a constant region (e.g., an N- or C-terminal fusion). Non-limiting examples of different forms of anti-TACI antibodies are provided herein.

[0210] In some embodiments, the anti-TACI antibody is a single-chain fragment variable region (scFv) that contains VH and VL in a single polypeptide chain. In some embodiments, the scFv contains any of the heavy chain CDRs, light chain CDRs, VH, and / or VL described herein on a single polypeptide chain. In some embodiments, the scFv contains VH linked to the N-terminus of the VL. In some embodiments, the scFv contains VL linked to the N-terminus of the VH. In some embodiments, the VH and VL are linked via a linker (e.g., a polypeptide linker). Any polypeptide linker can be used to link VH and VL in the scFv. The choice of linker sequence is within the ability of those skilled in the art.

[0211] In some embodiments, the scFv comprises a VH that contains a CDR-H1 having the amino acid sequence of SEQ ID NO:26 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:27 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:28 (according to the IMGT definition system); and a VL that contains a CDR-L1 having the amino acid sequence of SEQ ID NO:29 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the IMGT definition system), wherein the VH and VL are on a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0212] In some embodiments, the scFv comprises a VH that contains a CDR-H1 having the amino acid sequence of SEQ ID NO:32 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:33 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:34 (according to the Kabat definition system); and a VL that contains a CDR-L1 having the amino acid sequence of SEQ ID NO:35 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO:36 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO:31 (according to the Kabat definition system), wherein the VH and VL are on a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0213] In some embodiments, the scFv comprises a VH that contains a CDR-H1 having the amino acid sequence of SEQ ID NO:37 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:38 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:39 (according to the Chothia definition system); and a VL that contains a CDR-L1 having the amino acid sequence of SEQ ID NO:40 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO:30 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO:41 (according to the Chothia definition system), wherein the VH and VL are on a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0214] In some embodiments, the scFV comprises a VH that contains an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the VH shown in SEQ ID NO:1; and a VL (e.g., a humanized VL) that contains an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the VL shown in SEQ ID NO:2, wherein the VH and VL are in a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the scFv comprises an amino acid sequence that has at least 80% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0215] In some embodiments, the scFV comprises a VH that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH shown in SEQ ID NO:1, and a VL that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VL shown in SEQ ID NO:2, wherein the VH and VL are in a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the scFv comprises an amino acid sequence that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0216] In some embodiments, the scFV comprises a VH containing the amino acid sequence of SEQ ID NO:1 and a VL containing the amino acid sequence of SEQ ID NO:2, wherein the VH and VL are in a single polypeptide chain (e.g., linked via an amide bond or via a linker such as a peptide linker), and wherein the VH is linked to the N-terminus or C-terminus of the VL. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3).

[0217] In some embodiments, the scFv comprises a VH containing the amino acid sequence of SEQ ID NO:1 linked to the N-terminus of a VL containing the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3).

[0218] In some embodiments, the scFv comprises a VH having the amino acid sequence of SEQ ID NO:1 c-terminally linked to a VL having the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH and VL are linked via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3).

[0219] In some cases, the antibody binds to TACI with a K of about 5 nM or less (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 900 pM or less, about 875 pM or less, about 850 pM or less, about 825 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, or about 500 pM or less). D In one embodiment, the antibody binds to TACI with a K between about 500 pM and about 1 nM. D In one embodiment, the antibody binds to TACI with a K between about 700 pM and about 900 pM. D In one embodiment, the antibody binds to TACI with a K of about 861 pM. D In one embodiment, the antibody binds to TACI.

[0220] In some cases, the antibody comprises a heavy chain variable domain (VH) sequence having at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or having the sequence of SEQ ID NO:1. In other examples, the antibody comprises a light chain variable domain (VL) sequence having at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or having the sequence of SEQ ID NO:2. In some cases, the antibody comprises a VH having at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or having the sequence of SEQ ID NO:1; and a VL having at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or having the sequence of SEQ ID NO:2.

[0221] For example, such antibodies can be monoclonal, human, humanized or chimeric. The antibody can be a full-length antibody or an antibody fragment thereof (e.g., an antibody fragment that binds TACI). The antibody fragment can be selected from Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments. In some cases, the antibody is an IgG antibody (e.g., an IgG1 antibody). Such antibodies can have a half-life of ≥3 days (e.g., ≥1 week, e.g., ≥2 weeks, e.g., ≥1 month, e.g., ≥2 months, e.g., ≥3 months, e.g., ≥4 months, e.g., ≥5 months, e.g., ≥6 months).

[0222] In other aspects, an anti-TACI antibody according to any of the above embodiments can be incorporated alone or in combination into any feature described in the following article sections.

[0223] Antibody affinity

[0224] In certain embodiments, the antibodies provided herein can have a dissociation constant (K D ) of ≤10 μM, ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, or ≤0.01 nM. In some cases, the antibody binds to TACI with a K D of about 5 nM or lower (e.g., about 5 nM or lower, about 4 nM or lower, about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, about 900 pM or lower, about 875 pM or lower, about 850 pM or lower, about 825 pM or lower, about 800 pM or lower, about 700 pM or lower, about 600 pM or lower, or about 500 pM or lower).

[0225] In one embodiment, the antibody binds to TACI with a K D between about 500 pM and about 1 nM. In one embodiment, the antibody binds to TACI with a K D between about 700 pM and about 900 pM. In one embodiment, the antibody binds to TACI with a K D of about 861 pM.

[0226] In one embodiment, K D is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the Fab form of the antibody of interest and its antigen are used for the RIA. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen and then capturing the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865 - 881 (1999)). To establish the assay conditions, a multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked for 2 to 5 hours at room temperature (about 23 °C) with 2% (w / v) bovine serum albumin in PBS. In a non-adsorbing plate (Nunc #269620), 100 pM or 26 pM of 125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 (TWEEN- ) and washed 8 times. When the plate was dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and place the plate on TOPCOUNT TM Counting was performed on a gamma counter (Packard) for 10 min. The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for competitive binding assays.

[0227] According to another embodiment, using Surface plasmon resonance assay to measure K D For example, using an antigen-immobilized CM5 chip at 25°C at about 10 response units (RU) -3000 (BIAcore, Inc., Piscataway, NJ) for determination. In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Prior to injection at a flow rate of 5 μl / min, the antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) to obtain about 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected into a 20% PBS containing 0.05% polysorbate 20 (TWEEN-20) at 25°C at a flow rate of about 25 μl / min. TM ) surfactant in PBS (PBST). Using a simple one-to-one Langmuir binding model ( The association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams using the ELISA software version 3.2. on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) is calculated as the ratio k on / k offSee, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate measured by the above surface plasmon resonance assay exceeds 10 6 M- 1 s- 1 −1, the association rate can be determined by using fluorescence quenching techniques that measure the increase or decrease in the fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS at pH 7.2 in the presence of increasing concentrations of antigen at 25 °C (excitation = 295 nm; emission = 340 nm, 16 nm bandpass), as measured in a spectrometer with a stirred cuvette (e.g., a spectrophotometer equipped with a stop-flow meter (Aviv Instruments) or an 8000 series SLM-AMINCO TM spectrophotometer (ThermoSpectronic)).

[0228] According to another embodiment, the surface plasmon resonance assay described in Example 1 herein is used to measure K D D.

[0229] Antibody fragments

[0230] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments known in the art. Also included are diabodies, which have two antigen-binding sites that can be bivalent or bispecific, as known in the art. Triabodies and tetra-bodies are also known. Single-domain antibodies are also antibody fragments that comprise all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibodies are human single-domain antibodies.

[0231] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0232] Chimeric and humanized antibodies

[0233] In certain embodiments, the antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In a further example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0234] In certain embodiments, the chimeric antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises HVRs (e.g., CDRs) (or portions thereof) derived from a non-human antibody and FRs (or portions thereof) derived from one or more variable domains of human antibody sequences. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0235] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatic mutation-containing) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0236] Human antibodies

[0237] In certain embodiments, the antibodies provided herein are human antibodies (e.g., human monoclonal antibodies (HuMab), e.g., anti-TACI HuMab). Human antibodies can be produced using a variety of techniques known in the art.

[0238] Human monoclonal antibodies can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies can in principle also be used, such as viral or oncogenic transformation of B lymphocytes, phage display techniques using human antibody gene libraries.

[0239] In some cases, human antibodies are obtained by directly cloning the heavy and light chain genes from human B cells obtained from human subjects. The B cells are isolated from peripheral blood (e.g., by flow cytometry, e.g., FACS), stained for one or more B cell markers, and evaluated for antigen binding. RNA encoding the variable regions of the heavy and light chains (or the entire heavy and light chains) is extracted and reverse transcribed into DNA, from which the antibody genes are amplified (e.g., by PCR) and sequenced. The known antibody sequences can then be used to express recombinant human antibodies against known target antigens (e.g., TACI).

[0240] In some cases, human antibodies can be prepared by administering an immunogen (e.g., TACI) to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. The human variable regions from the intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0241] In some cases, human antibodies can also be prepared by hybridoma-based methods. The preferred animal system for generating hybridomas is the murine system, which produces human monoclonal antibodies. Hybridoma production in mice is well known in the art, including immunization protocols and techniques for isolating and fusing immune spleen cells. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies.

[0242] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a phage display library of human origin. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0243] Anti-TACI antibodies can also be generated using recombinant methods and compositions such as those described in, for example, U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-TACI antibody as described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In other embodiments, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In other embodiments, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector containing a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector containing a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of preparing an anti-TACI antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0244] For recombinant production of anti-TACI antibodies, a nucleic acid encoding an antibody as described above, for example, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such a nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the antibody heavy and light chains).

[0245] Suitable host cells for cloning or expressing vectors encoding antibodies include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0246] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with a partially or fully human glycosylation pattern.

[0247] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used in conjunction with insect cells have been identified, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts.

[0248] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth can be useful. Examples of other useful mammalian host cell lines are the simian kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (such as 293 or 293 cells as described, for example, by Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described, for example, by Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); TRI cells as described, for example, by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR - CHO cells) and myeloma cell lines (such as Y0, NS0, and Sp2 / 0).

[0249] Antibody variants

[0250] In certain embodiments, amino acid sequence variants of anti-TACI antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics (e.g., antigen binding).

[0251] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody, and the resulting product can be screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0252] Table 1. Exemplary and Preferred Amino Acid Substitutions

[0253]

[0254]

[0255] Amino acids can be grouped according to common side chain properties:

[0256] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0257] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0258] (3) Acidic: Asp, Glu;

[0259] (4) Basic: His, Lys, Arg;

[0260] (5) Residues affecting chain orientation: Gly, Pro;

[0261] (6) Aromatic: Trp, Tyr, Phe.

[0262] Non-conservative substitutions would require the exchange of a member of one of these classes for another.

[0263] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on a phage, and screened for a particular biological activity (e.g., binding affinity).

[0264] Changes (e.g., substitutions) can be made in the CDRs, e.g., to improve antibody affinity. Such changes can be made in CDR “hotspots,” i.e., residues encoded by codons that mutate at high frequency during somatic maturation and / or residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by construction and reselection from secondary libraries is known in the art. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants having the desired affinity. Another method for introducing diversity involves CDR-directed methods, where several CDR residues (e.g., 4–6 residues at a time) are randomized. The CDR residues involved in antigen binding can be specifically identified using, e.g., alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0265] In certain embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. For example, such changes can be outside of the antigen-contact residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0266] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called “alanine-scanning mutagenesis,” as described by Cunningham and Wells (1989) Science, 244:1081–1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen–antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they possess the desired properties.

[0267] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0268] In certain embodiments, the Fc region of an antibody can be altered. These alterations can be made alone or in combination with alterations of one or more antibody variable domains (i.e., VH or VL regions) or regions thereof (e.g., one or more CDRs or FRs). Alterations of the Fc region can, for example, result in enhanced antibody effector function (e.g., complement-dependent cytotoxicity (CDC)) by increasing the affinity of C1q for opsonized cells. Exemplary mutations that enhance CDC include, for example, the Fc mutations E345R, E430G, and S440Y. Thus, an anti-TACI antibody can contain one or more CDC-enhancing Fc mutations that promote IgG hexamer formation and subsequent recruitment and activation of the first complement component, C1.

[0269] In certain embodiments, alterations of the amino acid sequence of the Fc region of the antibody can alter the half-life of the antibody in a host. Certain mutations that alter binding to the neonatal Fc receptor (FcRn) can prolong the half-life of the antibody in serum. For example, an antibody having tyrosine at heavy chain position 252, threonine at position 254, and glutamate at heavy chain position 256 can have a significantly prolonged half-life in serum (see, e.g., U.S. Patent No. 7,083,784).

[0270] Characterization of Monoclonal Antibodies Against TACI

[0271] The sequence information of the human monoclonal antibodies of the invention can be determined using sequencing techniques well known in the art.

[0272] Similarly, standard techniques can also be used to evaluate the affinity of the antibody for TACI. For example, the affinity of a HuMab for TACI can be determined using a Biacore 3000. The HuMab is captured, for example, via amine coupling (Sensor Chip CM5) on the surface of a Biacore chip (GE Healthcare). The captured HuMab can be exposed to various concentrations of TACI in solution, and the affinity (K D ) of K on and K off .

