Compositions of IGHV-targeted engineered immune effector cells and methods of making and using the same

Abstract

Provided herein are engineered immune effector cells that include an engineered immune receptor, wherein the engineered immune receptor comprises an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein sequence present on a surface of a B cell.

Description

[0001] Attorney Docket No.: 44807-0494WO1

[0002] COMPOSITIONS OF IGHV-TARGETED ENGINEERED IMMUNE EFFECTOR

[0003] CELLS AND METHODS OF MAKING AND USING THE SAME

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Patent Application No. 63 / 693,394, filed on September 11, 2024, which is incorporated herein by reference in its entirety.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named 44807-0494WOl_SL_ST26.xml. The XML file, created on August 21, 2025, is 58,505 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] The present disclosure relates to cellular therapies that use an engineered synthetic immune receptor, a engineered chimeric antigen receptor (CAR) (e.g., engineered CAR-T cells) or an engineered chimeric T cell receptor (cTCR) (e.g., engineered cTCR-T cells) for precision targeting of immune cells such as, without limitation, 9G4 idiotope (9G4id) B cells in systemic lupus erythematosus (SLE), cold agglutinin disease, multiple sclerosis, or cancer.

[0010] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0011] This invention was made with government support under grant Al 176764 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0012] BACKGROUND

[0013] The autoreactive B cell compartment in systemic lupus erythematosus (SLE) is characterized by expansion B cells expressing immunoglobulin heavy variable gene 4-34 (IGHV4-34) B cell receptors (BCRs). This innately autoreactive B-cell population carries the 9G4 idiotope (9G4id) and is a major source of disease-relevant autoantibodies in lupus (e.g., anti-dsDNA, anti-DNAselL3, anticardiolipin, and ANA), cold agglutinin disease (CAD), and Attorney Docket No.: 44807-0494WO1 other autoimmune diseases. While CD19- and BCMA-targeted CAR-T cells have transformed the treatment of patients with severe or refractory SLE, pan-B cell depleting therapies carry an increased risk of infection and cytokine-related toxicities, precluding their use in mild or preclinical disease. In contrast, precision immunotherapies that deplete autoreactive B cells, while sparing most other cells, such as those provided herein, may treat SLE without causing immunosuppression.

[0014] SUMMARY

[0015] Provided herein are engineered immune effector cells comprising a synthetic immune receptor that includes an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell.

[0016] In some embodiments, the IGHV gene-encoded protein is derived from a IGHV4-34 allele (IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0017] In some embodiments, the antigen binding domain comprises an antibody fragment. In some embodiments, the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, a variable new antigen receptors (VNARs), or a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences. In some embodiments, the antigen binding domain is humanized. In some embodiments, the antigen binding domain comprises an engineered TCR alpha chain and / or an engineered TCR beta chain. In some embodiments, the antigen binding domain comprises an engineered TCR delta chain and / or an engineered TCR gamma chain.

[0018] In some embodiments, the immune effector cell is a human immune cell. In some embodiments, the immune effector cell is a human T cell, NK cell, orNK / T cell.

[0019] In some embodiments, the engineered immune effector cell further comprises an engineered intracellular co-stimulatory, immunomodulatory, or signaling domain. In some embodiments, the intracellular co-stimulatory or immunomodulatory domain is derived from full Attorney Docket No.: 44807-0494WO1 or partial sequences of CD28, 4-1BB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP10, DNAM-1, DAP12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, IT AM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD- 1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof. In some embodiments, the intracellular co- stimulatory domain is derived from MyD88 and CD40.

[0020] In some embodiments, an engineered immune effector cell is provided, wherein the synthetic immune receptor is a chimeric T-cell receptor (cTCR) comprising a variable heavy (VH) domain linked to a TCR beta chain and a variable light (VL) domain linked to a TCR alpha chain. In some embodiments, the engineered immune effector cell, when contacted by a 9G4 idiotope (9G4id)-expressing B cell, secretes a cytokine at a lower level than a 9G4-targeted CAR-T cell comparator under similar conditions. In some embodiments, the cytokine is interferon-gamma (IFN-y). In some embodiments, the engineered immune effector cell exhibits antigen-independent proliferation that is at least two-fold, at least three-fold, at least four-fold, or at least five-fold lower than a 9G4-targeted CAR-T comparator under similar conditions. In some embodiments, the engineered immune effector cell increases preservation of IgG+B-cells compared to a CD19-CAR-T treatment.