[0273] A variety of known techniques (such as ELISA, Western blotting, etc.) can also be used to characterize the binding of the human monoclonal antibodies of the present invention to TACI. Generally, the antibodies are initially characterized by ELISA. In some cases, an ELISA assay can be used to screen antibodies to screen for hybridomas that produce antibodies showing positive reactivity with the TACI immunogen. The hybridomas that bind to TACI, preferably with high affinity, can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells can then be selected (by ELISA) for the preparation of a cell bank and for antibody purification.

[0274] In some cases, a competition assay can be used to identify antibodies that compete with the anti-TACI antibodies of the present invention for binding to TACI. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the anti-TACI antibodies of the present invention. Detailed exemplary methods for mapping the epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, Volume 66 (Humana Press, Totowa, NJ).

[0275] Antibody-drug conjugate

[0276] In some embodiments, the anti-TACI antibodies of the present invention can be covalently attached to a drug moiety via a biodegradable stable linker (e.g., via a disulfide or non-cleavable thioether linker), i.e., as an antibody-drug conjugate. The drug covalently attached to the antibody can be cytotoxic or growth inhibitory when not conjugated to the antibody. The antibody-drug conjugate can be used to selectively deliver an effective dose of the cytotoxic agent to cells expressing TACI (e.g., to tumor tissue expressing TACI) to achieve greater selectivity and thus a lower effective dose. By achieving higher selectivity, the use of the antibody-drug conjugate can also reduce systemic exposure and increase the tolerance to the drug. Additionally, the antibody-drug conjugate can improve the bioavailability of the drug and / or the antibody compared to when the drug and / or the antibody are administered in their unconjugated forms.

[0277] Bispecific T cell engager (BiTE)

[0278] The present disclosure also provides bispecific antibodies referred to as bispecific T cell engagers (BiTEs). Such molecules can target T cells by binding to a T cell antigen (e.g., by binding CD3), as well as a target antigen (e.g., TACI). BiTEs can be used to enhance T cell responses, e.g., in the tumor microenvironment. The two components of the BiTE can optionally be separated from each other by a linker as described herein (e.g., a glycine / serine-based linker), and can also be joined in either orientation, e.g., where the anti-CD3 component is at the N-terminus of the anti-TACI component, or vice versa.

[0279] Exemplary BiTEs useful in the methods described herein include a CD3-binding domain and a TACI-binding domain. Such a BiTE can comprise an anti-CD3 scFv as the CD3-binding domain. The anti-CD3 scFv can be derived from any anti-CD3 antibody known in the art.

[0280] In some cases, the TACI-binding domain (e.g., an antibody) binds to TACI with a K D of about 5 nM or less (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 900 pM or less, about 875 pM or less, about 850 pM or less, about 825 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, or about 500 pM or less). D In one embodiment, the TACI-binding domain (e.g., an antibody) binds to TACI with a K D of between about 500 pM and about 1 nM. D In one embodiment, the TACI-binding domain (e.g., an antibody) binds to TACI with a K

[0281] In addition, the BiTE may comprise a TACI-binding domain derived from an anti-TACI antibody as described above. In some embodiments, the TACI-binding domain comprises a VH that has at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:1. In other embodiments, the TACI-binding domain comprises a VL that has at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:2. In certain embodiments, the TACI-binding domain comprises a VH that has at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:1; and a VL that has at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:2.

[0282] The TACI-binding domain may be located at the N-terminus of the CD3-binding domain, or the CD3-binding domain may be located at the N-terminus of the TACI-binding domain. The TACI-binding domain and the CD3-binding domain may optionally be linked via a linker sequence (e.g., a linker sequence of SEQ ID NO:3, 14, 15, 16 or 17 (described below) and any other linker described herein or known in the art).

[0283] Chimeric antigen receptor (CAR)

[0284] The techniques described herein provide improved chimeric antigen receptors (CARs) for use in immunotherapy. CARs and various improvements are discussed below.

[0285] A CAR refers to an engineered T cell receptor that confers ligand or antigen specificity onto cells engineered to express the CAR (e.g., T cells, natural killer (NK) cells, or induced pluripotent stem cells (iPSCs)). The CAR places a chimeric extracellular target-binding domain, which specifically binds a target (e.g., a polypeptide) expressed on the surface of a cell to be targeted to elicit a T cell response, onto a construct that includes a transmembrane domain of a T cell receptor molecule and one or more intracellular domains. In one embodiment, the chimeric extracellular target-binding domain includes one or more antigen-binding domains of an antibody that specifically binds an antigen expressed on the surface of a cell to be targeted for a T cell response. As is known in the art and as disclosed herein, the properties of one or more intracellular signaling domains of a CAR can vary, but when the chimeric target / antigen-binding domain binds the target / antigen on the surface of the targeted cell, one or more chimeric target / antigen-binding domains render the receptor sensitive to signaling activation.

[0286] With respect to the intracellular signaling domains, so-called "first-generation" CARs include those that provide only CD3zeta (CD3ζ) signaling upon antigen binding. So-called "second-generation" CARs include CARs that provide both a co-stimulatory (e.g., CD28 or CD137) and an activation (CD3ζ) domain, while so-called "third-generation" CARs include CARs that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3ζ). In various embodiments, a CAR is selected to have a high affinity or avidity for the target / antigen - for example, an antibody-derived target or antigen-binding domain will generally have a higher affinity and / or avidity for the target antigen compared to a naturally occurring T cell receptor. This property, combined with the high specificity with which an antibody can be selected, provides for highly specific T cell targeting by CAR T cells.

[0287] Extracellular target-binding domain

[0288] As used herein, the term "extracellular target-binding domain" refers to a polypeptide found external to a cell that is sufficient to promote binding to a target. The extracellular target-binding domain will specifically bind its binding partner, i.e., the target. As non-limiting examples, the extracellular target-binding domain can include an antibody or an antigen-binding domain of an antibody reagent or ligand that recognizes and binds a cognate binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the cell surface. Ligand:cognate partner binding can result in an alteration of the receptor bearing the ligand or activation of a physiological response, such as activation of a signaling pathway. In one embodiment, the ligand can be non-native to the genome. Optionally, the ligand has a conserved function across at least two species.

[0289] Antibody reagent

[0290] In various embodiments, the CARs described herein include an antibody reagent or an antigen-binding domain thereof as an extracellular target-binding domain.

[0291] As used herein, the term "antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds a given antigen. For example, antibody reagents that can be used in the CARs described herein include any of the antibodies described above. The antibody reagent can include an antibody or a polypeptide containing the antigen-binding domain of an antibody. In some embodiments in any aspect, the antibody reagent can include a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, an antibody can include VH and VL. In another example, an antibody contains two VH and two VL. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur. J. Immunol. 26(3):629-639, 1996; which is incorporated herein by reference in its entirety)) as well as intact antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (and their subtypes and combinations). Antibodies can be from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates) as well as primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, etc. Methods known to those of ordinary skill in the art can be used, e.g., to select intact human antibody-binding domains from phage display libraries. In addition, antibody reagents include single-domain antibodies, such as camelid antibodies.

[0292] In one embodiment, the extracellular target-binding domain of the CAR includes or consists essentially of a single-chain Fv (scFv) fragment, which is produced by fusing the VH and VL domains of an antibody (usually a monoclonal antibody) via a flexible linker peptide. In various embodiments, the scFv is fused to a transmembrane domain and fused to a T cell receptor intracellular signaling domain, such as the engineered intracellular signaling domain described herein. In another embodiment, the extracellular target-binding domain of the CAR includes a single-domain (camelid) antibody. Antibody-binding domains and the manner of selecting and cloning them are well known to those of ordinary skill in the art.

[0293] Target / antigen

[0294] Any cell surface moiety can be targeted by a CAR. Typically, the target is a cell surface polypeptide that is differentially or preferentially expressed on the cells that one wishes to target for a T cell response. As described herein, to target tumors or cancer cells, an antibody domain can be targeted, for example, to TACI. Targeting tumor antigens or tumor-associated antigens that are specific to tumors can provide a means of targeting tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues.

[0295] The CARs provided herein target TACI, which is a receptor that recognizes APRIL, BAFF, and CAML. TACI sequences are known for many species, such as human TACI (NCBI Gene ID: 23495) polypeptide (e.g., NCBI Ref Seq: NP_036584.1) and mRNA (e.g., NCBI Ref Seq: NM_012452.2). TACI can refer to human TACI, including its naturally occurring variants, molecules, and alleles. Those skilled in the art can readily identify homologs and / or orthologs of human TACI for such species, for example, using the NCBI ortholog search function or searching for sequences similar to the reference TACI sequence in the available sequence data for a given species.

[0296] In some examples, the CARs described herein can also target an additional second target. Non-limiting examples of additional targets such as tumor antigens, tumor-associated antigens, or other antigens of interest include BCMA, CD19, CD37, CEA, immature laminin receptor, TAG-72, HPV E6 and E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, BRCA1 / 2, CDK4, MART-2, p53, Ras, MUC1, TGF-βRII, IL-15, IL13Ra2, and CSF1R. In some embodiments, the second target is BCMA.

[0297] TACI binding domain

[0298] In some embodiments, the present invention provides an anti-TACI CAR that comprises a TACI-binding domain that includes an antibody reagent. In some cases, the TACI-binding domain binds to TACI with a Kd of about 5 nM or lower (e.g., about 5 nM or lower, about 4 nM or lower, about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, about 900 pM or lower, about 875 pM or lower, about 850 pM or lower, about 825 pM or lower, about 800 pM or lower, about 700 pM or lower, about 600 pM or lower, or about 500 pM or lower). D In one embodiment, the TACI-binding domain binds to TACI with a Kd between about 500 pM and about 1 nM. D In one embodiment, the TACI-binding domain binds to TACI with a Kd between about 700 pM and about 900 pM. D In one embodiment, the TACI-binding domain binds to TACI with a Kd of about 861 pM. D

[0299] ​For example, the antibody reagent can be an anti-TACI antibody or an antigen-binding fragment thereof, such as an anti-TACI scFv. In some embodiments, the anti-TACI scFv comprises a VH sequence as follows, which has at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:1. In other embodiments, the anti-TACI scFv comprises a VL sequence as follows, which has at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:2. In some cases, the anti-TACI scFv comprises a VH as follows, which has at least 80% sequence identity with SEQ ID NO:1 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:1; and a VL as follows, which has at least 80% sequence identity with SEQ ID NO:2 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) or has the sequence of SEQ ID NO:2. The VH can be located at the N-terminus of the VL (VH-VL), or the VL can be located at the N-terminus of the VH (VL-VH)

[0300] The VH and VL domains can be linked via a linker sequence. For example, linker sequences useful in the present invention include, but are not limited to, glycine / serine linkers such as GGGSGGGSGGGS (SEQ ID NO:14) and Gly4Ser (G4S) linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO:15)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:3)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO:16) as described by Whitlow et al., Protein Eng. 6(8):989-95, 1993 (the contents of which are incorporated herein by reference in their entirety); the linker sequence of GGSSRSSSSGGGGSGGGG (SEQ ID NO:17) as described by Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011(9), 2011 (the contents of which are incorporated herein by reference in their entirety); and linker sequences with added functionality (such as epitope tags or coding sequences containing Cre-Lox recombination sites) as described by Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000 (the contents of which are incorporated herein by reference in their entirety).

[0301] In a specific embodiment, the anti-TACI CAR described herein can comprise an anti-TACI scFv having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with SEQ ID NO:3 (VH-VL) or SEQ ID NO:4 (VL-VH) or having the sequence of SEQ ID NO:3 (VH-VL) or SEQ ID NO:4 (VL-VH).

[0302] Bispecific CAR

[0303] In another embodiment, a CAR useful in the techniques described herein comprises an extracellular target-binding domain containing at least two antigen-specific targeting regions, a transmembrane domain, and an intracellular signaling domain. In such embodiments, the two or more antigen-specific targeting regions target at least two different antigens and can be arranged in tandem and separated by a linker sequence. In another embodiment, the CAR is a bispecific CAR. The bispecific CAR is specific for two different antigens (e.g., TACI and any one of the additional targets described herein (e.g., BCMA)).

[0304] For example, a bispecific CAR can target TACI and BCMA and comprises a TACI binding domain and a BCMA binding domain. Such a bispecific CAR can comprise a TACI binding domain as described above (e.g., an anti-TACI scFv). In some embodiments, the BCMA binding domain comprises an anti-BCMA scFv. The anti-BCMA scFv can be derived from any anti-BCMA antibody known in the art. The TACI binding domain can be located at the N-terminus of the BCMA binding domain, or the BCMA binding domain can be located at the N-terminus of the TACI binding domain. The TACI binding domain and the BCMA binding domain can optionally be linked via a linker sequence (e.g., the linker sequences of SEQ ID NO: 3, 14, 15, 16, or 17 and any other linker described herein or known in the art).

[0305] In some embodiments, the bispecific CAR targeting TACI and BCMA described herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18 - 25. In some embodiments, the bispecific CAR targeting TACI and BCMA described herein comprises an amino acid sequence that is 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18 - 25. In some embodiments, the bispecific CAR targeting TACI and BCMA described herein comprises the amino acid sequence of any one of SEQ ID NOs: 18 - 25.

[0306] Hinge and transmembrane domains

[0307] Each CAR as described herein comprises a transmembrane domain, such as a hinge / transmembrane domain, that links the extracellular target binding domain to the intracellular signaling domain.

[0308] Optionally following the binding domain of the CAR is one or more "hinge domains" which function to position the antigen binding domain away from the effector cell surface to effect proper cell / cell contact, antigen binding, and activation. Optionally the CAR includes one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7, which may be the wild-type hinge regions from these molecules or may be altered. In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In one embodiment, the hinge domain comprises the CD8α hinge region.