[0021] In some embodiments, the antigen binding domain comprises a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises an antibody fragment, a linker, a transmembrane domain, a co-stimulatory domain, and a signaling domain. In some embodiments, the chimeric antigen receptor (CAR) comprises a hinge and / or linker domain, a transmembrane domain, at least one intracellular co-stimulatory or immunomodulatory domain, and at least one intracellular signaling / signal transducer domain.

[0022] In some embodiments, a chimeric antigen receptor (CAR) is provided, comprising an extracellular antigen-binding domain that specifically binds a 9G4 idiotope and a linker comprising an EAAAK motif. In some embodiments, the CAR further comprises a CD8a hinge or an IgG4 hinge. In some embodiments, the CAR comprises a CD28 transmembrane domain or Attorney Docket No.: 44807-0494WO1 a CD3(^ transmembrane domain. In some embodiments, the CAR comprises a CD28 intracellular co-stimulatory domain and a CD3(^ intracellular signaling domain.

[0023] Also provided herein are engineered T cells comprising any of the CARs described herein.

[0024] Also provided herein are engineered polypeptides that include an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell; and a signaling domain.

[0025] In some embodiments, the IGHV gene-encoded protein is derived from an IGHV4-34 allele (IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from an IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from an IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0026] In some embodiments, the antigen binding domain comprises an antibody fragment. In some embodiments, the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, a variable new antigen receptors (VNARs), or a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences. In some embodiments, the antigen binding domain is humanized. In some embodiments, the antigen binding domain comprises an engineered TCR alpha chain and / or an engineered TCR beta chain. In some embodiments, the antigen binding domain comprises an engineered TCR delta chain and / or an engineered TCR gamma chain.

[0027] In some embodiments, the immune effector cell is a human immune cell. In some embodiments, the immune effector cell is a human T cell, NK cell, or NK / T cell.

[0028] In some embodiments, the engineered polypeptide further comprises an intracellular costimulatory, immunomodulatory, or signaling domain. In some embodiments, the intracellular co-stimulatory domain or immunomodulatory domain is derived from full or partial sequences of CD28, 4-1BB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP10, DNAM-1, DAP12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, ITAM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, Attorney Docket No.: 44807-0494WO1

[0029] LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof. In some embodiments, the intracellular co- stimulatory domain is derived from MyD88 and CD40.

[0030] In some embodiments, the antigen binding domain comprises a scFv.

[0031] In some embodiments, the engineered polypeptide further comprises an antibody fragment, a linker, a transmembrane domain, a co-stimulatory domain, and a signaling domain. In some embodiments, the engineered polypeptide further comprises a hinge and / or linker domain, a transmembrane domain, at least one intracellular co-stimulatory or immunomodulatory domain, and at least one intracellular signaling / signal transducer domain.

[0032] Also provided herein are nucleic acid molecules encoding any of the engineered polypeptides described herein.

[0033] Also provided herein are recombinant vectors comprising any of the nucleic acids described herein.

[0034] Also provided herein are pharmaceutical compositions that include any one of the engineered immune effector cells, engineered T cells, engineered polypeptides, CARs, nucleic acid molecules, or recombinant vectors described herein, and a pharmaceutically acceptable carrier.

[0035] Also provided herein are methods of treating a disease in a subject that include administering to the subject any one of the engineered immune effector cells or the pharmaceutical compositions described herein. In some embodiments, the disease is an autoimmune disease, wherein the autoimmune disease is systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren's disease, scleroderma, rheumatoid arthritis, cold agglutinin disease, pemphigus vulgaris, or multiple sclerosis. In some embodiments, the disease is a B cell cancer, wherein the B cell cancer is a B cell lymphoma, Diffuse Large B cell Lymphoma (DLBCL), Chronic Lymphocytic Leukemia (CLL) / Small Lymphocytic lymphoma, Follicular lymphoma, Primary CNS lymphoma, primary vitreoretinal lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Hairy cell lymphoma, or a B cell acute lymphoblastic leukemia (ALL).

[0036] In some embodiments, a method of treating a disease in a subject is provided, comprising administering to the subject any one of the engineered immune effector cells or pharmaceutical Attorney Docket No.: 44807-0494WO1 compositions described herein. In some embodiments, the disease is an autoimmune disease, such as systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren’s disease, scleroderma, rheumatoid arthritis, cold agglutinin disease, pemphigus vulgaris, or multiple sclerosis. In some embodiments, the autoimmune disease is SLE, and the method reduces one or more autoantibodies selected from 9G4id antibodies and anti-dsDNA antibodies. In some embodiments, the autoimmune disease is cold agglutinin disease, and the method depletes or significantly reduces 9G4id B cells expressing cold agglutinin B-cell receptors without significantly depleting irrelevant B cells. In some embodiments, the method preserves total IgG+B-cell numbers relative to a CD19-CAR-T treatment.

[0037] Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0039] BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 is an exemplary schematic drawing of the framework for targeting 9G4id B cells in autoimmune diseases, including systemic lupus erythematosus (SLE), cold agglutinin disease, multiple sclerosis, and rheumatoid arthritis. A. Autoreactive 9G4id B cells are abnormally expanded in active SLE but do not significantly contribute to the normal B cell repertoire in health. B. Pan-B cell depletion therapies, such as CD19-, CD20-, or BCMA-targeted monoclonal antibodies, bispecific antibodies, or chimeric antigen receptor (CAR)-T cells, can achieve disease remission in SLE and other autoimmune diseases, but increase the risk of infection and impair vaccine responses. C. Precise depletion of 9G4id B cells may aid in restoring immune Attorney Docket No.: 44807-0494WO1 homeostasis in SLE without increasing the risk of infection, while also avoiding the depletion of regulatory B cell (Breg) populations.

[0041] FIG. 2 shows exemplary chimeric T cell receptor (cTCR) and chimeric antigen receptor (CAR) compositions for the depletion of 9G4id (IGHV4-34) B cells in patients with SLE, other autoimmune diseases, and B cell cancers. A T cell product is described where an antibody fragment (e.g., scFv, immunoglobulin variable heavy [VH], immunoglobulin variable light [VL] chain, or nanobody) is expressed as part of (human or murine) CD3 gamma (y), CD3 delta (8), CD3 epsilon (e), or any part of the TCR alpha constant region (Cu), TCR beta constant region (Cp), TCR gamma constant region (Cy), TCR delta constant region (C8), TCR alpha variable region (Va), TCR beta variable region (V ), TCR gamma variable region (Vy), TCR delta variable region (V8), under expression of either the endogenous promoter of the modified gene locus or under expression of an exogenous promoter (e.g., a promoter that is introduced in lieu of an endogenous promoter), with or without additional linker sequences, with or without additional intracellular and / or co-stimulatory and / or signaling domains, in addition to any needed selfcleaving peptides or sequences of equivalent function, in addition to any needed poly(A) sequences or alternative terminator sequences, and in addition to any needed Stop codons, when applicable. The figure shows an exemplary model representation of a native oPTCR (bottom left) compared to engineered cTCR-T cell constructs that link an antibody fragment (e.g., scFV, VH, or VL) to any parts of CD3y, CD38, CD3e, Cp, Cu, Cp and Ca, Vp, Vu, or VP and Va, respectively (bottom right). A peptide-human leukocyte antigen (HLA) complex (top left) compared to 9G4id BCRs of any isotype (IgG, IgM, IgA, IgE, or IgD) or subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2) on the cell surface of autoreactive B cells (top right) targeted by cTCR-T cells are shown.

[0042] FIG. 3 shows an exemplary model representation of a native y8TCR (bottom left) compared to engineered cTCR-T cell constructs that link an antibody fragment (e.g., scFV, VH, or VL) to any parts of C8, Cy, C8 and Cy, V8, Vy, or V8 and Vy, respectively (bottom left). In addition to y8- cTCR construct designs, variations applicable to any cTCR construct design comprising the addition of a linker / hinge domain (e.g., any natural or synthetic linker sequence(s) or binding / coupling domain(s), protein or other, that result in covalent or non-covalent linkage of the IGHV-targeted antibody fragment or parts thereof to any part of the TCR-CD3 complex such as (G4S)n linkers, (EAAAK)n linkers, immunoglobulin domains, protein tags, leucine zippers, or Attorney Docket No.: 44807-0494WO1 other dimerization domains) or the use of murine equivalents of any of the TCR-CD3 complex proteins (e.g., murine CP or murine Co.) are also described. Constructs expressing an anti-9G4 (IGHV4-34) antibody fragment (e.g., scFV, VH, or VL, nanobody) as part of a modified second or higher generation chimeric antigen receptor (CAR) construct, comprising a hinge and / or linker domain (e g., CD8, CD28, IgGl, IgG4, EAAAK, G4S, G3S, G2S, GS), a transmembrane domain (e.g., CD8a, CD3i^, CD4, CD28), at least one intracellular co-stimulatory or immunomodulatory domain (e.g., full or partial sequences of CD28, 4-1BB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP 10, DNAM-1, DAP 12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, IT AM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or their combination), and at least one intracellular signaling / signal transducer domain (e.g., CD3(^, ZAP70, LCK, FYN, PLCG1, LCP2) in addition to the extracellular autoantigenic peptide(s), are also described in an alternative composition (bottom right). 9G4id BCRs of any isotype (IgG, IgM, IgA, IgE, or IgD) or subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2) on the cell surface of autoreactive B cells (top) targeted by anti-9G4 cTCR-T cells or anti-9G4 CAR T cells are shown.