[0309] As used herein, a "transmembrane domain" (TM domain) refers to the portion of the CAR that optionally fuses the extracellular binding portion to the intracellular portion (e.g., co-stimulatory domain and intracellular signaling domain) via a hinge domain and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain is generally a hydrophobic region of the CAR that spans the cytoplasmic membrane. The TM domain can be the transmembrane region or a fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used in the examples, other transmembrane domains will be apparent to those skilled in the art and can be used in alternative embodiments of the technology. The transmembrane region or fragment thereof selected preferably does not interfere with the intended function of the CAR. As used with respect to the transmembrane domain of a protein or polypeptide, "a fragment thereof" refers to a portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0310] In some examples, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the transmembrane domains of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0311] As used herein, "hinge / transmembrane domain" refers to a domain that includes both a hinge domain and a transmembrane domain. For example, the hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In one embodiment, the hinge / transmembrane domain of the CAR or fragment thereof is derived from or includes the hinge / transmembrane domain of CD8.

[0312] CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and serves as a T cell coreceptor. CD8 is composed of an alpha (CD8α or CD8a) and a beta (CD8β or CD8b) chain. CD8a sequences of many species are known, for example, the human CD8a (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref SeqNP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 can refer to human CD8, including its naturally occurring variants, molecules, and alleles. In some embodiments in any aspect, for example, in veterinary applications, CD8 can refer to CD8 of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD8 can be readily identified for such species by those skilled in the art, for example, using the NCBI ortholog search function or available sequence data for searching for sequences similar to the reference CD8 sequence for a given species.

[0313] In some embodiments, the CD8 hinge and transmembrane sequences correspond to the amino acid sequence of SEQ ID NO:7; or comprise the sequence of SEQ ID NO:7; or comprise a sequence having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with the sequence of SEQ ID NO:7.

[0314] Costimulatory domain

[0315] Each CAR described herein optionally includes the intracellular domain of one or more costimulatory molecules, or a costimulatory domain. As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for the effective activation and function of T lymphocytes upon binding of an antigen. The costimulatory domain can be, for example, the costimulatory domain of 4-1BB, CD27, CD28, or OX40. Additional illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2CSLP76, TRIM, and ZAP70.

[0316] In one embodiment, the costimulatory domain is the costimulatory domain of 4-1BB. 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. The sequences of 4-1BB are known for many species, for example, human 4-1BB, also known as TNFRSF9 (NCBI Gene ID: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including its naturally occurring variants, molecules, and alleles. In some embodiments in any aspect, for example, in veterinary applications, 4-1BB can refer to 4-1BB of, for example, dogs, cats, cattle, horses, pigs, etc. Homologs and / or orthologs of human 4-1BB can be readily identified for such species by those skilled in the art, for example, using the NCBI ortholog search function or available sequence data for searching for sequences similar to the reference 4-1BB sequence for a given species.

[0317] In some embodiments, the costimulatory domain of 4-1BB corresponds to the amino acid sequence of SEQ ID NO:8; or comprises the sequence of SEQ ID NO:8; or comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:8.

[0318] Intracellular signaling domain

[0319] The CARs described herein include an intracellular signaling domain. An "intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in transducing information from the binding of an active CAR to a target antigen into the interior of an immune effector cell to initiate effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to a target cell bound by the CAR, or other cellular responses triggered after antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is from CD3ζ (see, e.g., below). Additional non-limiting examples of intracellular signaling domains containing immunoreceptor tyrosine-based activation motifs (ITAMs) that are particularly used in the art include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3η, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0320] CD3 is a T cell co-receptor that promotes T lymphocyte activation when also participating in appropriate co-stimulation (e.g., binding of co-stimulatory molecules). The CD3 complex consists of 4 different chains; mammalian CD3 consists of a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule called the T cell receptor (TCR) and CD3ζ to generate an activation signal in T lymphocytes. The complete TCR complex includes the TCR, CD3ζ, and the complete CD3 complex.

[0321] In some embodiments of any aspect, the CAR polypeptides described herein include an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif or ITAM from CD3 zeta (CD3ζ) (including variants of CD3ζ such as ITAM-mutated CD3ζ), CD3η, or CD3θ. In some embodiments of any aspect, the ITAM includes three motifs of the ITAM of CD3ζ (ITAM3). In some embodiments of any aspect, the three motifs of the ITAM of CD3ζ are not mutated and thus include a native or wild-type sequence. In some embodiments, the CD3ζ sequence includes the CD3ζ sequence as shown in the sequences provided herein, such as the CD3ζ sequence of SEQ ID NO:9 or a variant thereof.

[0322] For example, the CAR polypeptides described herein include the intracellular signaling domain of CD3ζ. In some embodiments, the CD3ζ intracellular signaling domain corresponds to the amino acid sequence of SEQ ID NO:9; or comprises the sequence of SEQ ID NO:9; or comprises a sequence having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) to the sequence of SEQ ID NO:9.

[0323] As will be appreciated by those of skill in the art, the individual CARs and other construct components described herein can be used together with one another and can be swapped in and out of the various constructs described herein. Each of these components can comprise or consist of any of the corresponding sequences shown herein or variants thereof.

[0324] A more detailed description of CARs and CAR T cells can be found in the following references: Maus et al., Blood 123:2624 - 2635, 2014; Reardon et al., Neuro-Oncology 16:1441 - 1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039 - 3047, 2014; Maher et al., Cancer Res 69:4559 - 4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436 - 6445, 2012; each of which is incorporated herein by reference in its entirety.

[0325] In some embodiments, the CAR polypeptides described herein include a signal peptide. The signal peptide can be derived from any protein having an extracellular domain or being secreted. The CAR polypeptides described herein can include any signal peptide known in the art. In some embodiments, the CAR polypeptide comprises a CD8 signal peptide, such as the CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO:6; or comprising the amino acid sequence of SEQ ID NO:6; or comprising a sequence having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) to the sequence of SEQ ID NO:6.

[0326] In other embodiments, the CAR polypeptides described herein may optionally not include one of the signal peptides described herein, e.g., the CD8 signal peptide of SEQ ID NO:6.

[0327] In one embodiment, the CAR further comprises a linker domain. As used herein, "linker domain" refers to an oligopeptide or polypeptide region that is about 2 to 100 amino acids in length and that joins together any domain / region of the CAR as described herein. In some embodiments, the linker may comprise or consist of flexible residues such as glycine and serine such that adjacent protein domains are free to move relative to each other. Linker sequences useful in the present invention may have a length of 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids, and include any suitable linker known in the art and / or described herein (e.g., the linker sequences of SEQ ID NOs: 3, 14, 15, 16, or 7). Longer linkers may be used when it is desired to ensure that there is no steric interference between two adjacent domains. Additionally, the linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A and T2A), 2A-like linkers, or functional equivalents thereof and combinations thereof. In various examples, a linker having a sequence as shown herein or a variant thereof is used. It should be understood that the indication of a particular linker at a particular position in a construct does not mean that only that linker can be used there. Rather, as can be determined by one of ordinary skill in the art, different linker sequences (e.g., P2A and T2A) may be exchanged with one another (e.g., in the context of the constructs of the present invention). In one embodiment, the linker region is T2A from Thosea asigna virus. Non-limiting examples of linkers that may be used in the art include T2A, P2A, E2A, BmCPV2A, and BmIFV2A.

[0328] In some embodiments, the CARs described herein optionally further comprise a reporter molecule, e.g., to allow non-invasive imaging (e.g., positron emission tomography PET scan). In bispecific CARs that include a reporter molecule, the first extracellular binding domain and the second extracellular binding domain may include different or the same reporter molecule. In bispecific CAR T cells, the first CAR and the second CAR may express different or the same reporter molecule. In another embodiment, the CARs described herein further include a molecule that can be imaged alone or in combination with a substrate or chemical (e.g., 9-[4- 18 F]fluoro-3-(hydroxymethyl)butyl]guanine( 18A reporter molecule (e.g., hygromycin phosphotransferase (hph)) for 64 Cu 2+ functionalized gold nanoparticles (GNPs)) that can be readily imaged using non-invasive techniques. A review of the labeling of CAR T cells for non-invasive imaging is provided, for example, in Bhatnagar et al., Integr. Biol. (Camb). 5(1):231-238, 2013, and Keu et al., Sci. Transl. Med. 18; 9(373), 2017, the disclosures of which are incorporated herein by reference in their entireties.

[0329] GFP and mCherry are shown herein to be fluorescent tags that can be used to image CARs expressed on T cells (e.g., CAR T cells). Any fluorescent protein that is substantially known in the art is expected to be useful as a fluorescent tag for this purpose. For clinical applications, a CAR need not include a fluorescent tag or fluorescent protein. Thus, in each case of the specific constructs provided herein, any label present in the construct can be removed. The invention includes constructs with or without labels. Thus, when a specific construct is referred to herein, it can be considered that the presence or absence of any marker or tag is included within the invention.

[0330] In some embodiments, the CAR polypeptide sequence corresponds to, comprises, or comprises a sequence having at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO: 10, 11, 12, or 13, optionally without the CD8 signal peptide as described herein. As can be determined by one of ordinary skill in the art, the various functionally similar or equivalent components of these CARs can be exchanged or substituted with one another, as well as with other similar or functionally equivalent components known in the art or listed herein.

[0331] Nucleic acid encoding the CAR

[0332] In some embodiments, any of the CAR polypeptides described herein (e.g., the CAR polypeptides of SEQ ID NO: 10, 11, 12, or 13) are encoded by a polynucleotide contained within a viral vector. Optionally, the polynucleotide encoding the CAR polypeptide as described herein can be codon optimized to enhance expression or stability. Codon optimization can be performed according to any standard method known in the art. In some embodiments, expression of the CAR can be driven by a constitutively expressed promoter (e.g., the EF1α promoter) or an inducibly expressed promoter (e.g., the NFAT response element).

[0333] In addition, the polynucleotides of the present invention can include the expression of a suicide gene. Doing so can facilitate externally drug-mediated control of the administered cells. For example, by using a suicide gene, in the case of, for example, adverse events, the modified cells can be depleted from the patient. In one example, an FK506 binding domain is fused to a caspase 9 apoptotic molecule. T cells engineered in this way are made sensitive to the immunosuppressive drug tacrolimus. Other examples of suicide genes are thymidine kinase (TK), CD20, thymidylate kinase, truncated prostate specific membrane antigen (PSMA), truncated low affinity nerve growth factor receptor (LNGFR), truncated CD19, and modified Fas, which can be triggered for conditional ablation by administration of a specific molecule (e.g., ganciclovir for TK+ cells) or an antibody or antibody-drug conjugate.

[0334] Retroviruses, such as lentiviruses, provide a convenient platform for delivering nucleic acid sequences encoding a gene of interest or a chimeric gene. The selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered, for example, in vitro or ex vivo, to cells. Retroviral systems are well known in the art and are described, for example, in U.S. Patent No. 5,219,740; Kurth and Bannert (2010) “Retroviruses: Molecular Biology, Genomics and Pathogenesis” Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512; the disclosures of which are incorporated herein by reference in their entirety. A lentiviral system for efficient DNA delivery can be purchased from OriGene of Rockville, Maryland. In alternative embodiments, the CAR polypeptide of any of the CARs described herein is expressed in mammalian cells via transfection or electroporation of an expression vector containing the nucleic acid encoding the CAR. Transfection or electroporation methods are known in the art.

[0335] Standard assays that detect nucleic acids encoding CAR, such as mRNA, DNA, or gene products (such as RT-PCR, FACS, Northern blot, Western blot, ELISA, or immunohistochemistry) can be used to assess the high-efficiency expression of the CAR polypeptides of any of the CAR polypeptides described herein. In some embodiments, the CAR polypeptides of any of the CAR polypeptides described herein are encoded by recombinant nucleic acid sequences.

[0336] Cell

[0337] Another aspect of the invention relates to a mammalian cell that comprises any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, the mammalian cell comprises an antibody, an antibody reagent, an antigen-binding portion thereof, or any of the CAR polypeptides described herein; or a nucleic acid encoding such an antibody, an antibody reagent, an antigen-binding portion thereof, or any of the CAR polypeptides described herein. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, but any other mammalian cell can be used. In a preferred embodiment of any aspect, the mammalian cell is human.

[0338] In one example, the mammalian cell is an induced pluripotent stem cell (iPSC), for example, as described by Takahashi K and Yamanaka S, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors," Cell. 126(4):663–76 (2006), which is incorporated herein by reference in its entirety. In other embodiments, the cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cell is a T cell; an NK cell; an NKT cell; a lymphocyte, such as a B cell and a T cell; and a myeloid cell, such as a monocyte, a macrophage, an eosinophil, a mast cell, a basophil, and a granulocyte. In one embodiment, the cell is a T cell or a natural killer (NK) cell.

[0339] The mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) can be obtained from a subject having or diagnosed with cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder. For example, the mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) can be obtained from a subject having cancer (e.g., multiple myeloma, smoldering myeloma, or Waldenström macroglobulinemia). In some embodiments, the mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) are obtained from a subject resistant to BCMA therapy. The cells can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells.

[0340] Mammalian cells (e.g., iPSCs or immune cells (e.g., T or NK cells)) that can be used in the present invention include autologous cells, which are obtained from a subject who will subsequently be administered the cells after ex vivo modification and expansion. For example, the mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) can be obtained from an individual having or diagnosed with cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder. Mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells. Mammalian cells that can be used in the present invention include, but are not limited to, iPSCs and immune cells (e.g., T or NK cells).