[0043] FIG. 4 shows agarose gel electrophoresis results of exemplary homology-directed repair template DNA encoding for anti-9G4 chimeric T cell receptors (cTCRs) or anti-9G4 chimeric antigen receptors (CARs) comprising homology arms specific for the targeted TCR gene loci, promoter sequences, stop codon, and 3' UTR for the editing of primary human T cells using CRISPR-Casl2 or Cas9.

[0044] FIG. 5 shows flow cytometry results showing editing rate of engineered T cells. Comparative expression of 9G4-CAR and 9G4-cTCR constructs in CRISPR / Cas HDR-edited primary human T cells as assessed by flow cytometry are shown.

[0045] FIG. 6 shows post-selection purity of engineered T cells. Surface expression of tNGFR (indicating expression of the transgene under control of the EFl -alpha), CD3e (indicating TCR Attorney Docket No.: 44807-0494WO1 surface expression or knock-out), and synthetic immune receptor binding to the 9G4id (clones 627A11, 75H12, 88F7, respectively) are shown.

[0046] FIG. 7 shows a summary of a representative co-culture experiment for anti-9G4 CAR-T cells (right panel), anti-9G4 VH-Ca-VL-Cp cTCR-T cells (cTCR90, middle panel), and anti-9G4 scFv-Ca cTCR-T cells (cTCR91, left panel) across different effectortarget cell [E:T] ratios. Graphs show the B cell viability at the end of a co-culture experiment of engineered T cells with either 9G4id target B cell lines and irrelevant non-9G4id B cells. 9G4id B cells were killed in a dose-dependent manner as the effector: target cell [E:T] ratios increased. No significant off-target killing against non-9G4id B cells was observed.

[0047] FIG. 8 shows a summary of a representative co-culture experiment of 9G4 CAR-T cells (triangle), 9G4 cTCR2-T cells (square), 9G4 cTCR2-T cells (diamond), and Control T cells (circle) and BCR627AH B cells, BCR75G12 B cells, BCRSSF? B cells, and non-9G4 BCR B cells. B cell viability and growth are quantified over time by monitoring the total green integrated intensity of GFP+ B cell lines.

[0048] FIG. 9 shows the quantification of B cells at the end of the co-culture experiments shown in FIG. 8. Both CAR-T cells and cTCR-T cells efficiently and potently eliminated 9G4id Ramos B cells, while not depleting non-9G4id B cells. Live-cell imaging (IncuCyte) was used to quantify the Green Integrated Intensity of GFP+ 9G4id or non-9G4 Ramos B cells in co-culture with 9G4-CAR-T cells, 9G4-cTCRl-T cells, 9G4-cTCR2-T cells, or control T cells over time. All data were normalized to day 0.

[0049] FIG. 10 shows the quantification of interferon-gamma release in a representative culture experiment of 9G4 synthetic immune receptor T cells (9G4-CAR-T cells, 9G4-cTCRl-T cells, or 9G4-cTCR2-T cells) co-cultured with the SLE 9G4id Ramos B cells (BCR627.AU, BCR75G12, BCRSSF?) or non-9G4 Ramos B cells (E:T ratio 5: 1, 62 hours). Conditioned supernatants were analyzed for IFN-y secretion by ELISA.

[0050] FIG. 11A shows flow cytometric analysis of CellTrace Violet dilution in stained engineered T cells to show the percentage of divided cells. 9G4-CAR-T cells compared to cTCR-T cells show an increased degree of proliferation in the presence (specific) and in the absence (unspecific) of 9G4id B cells.

[0051] FIG. 11B shows a T cell proliferation assay. CellTrace Violet (CTV)-labeled 9G4 synthetic immune receptor T cells (G4-CAR-T cells, 9G4-cTCRl-T cells, or 9G4-cTCR2-T cells) were Attorney Docket No.: 44807-0494WO1 cultured with SLE 9G4id Ramos B cells (BCR627A11, BCR75G12, BCRw?) or non-9G4 Ramos B cells, E:T ratio 5: 1. Flow cytometry was performed 108 hours after coincubation. Weighted average of generations was used to compute the statistical significance. Weighted average of generations = Sum of (T cell number in each generation x generation number) / sum of total T cell number, generation number= 0, 1, 2, 3, 4, 5, 6.

[0052] FIG. 12 shows abrogation of 9G4id antibody production in Experiment shown in Figures 8 and 9 (ELISA). 9G4id antibody levels in co-culture supernatants after treatment with 9G4-CAR-T cells, 9G4-cTCRl-T cells, 9G4-cTCR2-T cells, or control T cells were measured by ELISA. **** p<0.0001, ns (not significant), two-way ANOVA with Tukey’s multiple comparison test. FIG. 13 shows abrogation of anti-dsDNA antibody production in Experiment shown in Figures 8 and 9 (ELISA). Anti-dsDNA autoantibody levels in co-culture supernatants after treatment with 9G4-CAR-T cells, 9G4-cTCRl-T cells, 9G4-cTCR2-T cells, or control T cells were measured by ELISA. ****p<0.0001, ns (not significant), two-way ANOVA with Tukey’s multiple comparison test.

[0053] FIG. 14 shows PBMCs from SLE patients (100,000) were co-cultured with 9G4-CAR-T cells, 9G4-cTCRl-T cells, CD19-CAR-T cells, or control T cells (5,000). B cells were differentiated in stimulation media for 6 days. Equal numbers of B cells were transferred to Fluorospot plates coated with anti-9G4id (50,000 B cells / well), or to Fluorospot plates coated with anti-human IgG (5,000 B cells). Conjugated anti-human IgG was used to visualize all the antibody-secreting cells.

[0054] FIG. 15 shows the quantification of 9G4id autoantibody levels in co-culture supernatants of SKE patient PBMCs with anti-9G4 CAR-T cells (triangle), and anti-9G4 scFv-Ca cTCR-T cells (cTCR91, diamond), anti-CD19 CAR-T cells (hexagon), and mock-edited T cells (circle) showing that engineered anti-9G4 synthetic immune receptor T cells were able to deplete 9G4id autoantibody levels.

[0055] FIG. 16 shows an exemplary schematic of a summary of the characteristics of anti-9G4 CAR-T cells vs anti-9G4 cTCR-T cells for the treatment of autoimmune diseases and B cell cancers. Anti-9G4 cTCR-T cells show comparable potency but significantly lower cytokine release and reduce antigen-independent proliferation, reducing the risk of cytokine-related adverse events. FIG. 17 shows exemplary 9G4-CAR-T cells, 9G4-cTCRl-T cells, and c9G4-TCR2-T cells designs. 9G4-CAR in the subsequent experiments utilized an “EAAAK” linker, CD8a hinge, Attorney Docket No.: 44807-0494WO1

[0056] CD28 transmembrane domain, and CD28 co-stimulatory domain, followed by a CD3(^ intracellular domain. Ca and C denote TCR constant a and constant P chains, respectively; e, 5, y, and , denote the 8, 8, y and C, CD3 subunits, respectively; scFV VH and scFV VL, variable heavy and light chains of scFv, respectively; VH and VL, variable heavy and light chains of autoreactive BCR, respectively.

[0057] FIG. 18 shows exemplary co-culture of 9G4-CAR-T cells, 9G4-cTCRl-T cells, and 9G4- cTCR2-T cells with 9G4id, anti-dsDNA BCR Ramos B cells and non-9G4 control B cells. Representative flow cytometric staining. Edited T cells (9G4-CAR-T cells, 9G4-cTCRl-T cells, or 9G4-cTCR2-T cells) or control T cells were incubated at different E:T ratios with GFP+SLE 9G4id Ramos B cells (BCR627AII, BCR75G12, BCRSSF?) or non-9G4 B cells. Flow cytometry was performed at 4 days of co-incubation. DY-649 StrepTactin XT was used to detect Ramos B cells expressing engineered BCRs.

[0058] FIG. 19 shows differences in cytokine production by 9G4-CAR-T cells vs 9G4-cTCRl / 2-T cells. 9G4-cTCR-T cells have favorable safety characteristics (risk of cytokine-related toxicities) over 9G4-CAR-T cells for the treatment of autoimmune diseases.