[0341] Methods for obtaining T cells and NKs are known in the art and can be used for the engineered immune cells described herein. T cells and NK cells are typically obtained from peripheral blood, which is collected from a subject by, for example, venipuncture or by withdrawal through an implanted port or catheter. Optionally, blood can be obtained by a method including leukapheresis, in which leukocytes are obtained from the subject's blood while other blood components are returned to the subject. The blood or leukapheresis product (fresh or cryopreserved) is processed using methods known in the art to enrich for T cells or NK cells. For example, density gradient centrifugation (using, e.g., Ficoll) and / or counterflow centrifugal elutriation can be performed to enrich for monocytes (including T cells or NK cells). In one example, for T cells, a T cell stimulation step can be further performed using, e.g., CD3 / CD28 antibodies coated on magnetic beads or artificial antigen-presenting cells (aAPCs) expressing, e.g., cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below) to stimulate the T cells and deplete other cells (e.g., B cells). The T cells of the enriched T cell preparation can then be genetically modified.

[0342] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as sources of T cells and NK cells. The T cells and NK cells can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, but any other mammalian cells can be used. In certain embodiments in any aspect, the T or NK cells are human.

[0343] Mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) can be engineered to contain any of the CAR polypeptides described herein (e.g., the CAR polypeptides of SEQ ID NO: 10, 11, 12, or 13); or nucleic acids encoding any of the CAR polypeptides described herein (e.g., nucleic acids encoding the CAR polypeptides of SEQ ID NO: 10, 11, 12, or 13).

[0344] In addition, the present invention provides compositions and methods for treating and preventing diseases and disorders including, for example, cancer, autoimmune diseases or disorders, or plasma cell diseases or disorders. These methods include using mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells)) including immune cells (e.g., T cells or NK cells) containing a CAR polypeptide or nucleic acid encoding the CAR as described herein, and administering the modified mammalian cells to a subject to treat, for example, cancer. In some embodiments in any of the foregoing aspects, the modified mammalian cells (e.g., iPSCs or immune cells (e.g., T cells or NK cells) that contain one or more additional modifications as described herein) are stimulated and / or activated prior to administration to the subject.

[0345] Treatment methods

[0346] The anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein can be used to treat diseases or disorders in a subject, such as cancer, autoimmune disorders, or plasma cell diseases or disorders.

[0347] As used herein, the term "cancer" refers to the excessive proliferation of cells, the unique properties of which (loss of normal cell control) result in growth disorder, lack of differentiation, local tissue invasion, and metastasis. As used herein, the term "tumor" refers to the abnormal growth of cells or tissues, for example, of the malignant or benign type. Examples of cancers include, but are not limited to, glioblastoma, prostate cancer, glioma, leukemia, lymphoma, multiple myeloma, or solid tumors such as lung cancer and pancreatic cancer. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALL or CLL. Non-limiting examples of lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, hairy cell leukemia (HCL), and T-cell lymphoma (e.g., peripheral T-cell lymphoma (PTCL), including cutaneous T-cell lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)). In one embodiment, the cancer is DLBCL or follicular lymphoma. Non-limiting examples of solid tumors include adrenocortical tumor, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, endodermal sinus tumor, epitheloid hemangioendothelioma, Ewing sarcoma, germ cell tumor (solid tumor), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, kidney tumor, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms tumor. Solid tumors can be found in bone, muscle, or organs and can be sarcomas or carcinomas.

[0348] As used herein, an "autoimmune disease or disorder" is characterized by the patient's immune system being unable to distinguish foreign cells from healthy cells. This causes the patient's immune system to target their healthy cells, resulting in programmed cell death. Non-limiting examples of autoimmune diseases or disorders include inflammatory arthritis, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, SLE and vasculitis, allergic inflammation such as allergic asthma, atopic dermatitis, and contact hypersensitivity. Other examples of autoimmune diseases or disorders include, but are not limited to, transplant rejection, graft-versus-host disease, hemophilia with factor inhibitors, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis (hypothyroidism), celiac disease, Crohn's disease, and ulcerative colitis, Guillain-Barré syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjögren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), psoriasis, autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, canine polyarthritis, Reiter's syndrome, Takayasu's arteritis, warm antibody autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM).

[0349] Plasma cells are white blood cells produced by B lymphocytes, and their function is to generate and release antibodies required to resist infection. As used herein, a "plasma cell disease or disorder" is characterized by abnormal proliferation of plasma cells. Abnormal plasma cells can "squeeze out" healthy plasma cells, which results in a reduced ability to resist foreign substances such as viral or bacterial cells. Non-limiting examples of plasma cell diseases or disorders include plasma cell proliferative disorders, plasmacytomas, plasma cell leukemia, multiple myeloma, Waldenström's macroglobulinemia, solitary plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

[0350] Exemplary cancers treatable by the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein include cancers comprising cells that express TACI, such as multiple myeloma. Exemplary autoimmune diseases treatable by the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein include autoimmune diseases characterized by high titers of antibodies that contribute to the autoimmune disorder. For example, the autoimmune disease can be transplant rejection, graft-versus-host disease, or hemophilia with factor inhibitors. Plasma cell diseases or disorders treatable by the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein include plasmacytosis, plasmacytoma, plasma cell leukemia, multiple myeloma, Waldenström macroglobulinemia, solitary plasmacytoma, extramedullary plasmacytoma, sclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma. In certain embodiments, the subject is resistant to anti-BCMA therapy.

[0351] administer

[0352] In some embodiments, the methods described herein involve treating a subject having or diagnosed with cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder with mammalian cells comprising any of the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein. For example, the anti-TACI CARs described herein include mammalian cells that comprise any of the anti-TACI CAR polypeptides (and optional antibody reagents or cytokines) described herein or nucleic acids encoding any of the anti-TACI CAR polypeptides (and optional antibody reagents or cytokines) described herein. Subjects having cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder can be identified by a physician using current methods of diagnosing the condition. Signs and / or complications of the condition that are characteristic of and aid in the diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. Tests that can aid in the diagnosis of the condition, for example, include, but are not limited to, blood screening and bone marrow tests and are known in the art for a given condition. A family history of the condition or exposure to risk factors for the condition can also aid in determining whether a subject is likely to have the condition or aid in the diagnosis of the condition.

[0353] The compositions described herein can be administered to a subject having or diagnosed with a disorder. In some embodiments, the methods described herein include administering to a subject an effective amount of the activated CAR T cells described herein to alleviate the symptoms of the disorder. As used herein, "alleviating the symptoms of a disorder" is an improvement in any disorder or symptoms associated with a disorder. This reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique compared to an equivalent untreated control. A variety of ways of administering the compositions described herein are known to those of skill in the art and include oral, parenteral, intralung and intranasal, and (if local treatment is desired) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In one embodiment, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the tumor site. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulse infusion.

[0354] The effective amount, toxicity and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell culture or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. The therapeutically effective dose can initially be estimated from cell culture assays. Additionally, the dosage can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture or in a suitable animal model (i.e., the concentration of activated CAR T cells that achieves half-maximal inhibition of the symptoms). Plasma levels can be measured, for example, by high performance liquid chromatography. In particular, the effects of any particular dose can be monitored by suitable bioassays, such as assays for bone marrow testing. The dosage can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effects.

[0355] In one aspect of the present technology, the technologies described herein relate to pharmaceutical compositions comprising an activated CAR T cell as described herein, and optionally a pharmaceutically acceptable carrier. The active ingredient in the pharmaceutical composition comprises at least an activated CAR T cell as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of an activated CAR T cell as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of an activated CAR T cell as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffer solutions, Ringer's solution, and serum components (such as serum albumin, HDL, and LDL). Terms (such as "excipient", "carrier", "pharmaceutically acceptable carrier", etc.) are used interchangeably herein.

[0356] In some embodiments, a pharmaceutical composition comprising an activated CAR T cell as described herein can be a parenteral dosage form. Since the administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, components other than the CAR T cells themselves are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, ready-to-use injection solutions, anhydrous products that are readily soluble or suspendable in a pharmaceutically acceptable injectable vehicle, ready-to-use injection suspensions, and emulsions. Any of these can be added to the activated CAR T cell preparation prior to administration.

[0357] Suitable vehicles for parenteral dosage forms that can be used to provide the activated CAR T cells as disclosed herein are well known to those skilled in the art. Examples include, but are not limited to: saline solutions; glucose solutions; aqueous vehicles, including but not limited to sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0358] Dose

[0359] As used herein, the term "unit dosage form" refers to a dosage for a suitable single administration. By way of example, a unit dosage form can be a quantity of a therapeutic agent placed in a delivery device (e.g., a syringe or an intravenous drip bag). In one embodiment, the unit dosage form is administered as a single administration. In another embodiment, more than one unit dosage form can be administered simultaneously.

[0360] In some embodiments, the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACICARs described herein are administered as a single therapy, i.e., no other therapy for the disorder is administered concurrently to the subject.

[0361] As a general proposition, a therapeutically effective amount of an anti-TACI antibody, antibody-drug conjugate, and / or BiTE administered to a human will be in the range of about 0.01 to about 100 mg / kg patient body weight, whether administered as a single or multiple administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.1 to about 10 mg / kg, or about 1 to about 10 mg / kg, administered once (single administration) or multiple times (multiple administrations, e.g., daily administration). In one example, the antibody used is about 10 mg / kg, preferably administered orally. In one embodiment, the anti-TACI antibody, antibody-drug conjugate, and / or BiTE described herein is administered to a human at a flat dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose can be administered as a single dose or multiple doses (e.g., 2 or 3 doses), such as by infusion. For repeated administration over several days or longer, depending on the condition, treatment is typically continued until the desired disease symptom suppression occurs. An exemplary dose of the antibody will be in the range of about 0.01 mg / kg to about 10 mg / kg. Such doses can be administered, for example, intermittently weekly or every three weeks (e.g., such that the patient receives about two to about twenty or, for example, about six doses of the anti-TACI antibody, antibody-drug conjugate, and / or BiTE). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of this therapy can be easily monitored by conventional techniques and assays.

[0362] In another aspect, a pharmaceutical composition comprising mammalian cells containing the anti-TACI CAR described herein can generally be administered at 10 4 to 10 9 cells / kg body weight, and in some cases 10 5 to 10 6administered at a dose of cells / kg body weight (including all integer values within those ranges). If desired, the cell composition may also be administered multiple times at these doses. The cells may be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 319:1676, 1988). In some cases, it may be desirable to administer activated CAR T cells to a subject and then subsequently re-draw blood (or perform apheresis), activate T cells from the blood as described herein, and re-infuse these activated and expanded T cells into the patient. This process may be performed multiple times every few weeks. In certain aspects, T cells may be activated from a blood draw of 10 cc to 400 cc. In certain aspects, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. The mode of administration may include, for example, intravenous (i.v.) injection or infusion. The compositions described herein may be administered to a patient by arterial, intratumoral, intranodal, or intramedullary routes. In some embodiments, a composition of T cells may be directly injected into a tumor, lymph node, or site of infection. In one embodiment, the composition described herein is administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0363] In certain exemplary aspects, a subject may undergo leukapheresis, in which white blood cells are collected ex vivo, enriched or depleted to select and / or isolate cells of interest, such as T cells. These T cell isolates may be expanded by contact with artificial APCs (e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs) and processed such that one or more CAR constructs of the present technology can be introduced, thereby generating CAR T cells. A subject in need may then undergo standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. After or in parallel with the transplantation, the subject may receive an infusion of the expanded CAR T cells. In one embodiment, the expanded cells are administered before or after surgery.

[0364] In some embodiments, lymphodepletion is performed on a subject prior to administration of one or more CAR T cells as described herein. In such embodiments, lymphodepletion may include administration of one or more of melphalan, cyclophosphamide (cytoxan), cyclophosphamide, and fludarabine.

[0365] The dose of the above-described treatment to be administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment. Dosing may be scaled for human administration according to practices generally recognized in the art.

[0366] In some embodiments, a single treatment regimen is desired. In other cases, one or more subsequent doses or treatment regimens may be administered. For example, after a three-month treatment every two weeks, the treatment may be repeated monthly for six months or one year or longer. In some embodiments, no additional treatment is administered after the initial treatment.

[0367] The dosage of the compositions described herein can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effect. Regarding the duration and frequency of treatment, a skilled clinician typically monitors the subject to determine when the treatment provides a therapeutic benefit and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by those skilled in the art. In the event of any complications, the dosage can also be adjusted by the individual physician.

[0368] Combination therapy

[0369] As can be determined by those skilled in the art as appropriate, the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein can optionally be used in combination with each other and with other known agents and therapies. In one example, two or more different types of CARs targeting different cancer antigens (e.g., TACI and BCMA) can be administered in combination.

[0370] As used herein, "combinatorial" administration means the delivery of two (or more) different therapies to a subject during the course of a disorder in the subject, e.g., the two or more therapies are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has stopped for other reasons. In some embodiments, when delivery of the second therapy is initiated, delivery of one therapy is still ongoing such that there is an overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery". In other embodiments, delivery of one therapy ends before delivery of the other therapy begins. In some embodiments of either case, the therapies are more effective due to combinatorial administration. For example, the second therapy is more effective compared to administering the second therapy in the absence of the first therapy, e.g., equivalent effects are observed with less of the second therapy, or the second therapy alleviates symptoms to a greater extent, or a similar situation is observed with the first therapy. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with delivery of one therapy in the absence of the other therapy. The effects of the two therapies can be partially additive, fully additive, or more than additive. The delivery can be such that when the second therapy is delivered, the effect of the first therapy that has been delivered is still detectable. The anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the anti-TACI antibodies, antibody-drug conjugates, BiTEs, and / or anti-TACI CARs described herein can be administered first, and then the additional agent can be administered, or the order of administration can be reversed. The anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR therapies and / or other therapeutic agents, procedures, or modalities can be administered during an active disorder, or during a remission or less active period of the disease. The anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR therapies can be administered before, in parallel with, after, or during remission of the disorder of another therapy.

[0371] When administered in combination, the anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR therapy, and the additional agent(s) (e.g., a second or third agent) or all, can be administered in an amount or dose that is higher than, lower than, or equal to the amount or dose of each agent used alone (e.g., as a monotherapy). In certain embodiments, the amount or dose of the activated CAR T cells, additional agent(s) (e.g., a second or third agent) or all administered is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent used alone. In other embodiments, the amount or dose of the activated CAR T cells, additional agent(s) (e.g., a second or third agent) or all that achieves the desired effect (e.g., cancer treatment) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent required alone to achieve the same therapeutic effect. In further embodiments, the activated CAR T cells described herein in a treatment regimen can be used in combination with surgery, chemotherapy, radiation, mTOR pathway inhibitors, immunosuppressive agents (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies, or other immune depleting agents (such as CAMPATH), anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or peptide vaccines (such as those described in Izumoto et al., J. Neurosurg. 108:963-971, 2008).

[0372] In one example, the anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR described herein can be used in combination with checkpoint inhibitors. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors, anti-CTLA4 inhibitors, anti-PDL1 inhibitors, and anti-TIM3 inhibitors.

[0373] In another embodiment, the anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR described herein can be used in combination with chemotherapeutic agents.

[0374] Efficacy

[0375] The efficacy of the anti-TACI antibody, antibody-drug conjugate, BiTE, and / or anti-TACI CAR in treating, for example, the disorders described herein or inducing a response as described herein (such as cancer cell reduction) can be determined by a skilled clinician. However, if, after treatment according to the methods described herein, one or more signs or symptoms of the disorders described herein are altered in a beneficial manner, other clinically acceptable symptoms are improved or even alleviated, or the desired response is induced, for example, to an extent of at least 10%, the treatment is considered "effective treatment" (as the term is used herein). For example, efficacy can be evaluated by measuring markers, indicators, symptoms, and / or incidence rates of the disorder being treated according to the methods described herein or any other appropriate measurable parameter. Treatment according to the methods described herein can result in a reduction in the level of a marker or symptom of the disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0376] Efficacy can also be measured by failure of individual deterioration as evaluated by, for example, hospitalization or the need for medical intervention (i.e., halting disease progression). Methods for measuring these indicators are known to those of skill in the art and / or are described herein.

[0377] Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, e.g., preventing worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount sufficient to result in effective treatment of the disease when administered to a subject in need, as the term is defined herein. The efficacy of a pharmaceutical agent can be determined by evaluating physical indicators of the disorder or the desired response. Monitoring the efficacy of administration and / or treatment by measuring any one or any combination of such parameters is well within the ability of those of skill in the art. The efficacy of a given method can be evaluated in an animal model of the disorder described herein. When using an experimental animal model, the efficacy of treatment is demonstrated when a statistically significant change in a marker is observed.

[0378] All patents and other publications cited throughout this application; including references, issued patents, published patent applications, and co-pending patent applications are hereby expressly incorporated by reference for the purpose of description and disclosure. For example, the methods described in such publications may be used in combination with the techniques described herein. These publications are provided solely because they were disclosed prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior art or any other reason. All statements as to the dates or the content of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or the content of these documents.

[0379] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications will be recognized by those skilled in the relevant art as being within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order or the functions may be performed substantially in parallel. The teachings of the present disclosure provided herein may be suitably applied to other programs or methods. The various embodiments described herein may be combined to provide additional embodiments. If desired, aspects of the present disclosure may be modified to incorporate the components, functions, and concepts of the above references and applications to provide yet another embodiment of the present disclosure. Additionally, due to considerations of biological equivalence, some changes may be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes may be made to the disclosure in accordance with the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0380] The specific elements of any of the foregoing embodiments may be combined or substituted for elements in other embodiments. Additionally, while the advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need to exhibit such advantages to fall within the scope of the present disclosure.

[0381] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting.

[0382] Examples

[0383] The following are examples of useful methods and compositions. It should be understood that various other embodiments may be implemented in accordance with the description provided herein.

[0384] Example 1. Generation of Anti-TACI Antibodies

[0385] Monoclonal antibodies (mAbs) that specifically bind to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) were generated.

[0386] First, mAb-producing cells (i.e., hybridomas) were generated by fusing myeloma cells with B cells. After cell fusion, a large number of clones were screened and selected based on antigen specificity and immunoglobulin class. After identifying candidate hybridoma cell lines, the antibodies were verified and characterized using several downstream functional assays (Figure 1). The mAbs were sequenced and used to generate second-generation chimeric antigen receptors using variable heavy chain (VH) and variable light chain (VL).

[0387] Antibodies were analyzed by surface plasmon resonance according to the following protocol (Figure 2). The binding of his-tagged recombinant TACI to anti-TACI antibodies was captured on anti-mouse IgG to 120 RU (for TACI).

[0388] Capture: The antibody was brought to 1 mg / mL in running buffer and captured on the anti-mouse IgG surface (CM5 chip, 10,000 RU anti-IgG antibody immobilized by standard amine-directed chemistry according to the manufacturer's instructions, GE Healthcare, catalog number BR-1008-38) to reach 130 RU for TACI analysis.

[0389] Running buffer: PBS-P (10 mM sodium phosphate, 150 mM NaCl, 0.005% Tween 20, pH 7.4).

[0390] Regeneration buffer: 10 mM glycine, pH 2.1

[0391] Flow rate and injection protocol: Flow rate: 50 μL / min, 2 min contact time, 5 min dissociation

[0392] The results of the assay are shown in Figure 2. Results: k a (Association rate constant) = 1.407 x 10 6 1 / Ms, k d (Dissociation rate constant) = 0.00121 1 / s, K D (Equilibrium dissociation constant) = 8.61 x 10 -10 M, Rmax (maximum binding (fit)) = 31.28 RU and Chi 2 = 0.943 RU 2 . RU: Resonance unit.

[0393] Example 2. Design of anti-TACI chimeric antigen receptor (CAR)

[0394] Design the second-generation chimeric antigen receptor (CAR) as shown in Figure 3. Two anti-TACI CAR constructs were generated, one with VH at the N-terminus of the VL of the anti-TACI single-chain variable fragment (scFv) (anti-TACI (H / L)), and the other with VL at the N-terminus of the VH of the anti-TACI scFv (anti-TACI (L / H)). The VH and VL sequences included in the CAR were derived from the anti-TACI antibody described in Example 1. Each CAR also included a CD8 hinge / transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. The CAR was expressed in T cells according to standard methods (see Example 3 and subsequent sections).

[0395] To test the potential effect of the anti-TACI antibody on immune cells, peripheral blood mononuclear cells (PBMCs) were incubated with the anti-TACI antibody, and binding was measured by flow cytometry. The results demonstrated that the anti-TACI antibody did not bind to isolated T cells (resting or activated) or various other PBMCs (Figure 23). K562 overexpressing TACI was used as a positive control.

[0396] To evaluate the expression, binding efficacy, and binding specificity of the anti-TACI CAR, normal donor T cells were transduced with anti-TACI H-L, anti-TACI L-H, or anti-BCMA, and the transduction efficiency was evaluated by measuring the percentage of each T cell population that was positive for mCherry expression by flow cytometry. The results demonstrated that the transduction efficiency of both anti-TACI CAR constructs was high and similar to that of the anti-BCMA CAR (Figure 24). To compare the binding efficiency and specificity of different CARs, T cells expressing each CAR construct were incubated with fluorescently labeled soluble TACI and analyzed by flow cytometry. The results demonstrated that the anti-TACI L-H CAR had a higher binding affinity for soluble TACI than the anti-TACI H-L CAR, and the anti-BCMA CAR showed no binding to the soluble TACI protein (Figure 25A and Figure 25B).

[0397] To further evaluate the specificity of anti-TACI CAR for TACI, MM1S cancer cells expressing Cas9 were generated. Cells expressing TACI or lacking TACI via Cas9 knockout were generated. Figure 27A shows that Cas9 knockout of TACI was successful. MM1S Cas9 and MM1S Cas9 TACI knockout (KO) cells were incubated with T cells that were untransduced or transduced with anti-TACI H-L, anti-TACI L-H, or anti-BCMA, and cytotoxicity was measured. The results demonstrated that anti-TACI CAR was specific for TACI and that T cells expressing it had little efficacy against TACI KO cells, and only the positive control anti-BCMA CAR T cells had significant cytotoxicity against TACI KO cells (Figure 27B).

[0398] Example 3. BCMA and TACI Antigen Expression

[0399] The multiple myeloma cell line MM1S was used in the experiments described herein. MM1S expresses both TACI and B cell maturation antigen (BCMA). A BCMA knockout MM1S cell line, as well as a K562 cell line expressing TACI and a K562 cell line expressing BCMA, were generated (Figure 4).

[0400] Since TACI is expressed in activated T cells, it was evaluated whether resting CAR T cells or activated CAR T cells (activated with beads) expressed TACI or upregulated the expression of TACI. As shown in Figure 5, there was some expression on resting cells, but the change was very small when CAR T cells were activated.

[0401] Example 4. Transfection Efficiency and Killing Activity of Anti-TACI CAR T Cells

[0402] The transfection efficiency of anti-TACI CAR T cells was measured by quantification of mCherry-positive cells, since CAR T cells had been engineered to express mCherry (see Figure 3). As shown in Figure 6, the transfection efficiency of anti-BCMA-BBz CAR was very similar to that of the two TACI CARs. The target multiplicity of infection (MOI) = 10.

[0403] Cytotoxicity assays were performed to evaluate the activity of anti-TACI (H / L) and (L / H) CAR T cells compared to anti-BCMA CAR and untransduced (UTD) T cells. The results of the assays are shown in Figure 7. The CAR T cells were incubated overnight with target cells at different effector:target (E:T) ratios as shown in Figure 7. The target cells used were MM1S cells that express both BCMA and TACI. The results demonstrated that both anti-BCMA CAR and anti-TACI CAR T cells are excellent killers. At low E:T ratios, anti-TACI (L / H) CAR T cells appeared to be more effective.

[0404] A similar cytotoxicity assay was performed using the MM1S BCMA knockout cell line generated in Example 3 as the target cell with anti-BCMA CAR, anti-TACI (H / L) CAR, anti-TACI (L / H) CAR, and UTD T cells as described above. It was observed that anti-BCMA CAR T cells were unable to kill the MM1S BCMA knockout cell line, while both anti-TACI CAR T cells specifically recognized TACI and thus killed the target (Figure 8).

[0405] Example 5. Immunophenotypic analysis of CAR T cells

[0406] Figure 9 shows T cells labeled for CD45RO and CCR7, which allows the characterization of 4 different functional populations: naive, central memory (CM), effector memory (EM), and late effector (EMRA) T cells. As is commonly observed with second-generation 4-1BB CAR, the CM population of T cells was enriched. Similar results were observed when anti-BCMA CAR was compared to the two anti-TACI CARs.

[0407] The population important for long-term memory in the immune system described previously is the T stem cell memory (TSCM) population, which is defined by specific markers from the naive population (CD45-CCR7+) shown in Figure 9. The markers unique to the TSCM population are CD95 and CD127. Figure 10 shows that anti-TACI CAR T cells have the TSCM population.

[0408] Example 6. Degranulation and specific binding of CAR T cells

[0409] BCMA and TACI CAR T cells were stained with anti-CD107a antibody (protein transport inhibitor added) and placed separately (negative control: -CL), co-incubated with different targets, or activated with PMA / ionomycin (positive control: +CL) for 6 hours. Flow cytometry was performed and live CAR T cells were gated to evaluate the expression of CD107a, a marker of cytotoxic degranulation. The results are shown in Figure 11. Specific degranulation of anti-TACI CAR T cells in response to TACI+ targets was observed, but not for BCMA+ / TACI- targets.

[0410] The ability of anti-BCMA, anti-TACI (H / L), and anti-TACI (L / H) CAR T cells to bind soluble TACI (sTACI), soluble BCMA (sBCMA), and soluble APRIL (sAPRIL) was evaluated. T cells were washed 4 times in PBS 4% BSA and incubated with the labeled proteins on ice at 4°C for 40 min. After incubation, the cells were washed twice with FACS buffer and flow cytometry was performed with gating on live CAR T cells to calculate protein binding. The results are shown in Figures 12A - 12C. As shown in Figure 12A, the binding efficiency of anti-TACI (L / H) CAR T cells to soluble TACI was higher than that of anti-TACI (H / L) CAR T cells. In Figure 12B, negligible binding of both anti-TACI CAR T cells to BCMA was shown. Finally, Figure 12C shows similar binding efficiencies of anti-BCMA and both anti-TACI CAR T cells to soluble APRIL.