[0059] FIG. 20 shows total IgG+ B cell numbers in PBMCs from patients with SLE treated with 9G4- CAR-T cells ,9G4-cTCR-T cells, CD19-CAR-T cells, or control T cells as determined by IgG B cell fluorospot. Total IgG+ B cells are preserved by treatment with 9G4-targeted T cells and control T cells but eliminated by conventional CD19-CAR-T cells.

[0060] FIG. 21 shows a schematic representation of BCR repertoire sequencing showing the depletion of VH4-34+ B cells in SLE patient PBMCs with CAR-T cells vs cTCRl-T cells. mRNA bulk BCR repertoire sequencing was performed on SLE patient PBMCs after treatment with autologous engineered anti-9G4 T cells (9G4-CAR-T cells or 9G4-cTCRl-T cells) or control T cells. Chord diagrams summarizing IGHV and IGHC usage by BCRs are shown. IGHV4-34 BCRs are depleted with 9G4-CAR-T cells and 9G4-cTCRl-T cells.

[0061] FIG. 22 shows analysis of bulk mRNA B cell receptor (BCR) repertoire data showing significant depletion of VH4-34+(9G4id) B cells and preservation of other compartments. The heatmap shows the percentage of IGHV gene usage (%IGHV) in total B cells contained in SLE patient PBMCs. Forest plots on the right show mean odds ratios and SD for each IGHV gene in PBMCs treated with 9G4-CAR-T cells or 9G4-cTCRl-T cells as compared to untreated PBMCs. Attorney Docket No.: 44807-0494WO1

[0062] FIG. 23 shows marked reduction of cytokine secretion is observed with 9G4-targeted cellular therapies as compared to CD19-CAR-T cell therapy. PBMCs (100,000) from patients with SLE were co-cultured with 9G4-CAR-T cells, 9G4-cTCRl-T cells, CD19-CAR-T cells, or control T cells (5,000) in B cell expansion and differentiation media for 48 hours. Then conditioned supernatant was then analyzed by MesoScale U-PLEX for secreted cytokines. Data are shown as mean ± SD of three technical replicates.

[0063] FIG. 24 shows depletion of primary human 9G4id B cells from SLE patients with 9G4-CAR-T cells and 9G4-cTCR-T cells is potent and selective, as evaluated by flow cytometry. B cells (100,000) from SLE patient PBMCs were co-cultured with 50,000 (E:T=10: l), 25,000 (E:T=5:1), 12,500 (E:T=2.5: 1), 6,250 (E:T=1.25: 1), 3,125 (E:T=0.63:l), or 1,563 (E:T=0.3: l) 9G4-CAR-T cells, 9G4-cTCRl-T cells, or control T cells for 65 hours. Flow cytometric staining with anti-CD20 and anti-9G4id was used to quantify the absolute number of viable 9G4id B cells or non-9G4 B cells, which was then used to calculate the percentage of viable B cells.

[0064] FIG. 25 shows a schematic representation of alternative 9G4-CAR-T cell designs tested. Illustrations of alternative 9G4-CAR-T cell constructs. The CAR construct “EAAAK-CD8a Hinge” incorporates a EAAAK linker, CD8a hinge, CD28 transmembrane domain. The CAR construct “G4S-IgG4 Hinge” incorporates a G4S linker, IgG4 hinge, CD28 transmembrane domain. The CAR construct “EAAAK -IgG4 Hinge” incorporates a EAAAK linker, IgG4 hinge, CD28 transmembrane domain. The CAR construct “(EAAAK)3” incorporates a EAAAKEAAAKEAAAK linker, no additional hinge, CD28 transmembrane domain. The CAR construct “EAAAK-CD3^ Hinge” incorporates a EAAAK linker, CD3(^ hinge, CD3^ transmembrane domain. All CAR constructs incorporate a CD28 co-stimulatory domain, followed by a CD3(^ intracellular signaling domain.

[0065] FIG. 26 shows comparative expression of different 9G4-CAR constructs in CRISPR / Cas HDR- edited primary human T cells as assessed by flow cytometry. Surface expression of tNGFR (indicating expression of the CAR-tNGFR transgene under control of the EFl -alpha), CD3e (indicating TCR surface expression or knock-out), and CAR binding to the 9G4id (clones 627A11, 75H12, 88F7, respectively) are shown. All CARs were found to be surface expressed in primary human T cells, lacked endogenous TCRs expression, and bound the 9G4id expressed by different BCRs. Attorney Docket No.: 44807-0494WO1

[0066] FIG. 27 shows cytotoxicity of different 9G4-CAR-T cell constructs against SLE 9G4id Ramos B cells and control B cells. Different engineered 9G4-CAR-T cells (6xl03, 1.25xl04, 2.5xl04, 5xl04) were incubated with SLE 9G4id Ramos B cells (IxlO4) for 65 hours, followed by assessment by flow cytometry to quantify live 9G4id B cells. Viability was calculated from absolute B cell counts by normalizing to untreated conditions (E:T 0: 1). Data are shown as mean ± SD of technical replicates for all conditions. 9G4-CAR-T cells demonstrated potent killing of target 9G4id Ramos B cells without depletion of irrelevant B cells.