[0411] Example 7. Cytokine production and long-term proliferation of anti-TACI CAR T cells

[0412] The cytokine production of anti-BCMA and two anti-TACI CAR T cells was evaluated by incubating the CAR T cells with the designated targets (MM1S or MM1S BCMA knockout cell lines). Untransduced cells were used as controls. The levels of cytokine production are shown in FIGS. 13A and 13B. It was shown that the anti-TACI CAR T cells produced cytokines in response to MM1S and MM1S BCMA knockout cells. Similarly, the cytokine production of anti-BCMA and two anti-TACI CAR T cells was evaluated, this time using the K562-TACI and K562-BCMA cell lines as targets (FIGS. 13C and 13D). The two anti-TACI CAR T cells produced more cytokines in response to the TACI-expressing target than they did in response to the BCMA-expressing target. Finally, the cytokine production of the CAR T cells was evaluated using another multiple myeloma cell line, U266 cells. The results are shown in FIG. 13E.

[0413] In addition, it has been demonstrated that anti-TACI CAR T cells can undergo long-term proliferation in vitro when stimulated with K562 target cells. The CAR T cells were stimulated on the days indicated by the arrows, and the population doublings of the CAR T cells are shown in FIG. 14.

[0414] Example 8. Anti-TACI CAR T Cells In Vivo

[0415] The experimental design of the in vivo experiment using anti-TACI CAR T cells is shown in FIG. 15. One million MM1S cells were injected intravenously (i.v.) into mice. Two weeks later, 2 million CAR T cells or UTD cells were injected into the mice. Images were taken twice a week and bioluminescence was quantified. The bioluminescence images are shown in FIGS. 16A and 16B. In FIG. 17, the bioluminescence was quantified in photons / second and presented in four graphs for each type of CAR T cell used.

[0416] As shown in FIG. 18, anti-TACI (L / H) CAR T cells were as effective as anti-BCMA CAR T cells in treating BCMA+ / TACI+ multiple myeloma. FIG. 19 shows the ability of anti-TACI CAR T cells to cure TACI+ multiple myeloma.

[0417] The design of an additional in vivo experiment using anti-TACI CAR T cells is shown in FIG. 26A. 5 x 10 6 RPMI8226 cells were implanted subcutaneously into mice. Two weeks later, 2 x 10 6CAR T cells or untransduced cells, or no treatment of the mice. At 4, 7, 10, and 14 days after CAR T treatment, tumor volume was measured by caliper. Individual tumor growth curves are shown in FIGS. 26B, 26C, 26D, 26E, and 26F. FIG. 26G shows the survival of mice from each treatment group. The results demonstrated that anti-TACI CAR T cells effectively inhibited tumor growth and prolonged survival in vivo in a subcutaneous tumor growth model, and indicated that the L-H construct might be more effective than the H-L construct. FIG. 26H further shows that anti-TACI L-H CAR T cells showed expansion in the blood at day 21. Anti-BCMA CAR T cells served as a positive control, and untransduced and tumor-only groups served as negative controls.

[0418] Example 9. Overcoming Antigen Loss in Hematological Malignancies with CAR T Cell Therapy

[0419] Currently, in addition to CAR19 (CAR targeting CD19), one of the most promising candidates for CAR T cell therapy is the treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA), a member of the TNF receptor superfamily. Multiple myeloma accounts for 13% of all hematological malignancies, and the clinical need for new therapies is unmet. Currently, the reported median progression-free survival of BCMA CARs in the clinic is 11.8 months, indicating that targeting BCMA alone may not be sufficient. Similar to what has been observed with CAR19 treatment and CD19-negative relapses, there is evidence that patients treated with BCMA-targeted therapies may be hindered by BCMA-negative relapses. In CD19+ malignancies, there have been many approaches to overcome CD19-negative malignancies, including tandem CAR T cells targeting two antigens such as CD19 and CD20 or CD19 and CD22. In multiple myeloma, a possible target in addition to BCMA is transmembrane activator and CAML interactor (TACI). Like BCMA, TACI is also a member of the TNF receptor superfamily and provides a survival signal to plasma cells. This antigen has been targeted with CAR T cells using its natural ligand, a proliferation-inducing ligand (APRIL), which also recognizes BCMA. There are currently no published CAR T cells that target only TACI. To date, there has been no direct comparison of scFv-based dual-targeting CAR T cells with natural ligand designs.

[0420] Generation of anti-TACI CAR by immunizing mice and constructing from the resulting hybridomas A novel CAR T cell targeting TACI is designed and provided in the present disclosure. Based on the orientation of the variable heavy chain and variable light chain, there are two forms of anti-TACI CAR (Figure 20A). Anti-TACI CAR is functional in vitro against multiple myeloma lines MM1S and RPMI-8226 that are positive for both BCMA and TACI, and has much lower activity against the TACI-low myeloma line U266 (Figures 20B - 20C). In a transgenic model of multiple myeloma, the L-H orientation of the scFv cures faster, and the L-H CAR binds soluble TACI more effectively in vitro (Figures 20D - 20E).

[0421] Although anti-TACI CAR is functional in the presence of the TACI antigen, they lose efficacy upon antigen loss, similar to BCMA CAR (Figures 21D - 21E). To overcome the potential loss of antigen, which was the root cause of CAR19 failure, tandem bispecific CAR T cells were designed that target both BCMA and TACI using an anti-TACI scFv (VH-VL configuration). The modified bispecific BCMA and TACI CARs were also designed based on their natural ligand APRIL (Figure 21A). This construct, named TriPRIL, has three repeats of a truncated APRIL ligand to form a trimer, which has been shown to be its secreted form. It is not clear which design of the bispecific CAR, tandem bispecific CAR, or natural ligand will be a more effective therapy. Despite the large vector size, the transduction efficiency of the bispecific CAR construct is comparable to that of the single-chain scFv in normal donor T cells (Figure 21B). In vitro, the cytotoxicity of the tandem bispecific CAR and TriPRIL against the MM1S and RPMI-8226 myeloma lines is comparable (Figure 21C). The anti-TACI-anti-BCMA bispecific and TriPRIL showed the same functionality against the multiple myeloma line U266 with low TACI expression (Figure 20B). The anti-BCMA-anti-TACI bispecific was less effective. For the case of antigen loss, the loss of BCMA showed the greatest difference, where the anti-TACI-anti-BCMA bispecific lost some efficacy (Figures 21D - 21E). Similarly, supernatants were harvested from these assays to evaluate cytokine production.

[0422] Example 10. Bispecific CAR T Cells Against Multiple Myeloma: Comparison of Natural Ligand and Tandem scFv Design

[0423] Example 9 showed that bispecific BCMA- and TACI-targeting CARs designed based on tandem scFv or native ligands had similar efficacy against wild-type multiple myeloma models. However, this changed in the case of single antigen loss. The proliferation (population doublings) and activation capacity (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotypes (PD-1, Tim3, LAG-3), were characterized upon long-term exposure to a single antigen. The results showed that tandem bispecific CARs and native ligand CARs differed in their sensitivity to antigen density. The results indicated that the structural differences between bispecific CAR T cells affected their function.

[0424] To test the efficacy of bispecific CAR T cells in binding their respective antigens, T cells were either not transduced or transduced with anti-TACI H-L CAR, anti-TACI L-H CAR, anti-BCMA CAR, anti-TACI / anti-BCMA bispecific CAR, or anti-BCMA / anti-TACI bispecific CAR, and their binding to soluble antigens was tested. The results demonstrated that bispecific CARs had lower binding affinities for their soluble antigens compared to their monospecific counterparts, and the orientation of the two components of the bispecific CARs tested did not affect their binding (Figure 28).

[0425] To test the effect of bispecific CARs on cytokine production in cancer cell / T cell co-cultures, MM1S or RPMI8226 multiple myeloma cells were cultured alone or co-cultured with untransduced T cells, T cells transduced with anti-TACI / anti-BCMA bispecific CAR, or T cells transduced with anti-BCMA / anti-TACI bispecific CAR, and cytokines were measured in the culture supernatants. The results showed that bispecific CAR T cells produced interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor α (TNF-α) in response to multiple myeloma cell lines in vitro (Figure 29).

[0426] The design of additional experiments using bispecific CAR T cells in vivo is shown in Figure 30A. First, 1 x 10 6 MM1S multiple myeloma cells were injected intravenously (i.v.) into mice. Then, 2 weeks later, 2 x 10 6CAR T cells were used and bioluminescence imaging was performed to detect the viability of MM1S cells over time. For T cells from three different donors, measurements of bioluminescence flux over time in mice untreated or treated with untransduced T cells or T cells transduced with an anti-TACI / anti-BCMA bispecific CAR or an anti-BCMA / anti-TACI bispecific CAR are shown in FIGS. 30B, 30C, and 30D. Representative bioluminescence images from donor 1 are shown in FIG. 30E. The results indicate that bispecific CAR T cells are functional against MM1S in vivo, and that anti-BCMA / anti-TACI CAR T cells from 2 out of 3 donors are effective, and anti-TACI / anti-BCMA CAR T cells from all 3 donors are effective.

[0427] Bispecific CAR T cells showed further efficacy in a subcutaneous tumor model of multiple myeloma. FIG. 31A shows the experimental design, in which 5 x 10 6 RPMI8226 cells were implanted subcutaneously into mice, and then CAR T cells were injected intravenously 2 weeks later. Tumor volume was measured over time, and CAR T cells in the blood were counted on days 14 and 21 after injection. The results demonstrated that anti-BCMA / anti-TACI CAR T cells expanded more in vivo than anti-TACI / anti-BCMA CAR T cells or untransduced T cells (FIG. 31B), and that both CAR T cell types inhibited subcutaneous tumor growth relative to untransduced T cells or tumors in untreated mice (FIGS. 31C, 31D, 31E, and 31F). T cell exhaustion can lead to ineffectiveness of CAR T cells. To evaluate the exhaustion of bispecific CAR T cells, experiments were conducted according to the timeline shown in FIG. 32A. First, 1 x 10 6 untransduced or T cells transduced with an anti-BCMA CAR, an anti-TACI / anti-BCMA bispecific CAR, or an anti-BCMA / anti-TACI bispecific CAR construct were plated. Flow cytometry analysis of Tim-3 and Lag3 exhaustion markers was performed on days 0, 7, 14, 21, and 28 after initial plating. At each time point, the T cells were counted, plated at 1 x 10 6 cells per condition, and (re)stimulated with irradiated K562 cells overexpressing BCMA. The results showed that anti-BCMA / anti-TACI bispecific CAR T cells showed lower expression of the exhaustion markers Tim-3 and Lag3 upon repeated stimulation compared to anti-BCMA and anti-TACI / anti-BCMA bispecific CAR T cells, which may result in stronger efficacy against a single antigen.

[0428] Evaluate the T cell phenotype within the CAR T cell population according to the experimental design summarized in Figure 33A. CAR T cells expressing anti-BCMACAR, anti-TACI / anti-BCMA bispecific CAR, or anti-BCMA / anti-TACI bispecific CAR were counted and plated at 1x 10 6 cells, and the memory phenotype was quantified by flow cytometry weekly. CAR T cells were characterized according to the four phenotypes shown on the left side of Figure 33B. If the cells were positive for CCR7 and negative for CD45RA, the cells were classified as central memory cells (CM); if they were negative for both CCR7 and CD45RA, they were classified as effector memory cells (EM); if they were negative for CCR7 and positive for CD45RA, they were classified as terminally differentiated effector cells (TDE); or if they were positive for both CCR7 and CD45RA, they were classified as naive cells. See X. Wang et al., Blood (2016) 127(24):2980-2990; L. Gattinoni et al., Nat. Med. (2011) 17(10):1290-1297; and L. Biaseo et al., Sci. Transl. Med. (2015) 7(273):273ra213. The results showed that a greater proportion of anti-BCMA / anti-TACI bispecific CAR T cells retained the central memory phenotype compared to anti-TACI / anti-BCMA bispecific CAR T cells or anti-BCMA CAR T cells, while they converted to the effector phenotype within the same time frame (Figure 33B). Little difference in expansion was observed between the groups (Figure 33C).

[0429] Table 2. Amino acid sequences

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437] Table 3. Complementary determining region (CDR) definitions

[0438] <![CDATA[IMGT 1 > <![CDATA[Kabat 2 > <![CDATA[Chothia 3 > CDR-H1 27-38 31-35 26-32 CDR-H2 56-65 50-65 53-55 CDR-H3 105-116 / 117 95-102 96-101 CDR-L1 27-38 24-34 26-32 CDR-L2 56-65 50-56 50-52 CDR-L3 105-116 / 117 89-97 91-96

[0439] 1 the international ImMunoGeneTics information imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)

[0440] 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0441] 3 Chothia et al., J. Mol. Biol. 196:901-917 (1987)).

[0442] Table 4. Anti-TACI complementary determining region (CDR) amino acid sequence

[0443]

[0444] Some embodiments of the technology described herein may be defined according to any of the following numbered paragraphs:

[0445] 1. An isolated antibody that specifically binds to a transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI), wherein the antibody binds to the TACI with a K of about 2 nM or less. D Combined with TACI.

[0446] 2. The antibody of claim 1, wherein the antibody has a K between about 500 pM and about 1 nM. D Combined with TACI.

[0447] 3. The antibody according to paragraph 1 or 2, wherein the antibody has a K between about 700 pM and about 900 pM. D Combined with TACI.

[0448] 4. The antibody according to any one of paragraphs 1-3, wherein the antibody has a K of about 861 pM. D Combined with TACI.

[0449] 5. An isolated antibody that specifically binds to TACI, wherein the antibody comprises a heavy chain variable domain (VH) that contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; and / or a light chain variable domain (VL) that contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0450] 6. The antibody according to paragraph 5, wherein the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0451] 7. The antibody according to paragraph 5, wherein the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0452] 8. The antibody according to paragraph 5, wherein the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0453] 9. The antibody according to paragraph 5, wherein the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0454] 10. An isolated antibody that specifically binds to TACI, wherein the antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 2.

[0455] 11. The antibody according to any one of paragraphs 1-10, wherein the antibody is a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.

[0456] 12. The antibody according to paragraph 11, wherein the antibody is a monoclonal antibody.

[0457] 13. The antibody according to any one of paragraphs 1-12, wherein the antibody is a full-length antibody.

[0458] 14. An antibody according to any one of paragraphs 1-12, wherein the antibody is an antibody fragment that specifically binds to TACI.

[0459] 15. An antibody according to paragraph 14, wherein the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0460] 16. An antibody according to any one of paragraphs 1-15, wherein the antibody is an IgG antibody.

[0461] 17. An antibody according to paragraph 16, wherein the antibody is an IgG1 antibody.

[0462] 18. A composition comprising an antibody according to any one of paragraphs 1-17.

[0463] 19. A polynucleotide encoding an antibody according to any one of paragraphs 1-17.

[0464] 20. A vector comprising the polynucleotide according to paragraph 19.

[0465] 21. A host cell comprising the vector according to paragraph 16.

[0466] 22. A host cell according to paragraph 21, wherein the host cell is a mammalian cell.

[0467] 23. A host cell according to paragraph 22, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0468] 24. A host cell according to paragraph 21, wherein the host cell is a prokaryotic cell.

[0469] 25. A host cell according to paragraph 24, wherein the prokaryotic cell is Escherichia coli.

[0470] 26. A method for producing an antibody that specifically binds to TACI, the method comprising culturing a host cell according to any one of paragraphs 21-25 in a medium.

[0471] 27. A method according to paragraph 22, wherein the method further comprises recovering the antibody from the host cell or the medium.

[0472] 28. An antibody-drug conjugate comprising an antibody according to any one of paragraphs 1-27.

[0473] 29. A chimeric antigen receptor (CAR) polypeptide, the chimeric antigen receptor polypeptide comprising an extracellular target-binding domain, wherein the extracellular target-binding domain comprises a TACI-binding domain.

[0474] 30. The CAR polypeptide according to paragraph 29, wherein the CAR polypeptide comprises a transmembrane domain and an intracellular signaling domain.

[0475] 31. The CAR polypeptide according to paragraph 29 or 30, wherein the CAR polypeptide further comprises one or more co-stimulatory domains.

[0476] 32. The CAR polypeptide according to any one of paragraphs 29-31, wherein the TACI binding domain does not comprise APRIL, BAFF, CAMLG or a part thereof.

[0477] 33. The CAR polypeptide according to any one of paragraphs 29-32, wherein the TACI binding domain binds to TACI with a K D of about 2 nM or lower.

[0478] 34. The CAR polypeptide according to any one of paragraphs 29-33, wherein the TACI binding domain binds to TACI with a K D in the range of about 500 pM to about 1 nM.

[0479] 35. The CAR polypeptide according to any one of paragraphs 29-34, wherein the TACI binding domain binds to TACI with a K D in the range of about 700 pM to about 900 pM.

[0480] 36. The CAR polypeptide according to any one of paragraphs 29-35, wherein the TACI binding domain binds to TACI with a K D of about 861 pM.

[0481] 37. The CAR polypeptide according to any one of paragraphs 29-36, wherein the TACI binding domain comprises an antibody or an antigen-binding fragment thereof.

[0482] 38. A CAR polypeptide comprising an extracellular target-binding domain, wherein the extracellular target-binding domain comprises an antibody or an antigen-binding fragment thereof according to any one of paragraphs 1-17.

[0483] 39. The CAR polypeptide according to any one of paragraphs 29-38, wherein the TACI binding domain comprises an anti-TACI single-chain variable fragment (scFv).

[0484] 40. The CAR polypeptide according to paragraph 39, wherein the anti-TACI scFv comprises a heavy-chain variable domain (VH) that contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0485] 41. The CAR polypeptide according to paragraph 40, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1.

[0486] 42. The CAR polypeptide according to any one of paragraphs 39-41, wherein the anti-TACI scFv comprises a light chain variable domain (VL) that contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0487] 43. The CAR polypeptide according to paragraph 42, wherein the VL comprises the amino acid sequence of SEQ ID NO: 2.

[0488] 44. The CAR polypeptide according to any one of paragraphs 39-43, wherein the anti-TACI scFv comprises a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 2.

[0489] 45. The CAR polypeptide according to any one of paragraphs 40-44, wherein the VH is located at the N-terminus of the VL.

[0490] 46. The CAR polypeptide according to any one of paragraphs 40-44, wherein the VL is located at the N-terminus of the VH.

[0491] 47. The CAR polypeptide according to any one of paragraphs 40-46, wherein the VH and the VL are connected via a linker sequence.

[0492] 48. The CAR polypeptide according to paragraph 47, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 3, 14, 15, 16 or 17.

[0493] 49. The CAR polypeptide according to paragraph 48, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 3.

[0494] 50. The CAR polypeptide according to any one of paragraphs 29-49, wherein the TACI binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4 or 5.

[0495] 51. The CAR polypeptide according to paragraph 49 or 50, wherein the TACI binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0496] 52. The CAR polypeptide according to any one of paragraphs 29-51, wherein the transmembrane domain comprises a hinge / transmembrane domain.

[0497] 53. The CAR polypeptide according to paragraph 52, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of the immunoglobulin-like proteins CD28, CD8 or 4-1BB.

[0498] 54. The CAR polypeptide according to paragraph 53, wherein the hinge / transmembrane domain is the hinge / transmembrane domain of CD8, optionally wherein the hinge / transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO:7.

[0499] 55. The CAR polypeptide according to any one of paragraphs 29-54, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, CD3ε or CD3θ.

[0500] 56. The CAR polypeptide according to paragraph 55, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, optionally wherein the intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO:9.

[0501] 57. The CAR polypeptide according to any one of paragraphs 31-56, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, CD28, CD27, ICOS or OX40.

[0502] 58. The CAR polypeptide according to paragraph 57, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, optionally wherein the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO:8.

[0503] 59. The CAR polypeptide according to any one of paragraphs 29-58, wherein the CAR polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:10, 11, 12 or 13.

[0504] 60. The CAR polypeptide according to any one of paragraphs 29-58, wherein the extracellular target-binding domain further comprises a target-binding domain that binds to a second target other than TACI.

[0505] 61. The CAR polypeptide according to paragraph 60, wherein the second target is B cell maturation antigen (BCMA).

[0506] 62. The CAR polypeptide according to paragraph 60 or 61, wherein the target-binding domain comprises a ligand of the second target.

[0507] 63. The CAR polypeptide according to paragraph 60 or 61, wherein the target binding domain comprises an antibody or an antigen-binding fragment thereof.

[0508] 64. The CAR polypeptide according to paragraph 63, wherein the antibody or the antigen-binding fragment thereof comprises a scFv.

[0509] 65. The CAR polypeptide according to paragraph 64, wherein the scFv is an anti-BCMA scFv.

[0510] 66. The CAR polypeptide according to paragraph 65, wherein the anti-BCMA scFv is located at the N-terminus of the anti-TACI scFv.

[0511] 67. The CAR polypeptide according to paragraph 65, wherein the anti-TACI scFv is located at the N-terminus of the anti-BCMA scFv.

[0512] 68. A CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 10.

[0513] 69. A CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0514] 70. A CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

[0515] 71. A CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 13.

[0516] 72. A polynucleotide encoding the CAR polypeptide according to any one of paragraphs 29-71.

[0517] 73. The polynucleotide according to paragraph 72, further comprising a suicide gene.

[0518] 74. The polynucleotide according to paragraph 72 or 73, further comprising a sequence encoding a signal sequence.

[0519] 75. A mammalian cell comprising the CAR polypeptide according to any one of paragraphs 29-71 and / or the polynucleotide according to any one of paragraphs 72-74.

[0520] 76. A mammalian cell according to paragraph 75, wherein the mammalian cell is an induced pluripotent stem cell (iPSC).

[0521] 77. The mammalian cell according to paragraph 75, wherein the mammalian cell is an immune cell.

[0522] 78. The mammalian cell according to paragraph 77, wherein the immune cell is a T cell or a natural killer (NK) cell.

[0523] 79. The mammalian cell according to any one of paragraphs 75 - 78, wherein the mammalian cell is a human cell.

[0524] 80. A bispecific antibody that binds to TACI and CD3, wherein the bispecific antibody comprises a TACI - binding domain and a CD3 - binding domain.

[0525] 81. The bispecific antibody according to paragraph 80, wherein the TACI - binding domain binds to TACI with a K D of about 2 nM or less.

[0526] 82. The bispecific antibody according to paragraph 80 or 81, wherein the TACI - binding domain binds to TACI with a K D between about 500 pM and about 1 nM.

[0527] 83. The bispecific antibody according to any one of paragraphs 80 - 82, wherein the TACI - binding domain binds to TACI with a K D between about 700 pM and about 900 pM.

[0528] 84. The bispecific antibody according to any one of paragraphs 80 - 83, wherein the TACI - binding domain binds to TACI with a K D of about 861 pM.

[0529] 85. The bispecific antibody according to any one of paragraphs 80 - 84, wherein the TACI - binding domain comprises a VH containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1; and / or a VL containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0530] 86. A bispecific antibody that specifically binds to TACI and CD3, wherein the bispecific antibody comprises a TACI - binding domain and a CD3 - binding domain, and the TACI - binding domain comprises a VH containing the amino acid sequence of SEQ ID NO:1 and a VL containing the amino acid sequence of SEQ ID NO:2.

[0531] 87. The bispecific antibody according to any one of paragraphs 80 - 86, wherein the TACI - binding domain is located at the N - terminus of the CD3 - binding domain.

[0532] 88. The bispecific antibody according to any one of paragraphs 80 - 86, wherein the CD3 - binding domain is located at the N - terminus of the TACI - binding domain.

[0533] 89. The bispecific antibody according to any one of paragraphs 80 - 88, wherein the TACI - binding domain and the CD3 - binding domain are connected by a linker sequence.

[0534] 90. The bispecific antibody according to paragraph 89, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO:3, 14, 15, 16 or 17.

[0535] 91. The bispecific antibody according to any one of paragraphs 80 - 90, wherein the bispecific antibody is a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.

[0536] 92. The bispecific antibody according to paragraph 91, wherein the bispecific antibody is a monoclonal antibody.

[0537] 93. The bispecific antibody according to any one of paragraphs 80 - 92, wherein the bispecific antibody is a full - length antibody.

[0538] 94. The bispecific antibody according to any one of paragraphs 80 - 92, wherein the bispecific antibody is an antibody fragment that specifically binds to TACI and CD3.

[0539] 95. The antibody according to paragraph 94, wherein the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv and (Fab')2 fragments.

[0540] 96. The bispecific antibody according to any one of paragraphs 80 - 95, wherein the bispecific antibody is an IgG antibody.

[0541] 97. The bispecific antibody according to paragraph 96, wherein the bispecific antibody is an IgG1 antibody.

[0542] 98. A composition comprising the bispecific antibody according to any one of paragraphs 80 - 97.

[0543] 99. A polynucleotide encoding the bispecific antibody according to any one of paragraphs 80 - 97.

[0544] 100. A vector comprising the polynucleotide according to paragraph 99.

[0545] 101. A host cell comprising the vector according to paragraph 100.

[0546] 102. The host cell according to paragraph 101, wherein the host cell is a mammalian cell.

[0547] 103. The host cell according to paragraph 102, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0548] 104. The host cell according to paragraph 101, wherein the host cell is a prokaryotic cell.

[0549] 105. The host cell according to paragraph 104, wherein the prokaryotic cell is Escherichia coli.

[0550] 106. A method for producing a bispecific antibody that specifically binds TACI and CD3, the method comprising culturing the host cell according to any one of paragraphs 101-105 in a medium.

[0551] 107. The method according to paragraph 106, wherein the method further comprises recovering the bispecific antibody from the host cell or the medium.

[0552] 108. A method for treating a disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject one or more of the following: (i) a CAR polypeptide according to any one of paragraphs 29-71, a polynucleotide according to any one of paragraphs 19, 72-74, and 99, and / or a mammalian cell according to any one of paragraphs 22, 23, 75-79, 102, and 103; (ii) an antibody according to any one of paragraphs 1-17; (iii) an antibody-drug conjugate according to paragraph 28; and (iv) a bispecific antibody according to any one of paragraphs 80-97.

[0553] 109. The method according to paragraph 108, wherein the disease or disorder is cancer, an autoimmune disorder, or a plasma cell disease or disorder.

[0554] 110. The method according to paragraph 109, wherein the disease or disorder is cancer.

[0555] 111. The method according to paragraph 110, wherein the cancer comprises cells that express TACI.

[0556] 112. The method according to paragraph 111, wherein the cancer is multiple myeloma.

[0557] 113. The method according to any one of paragraphs 110-112, wherein the subject is resistant to anti-BCMA therapy.

[0558] 114. The method according to paragraph 109, wherein the disease or disorder is an autoimmune disease or disorder.

[0559] 115. The method according to paragraph 114, wherein the autoimmune disease or disorder is characterized by a high titer of antibodies that contribute to the autoimmune disorder.