[0067] FIG. 28 shows application of 9G4-CAR-T cells and 9G4-cTCR-T cells to other disease states. Here, engineered cellular therapies targeting 9G4id show selective depletion of 9G4id B cells that express and secrete cold agglutinins (CAs) derived from patients with cold agglutinin disease (CAD). Engineered and control T cells were incubated with GFP+ Ramos B cells expressing CA+ BCRs from patients with CAD (top row) or non-9G4 Ramos B cells expressing irrelevant BCRs (bottom row). Flow cytometry was performed after 3 days co-incubation to quantify the number of viable, single B cells (Q2).

[0068] FIG. 29 shows engineered T cells (CAR-T cell or cTCRl-T cells) or control T cells were incubated at different E:T ratios (E:T 0:1, 1 : 1, 2.5: 1, 5:1) with one of three Ramos B cell lines expressing / secreting CA+ BCRs / antibodies (clones C4, C5, C6) or non-9G4 Ramos B cells (negative control). Flow cytometry was performed at the end of co-incubation to quantify the number of viable, single B cells. Percent viability for B cells was calculated compared to untreated B cells (E:T 0:1 = 100%). Both 9G4-CAR-T cells and 9G4-cTCRl-T cells eliminated CAD 9G4id Ramos B cells, but not irrelevant Ramos B cells.

[0069] FIG. 30 shows IFN -gamma secretion by 9G4-CAR-T cells, 9G4-cTCRl-T cells, and control T cells in co-culture with 9G4id Ramos B cells expressing CA+ BCRs from patients with cold agglutinin disease. Analogous to co-culture of SLE 9G4id Ramos B cells, 9G4-cTCRl-T cells showed mitigated cytokine secretion compared to 9G4-CAR-T cells.

[0070] DETAILED DESCRIPTION

[0071] This disclosure describes the use of engineered immune effector cells, including chimeric T cell receptor (cTCR)-T cells and chimeric antigen receptor (CAR)-T cells, targeting B cell receptor variable or light chains (IGHV, IGHJ, IGKV, IGLV, IGKJ, IGLJ alleles / genes) or B cell Attorney Docket No.: 44807-0494WO1 receptor constant light chains (IGKC, IGLC alleles / genes) for the selective depletion of B cells in cancer, autoimmune diseases, and other B-cell mediated diseases.

[0072] Provided herein are engineered immune effector cells comprising a synthetic immune receptor, wherein the synthetic immune receptor includes an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV4-34 allele (e.g., IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene- encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0073] Also provided herein are engineered polypeptides that include an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV4-34 allele (e.g., IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3- 7, or IGHV4-39 allele.

[0074] This disclosure also provides nucleic acids, vectors, pharmaceutical compositions, and methods of treatment using the engineered immune effector cells or the engineered polypeptides that comprise an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV4-34 allele (e.g., IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0075] Various non-limiting aspects of these engineered immune effector cells are described herein, and can be used in any combination without limitation. Additional aspects of various components of methods of making and using the engineered immune effector cells are known in the art. Attorney Docket No.: 44807-0494WO1

[0076] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0077] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0078] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, transdermal, etc.), enteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, rectal, subcutaneous, sublingual, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0079] As used herein, the term “antibody” refers to an immunoglobulin (human, mammalian, non-mammalian, or synthetic) molecule that includes one or more antigen-binding domains that specifically bind to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin (Ig) structural elements sufficient to confer specific binding. Exemplary antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody may include one or more sequence elements that are humanized, primatized, chimeric, etc., as is known in the art. In some embodiments, the term “antibody” is used to refer to one or Attorney Docket No.: 44807-0494WO1 more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with the present disclosure can be in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain variable fragments (scFvs); polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies or fragments thereof (e g., VHHs); masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); Anticalins®; Nanobodies®; minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR). In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody is a polypeptide protein Attorney Docket No.: 44807-0494WO1 having a binding domain which is homologous or largely homologous to an immunoglobulin- binding domain.