[0560] 116. The method according to paragraph 114 or 115, wherein the autoimmune disease or disorder is transplant rejection, graft-versus-host disease, or hemophilia with factor inhibitors.

[0561] 117. The method according to paragraph 109, wherein the disease or disorder is a plasma cell disease or disorder.

[0562] 118. The method according to paragraph 117, wherein the plasma cell disease or disorder is plasmocytosis, plasmacytoma, plasma cell leukemia, multiple myeloma, Waldenström macroglobulinemia, solitary plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

[0563] Other embodiments

[0564] Although the foregoing invention has been described in detail for purposes of clarity of understanding by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0565] Equivalents and scope

[0566] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0567] Unless the context clearly dictates otherwise or has some other meaning, articles such as "a," "an," and "the" may mean one or more than one. Unless the context clearly dictates otherwise or has some other meaning, a claim or specification that includes "or" between two or more members of a group is considered to satisfy the condition that there be one, more than one, or all of the members of the group. The disclosure of a group that includes "or" between two or more members provides embodiments in which exactly one member of the group exists, embodiments in which more than one member of the group exists, and embodiments in which all of the members of the group exist. For purposes of brevity, those embodiments are not set forth separately herein, but it should be understood that each of those embodiments is provided herein and may be specifically claimed or disclaimed.

[0568] It should be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or from one or more relevant portions of the specification are introduced into another claim. For example, a claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same base claim. Additionally, when a claim recites a composition, it should be understood that, unless it would be apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise, methods of making or using the composition according to any of the methods disclosed herein or according to methods known in the art (if any) are included.

[0569] In cases where elements are presented in a list form (e.g., in the form of a Markush group), it should be understood that every possible subgroup of the elements is also disclosed, and any element or subgroup of elements may be removed from the group. It should also be noted that the term "comprising" is intended to be open-ended and permits the inclusion of other elements or steps. It should be understood that generally, when an embodiment, product, or method is said to comprise a particular element, feature, or step, embodiments, products, or methods consisting of or consisting essentially of such element, feature, or step are also provided. For the sake of brevity, those embodiments are not set forth separately herein, but it should be understood that each of these embodiments is provided herein and may be particularly claimed or disclaimed.

[0570] When ranges are given, the endpoints are included. Additionally, it should be understood that, unless otherwise indicated by the context and / or the understanding of one of ordinary skill in the art or there is some other clear meaning, a value expressed as a range in the same embodiment may assume any specific value within the range, accurate to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For the sake of brevity, the values within each range are not set forth separately herein, but it should be understood that each of these values is provided herein and may be particularly claimed or disclaimed. It should also be understood that, unless otherwise indicated by the context and / or the understanding of one of ordinary skill in the art or there is some other clear meaning, a value expressed as a range may assume any sub-range within the given range, where the endpoints of the sub-range are expressed with the same precision: one-tenth of the unit of the lower limit of the range.

[0571] In cases where a website is provided, the URL address is provided in a non-browser-executable code form, and in parentheses is the period of the corresponding website address. The actual website address does not include the parentheses.

[0572] In addition, it should be understood that any particular embodiment of the present disclosure may be expressly excluded from any one or more of the claims. Where a range is given, any value within the range may be expressly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more of the claims. For the sake of brevity, all embodiments that exclude one or more elements, features, objects, or aspects are not expressly set forth herein.

Claims

1. An antibody that specifically binds to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), wherein the antibody comprises: (i) CDR-H1 consisting of SEQ ID NO: 26, CDR-H2 consisting of SEQ ID NO: 27, and CDR-H3 consisting of SEQ ID NO: 28; CDR-L1 consisting of SEQ ID NO: 29, CDR-L2 consisting of SEQ ID NO: 30, and CDR-L3 consisting of SEQ ID NO: 31; (ii) CDR-H1 consisting of SEQ ID NO: 32, CDR-H2 consisting of SEQ ID NO: 33, and CDR-H3 consisting of SEQ ID NO: 34; CDR-L1 consisting of SEQ ID NO: 35, CDR-L2 consisting of SEQ ID NO: 36, and CDR-L3 consisting of SEQ ID NO: 31; or (iii) CDR-H1 consisting of SEQ ID NO: 37, CDR-H2 consisting of SEQ ID NO: 38, and CDR-H3 consisting of SEQ ID NO: 39; CDR-L1 consisting of SEQ ID NO: 40, CDR-L2 consisting of SEQ ID NO: 30, and CDR-L3 consisting of SEQ ID NO:

41.

2. The antibody according to claim 1, wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 1 and VL containing the amino acid sequence of SEQ ID NO:

2.

3. The antibody according to claim 1, wherein the antibody is a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody.

4. The antibody according to claim 3, wherein the antibody is a monoclonal antibody.

5. The antibody according to claim 1, wherein the antibody is a full-length antibody.

6. The antibody according to claim 1, wherein the antibody is an antibody fragment that specifically binds to TACI.

7. The antibody according to claim 6, wherein the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2 fragments.

8. The antibody according to claim 1, wherein the antibody is an IgG antibody.

9. The antibody according to claim 1, wherein the antibody is an IgG1 antibody.

10. The antibody according to claim 7, wherein the antibody is an scFv.

11. The antibody according to claim 10, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:

5.

12. The antibody according to claim 10 or 11, wherein the scFv is fused to Fc.

13. A composition comprising the antibody according to any one of claims 1-12.

14. A polynucleotide encoding an antibody according to any one of claims 1-12.

15. A vector comprising the polynucleotide according to claim 14.

16. A host cell comprising the vector according to claim 15.

17. The host cell according to claim 16, wherein the host cell is a mammalian cell.

18. The host cell according to claim 17, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

19. The host cell according to claim 16, wherein the host cell is a prokaryotic cell.

20. The host cell according to claim 19, wherein the prokaryotic cell is Escherichia coli.

21. A method of producing an antibody that specifically binds to TACI, the method comprising culturing the host cell according to any one of claims 16-20 in a medium.

22. The method according to claim 21, wherein the method further comprises recovering the antibody from the host cell or the medium.

23. An antibody-drug conjugate comprising an antibody according to any one of claims 1-12.

24. A CAR polypeptide comprising an extracellular target-binding domain, the extracellular target-binding domain comprising an antibody according to any one of claims 1-12.

25. The CAR polypeptide according to claim 24, wherein the CAR polypeptide comprises a transmembrane domain and an intracellular signaling domain.

26. The CAR polypeptide according to claim 24 or 25, further comprising one or more co-stimulatory domains.

27. The CAR polypeptide according to claim 26, wherein the antibody comprises VH of SEQ ID NO: 1 and VL of SEQ ID NO:

2.

28. The CAR polypeptide according to claim 27, wherein the antibody comprises VH at a position N-terminal to VL.

29. The CAR polypeptide according to claim 27, wherein the antibody comprises VL at a position N-terminal to VH.

30. The CAR polypeptide according to any one of claims 27-29, wherein VH and VL are linked via a linker sequence.

31. The CAR polypeptide according to claim 30, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 3, 14, 15, 16 or 17.

32. The CAR polypeptide according to claim 31, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO:

3.

33. The CAR polypeptide according to claim 25, wherein the transmembrane domain comprises a hinge / transmembrane domain.

34. The CAR polypeptide according to claim 33, the hinge / transmembrane domain comprising the hinge / transmembrane domain of an immunoglobulin-like protein, CD28, CD8 or 4-1BB.

35. The CAR polypeptide according to claim 34, wherein the hinge / transmembrane domain is the hinge / transmembrane domain of CD8.

36. The CAR polypeptide according to claim 35, wherein the hinge / transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO:

7.

37. The CAR polypeptide according to claim 25, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, CD3ε or CD3θ.

38. The CAR polypeptide according to claim 37, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ.

39. The CAR polypeptide according to claim 38, wherein the intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO:

9.

40. The CAR polypeptide according to claim 26, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, CD28, CD27, ICOS or OX40.

41. The CAR polypeptide according to claim 40, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB.

42. The CAR polypeptide according to claim 41, wherein the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO:

8.

43. The CAR polypeptide according to claim 24 or 25, wherein the CAR polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 10, 11, 12 or 13.

44. The CAR polypeptide according to claim 24 or 25, wherein the extracellular target-binding domain further comprises a target-binding domain that binds to a second target other than TACI.

45. The CAR polypeptide according to claim 44, wherein the second target is B cell maturation antigen (BCMA).

46. The CAR polypeptide according to claim 44, wherein the target-binding domain comprises a ligand of the second target.

47. The CAR polypeptide according to claim 44, wherein the target-binding domain comprises an antibody or an antigen-binding fragment thereof.

48. The CAR polypeptide according to claim 47, wherein the antibody or the antigen-binding fragment thereof comprises an scFv.

49. The CAR polypeptide according to claim 48, wherein the scFv is an anti-BCMA scFv.

50. The CAR polypeptide according to claim 49, wherein the anti-BCMA scFv is located at the N-terminus of the anti-TACI scFv.

51. The CAR polypeptide according to claim 49, wherein the anti-TACI scFv is located at the N-terminus of the anti-BCMA scFv.

52. A CAR polypeptide comprising an amino acid sequence consisting of SEQ ID NO: 10 or SEQ ID NO:

11.

53. A CAR polypeptide comprising an amino acid sequence consisting of SEQ ID NO: 12 or SEQ ID NO:

13.

54. A CAR polypeptide comprising an amino acid sequence consisting of any one of SEQ ID NO: 18-21.

55. A CAR polypeptide comprising an amino acid sequence consisting of any one of SEQ ID NO: 22 - 25.

56. A polynucleotide encoding the CAR polypeptide according to any one of claims 24 - 55.

57. The polynucleotide according to claim 56, wherein the polynucleotide further comprises a suicide gene.

58. The polynucleotide according to claim 56 or 57, wherein the polynucleotide further comprises a sequence encoding a signal sequence.

59. A mammalian cell comprising the CAR polypeptide according to any one of claims 24 - 55 and / or the polynucleotide according to any one of claims 56 - 58.

60. The mammalian cell according to claim 59, wherein the mammalian cell is an induced pluripotent stem cell (iPSC).

61. The mammalian cell according to claim 59, wherein the mammalian cell is an immune cell.

62. The mammalian cell according to claim 61, wherein the immune cell is a T cell or a natural killer (NK) cell.

63. The mammalian cell according to any one of claims 59 - 62, wherein the mammalian cell is a human cell.

64. A bispecific antibody that binds to TACI and CD3, wherein the bispecific antibody comprises a TACI - binding domain and a CD3 - binding domain, and the TACI - binding domain comprises the antibody according to any one of claims 1 - 12.

65. The bispecific antibody according to claim 64, wherein the TACI - binding domain is located at the N - terminus of the CD3 - binding domain.

66. The bispecific antibody according to claim 64 or 65, wherein the CD3 - binding domain is located at the N - terminus of the TACI - binding domain.

67. The bispecific antibody according to claim 64 or 65, wherein the TACI - binding domain and the CD3 - binding domain are linked by a linker sequence.

68. The bispecific antibody according to claim 67, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 3, 14, 15, 16 or 17.

69. The bispecific antibody according to claim 64 or 65, wherein the bispecific antibody is a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.

70. The bispecific antibody according to claim 69, wherein the bispecific antibody is a monoclonal antibody.

71. The bispecific antibody according to claim 64 or 65, wherein the bispecific antibody is a full - length antibody.

72. The bispecific antibody according to claim 64 or 65, wherein the bispecific antibody is an antibody fragment that specifically binds to TACI and CD3.

73. The bispecific antibody according to claim 72, wherein the antibody fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv and (Fab')2 fragments.

74. The bispecific antibody according to claim 64 or 65, wherein the bispecific antibody is an IgG antibody.

75. The bispecific antibody according to claim 74, wherein the bispecific antibody is an IgG1 antibody.

76. A composition comprising the bispecific antibody according to any one of claims 64-75.

77. A polynucleotide encoding the bispecific antibody according to any one of claims 64-75.

78. A vector comprising the polynucleotide according to claim 77.

79. A host cell comprising the vector according to claim 78.

80. The host cell according to claim 79, wherein the host cell is a mammalian cell.

81. The host cell according to claim 80, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

82. The host cell according to claim 79, wherein the host cell is a prokaryotic cell.

83. The host cell according to claim 82, wherein the prokaryotic cell is Escherichia coli.

84. A method for producing a bispecific antibody that specifically binds to TACI and CD3, the method comprising culturing the host cell according to any one of claims 79-83 in a medium.

85. The method according to claim 84, wherein the method further comprises recovering the bispecific antibody from the host cell or the medium.

86. Use of one or more of the following in the preparation of a medicament for treating a plasma cell disease or disorder in a subject in need thereof: (i) The CAR polypeptide according to any one of claims 24-55, the polynucleotide according to any one of claims 14, 56-58 and 77, and / or the mammalian cell according to any one of claims 17, 59-63 and 80; (ii) The antibody according to any one of claims 1-12; (iii) The antibody-drug conjugate according to claim 23; and (iv) The bispecific antibody according to any one of claims 64-75, wherein the plasma cell disease or disorder is plasmacytosis, plasmacytoma, plasma cell leukemia, multiple myeloma, Waldenström macroglobulinemia, osteosclerotic myeloma, heavy chain disease or monoclonal gammopathy of undetermined significance.

87. The use according to claim 86, wherein the plasma cell disease or disorder is solitary plasmacytoma, extramedullary plasmacytoma and smoldering multiple myeloma.

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