[0080] As used herein, the term “antigen” refers to a molecule or molecular structure that binds to a specific antibody, B-cell receptor, or T-cell receptor. As used herein, the term “autoantigen” refers to a human molecule or molecular structure that is a normal bodily constituent in health or disease and binds to a specific antibody, B-cell receptor, or T-cell receptor. In some embodiments, an (auto)antigen binds to an antibody, B-cell receptor, or T-cell receptor and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid sequence (e.g., DNA or RNA), a peptide, a polypeptide, a protein, a carbohydrate, a glycoprotein, a lipid or phospholipid, a lipoprotein, a polymer (including biologic polymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biologic polymers [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. In some embodiments, an antigen is or comprises a lipid. In some certain embodiments, an antigen is present in a cellular context (e.g., an antigen is expressed on the surface of a cell or expressed in a cell).

[0081] As used herein, an “antigen-binding domain” refers to a fusion protein or portion thereof that specifically binds to a target moiety or entity (e.g., a B cell receptor or a T cell receptor). Typically, the interaction between an antigen-binding domain and its target is non-covalent. In some embodiments, a target moiety or entity can be of any chemical class including, for example, a carbohydrate, a lipid, a nucleic acid, a metal, a polypeptide, or a small molecule. In some embodiments, an antigen binding domain may be or comprise a polypeptide (or complex thereof). In some embodiments, an antigen binding domain is part of a fusion polypeptide.

[0082] It will be understood that the term “binding”, as used herein, typically refers to a non- covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moi eties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). Attorney Docket No.: 44807-0494WO1

[0083] As used herein, in general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered” when the polypeptide sequence manipulated by the hand of man. For example, in some embodiments of the present invention, an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, truncations, deletions, and / or insertions that have been introduced by the hand of man into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (e.g., covalently linked) to one or more additional polypeptides by the hand of man, to form a fusion polypeptide that would not naturally occur in vivo. Comparably, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, electroporation, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, derivatives and / or progeny of an engineered polypeptide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0084] As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0085] As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to “bind specifically” to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative Attorney Docket No.: 44807-0494WO1 interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0086] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more signs or symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0087] As used herein, a “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Attorney Docket No.: 44807-0494WO1

[0088] Engineered Immune Effector Cells

[0089] Provided herein are engineered immune effector cells comprising a synthetic immune receptor, wherein the synthetic immune receptor includes an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell. In some embodiments, the IGVH gene-encoded protein is derived from an IGHV4-34 allele (e.g., IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34* 10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from an IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from an IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0090] As used herein, “immune cells” refer to cells of the immune system which can be categorized as lymphocytes (e.g., T cells, B cells, NK cells and NKT cells), neutrophils, and monocytes / macrophages. In some embodiments, the immune cell is a T cell. In some embodiments, an immune cell is an engineered immune cell, which means the immune cell has been genetically modified to express a non-naturally occurring protein (e.g., a chimeric antigen receptor) or to include an exogenous nucleic acid.

[0091] The immune cells (e.g., T cells) may be modified in one or more than one manner. Immune cells (e.g., T cells) may express at least one non-natural molecule that is a receptor for an antigen that is present on the surface of one or more types of cells. In some embodiments, immune cells, include immune cells (e.g., T cells) that are not found in nature because they are engineered to comprise or express at least one synthetic molecule that is not found in nature.

[0092] In specific embodiments, the immune cell can be, without limitation, a T cell, e.g., a CD4+ T cell, a CD8+ T cell, a Treg cell, a Thl T cell, a Th2 T cell, a Thl7 T cell, an unspecific T cell, or a population of T cells that comprises a combination of any of the foregoing, a natural killer T (NKT) cell, a natural killer (NK) cell, or a macrophage.

[0093] As used herein, an “immune effector cell” refers to an immune cell that provides a specific response against a stimulation and / or pathogen. In the immune system, immune effector cells are relatively short-lived activated cells that defend the body in an immune response. For example, effector B cells are called plasma cells and secrete antibodies, and activated T cells include cytotoxic T cells and helper T cells, which carry out cell-mediated responses. In some embodiments, an immune effector cell can refer to specific cells in the immune system (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil). In some embodiments, an Attorney Docket No.: 44807-0494WO1 engineered immune effector cell provided herein comprises an engineered T cell. In some embodiments, the immune effector cell in a human immune cell. In some embodiments, the immune effector cell is a human T cell, NK cell, or NK / T cell.

[0094] In some embodiments, engineered immune effector cells provided herein are derived from autologous T cells. In some embodiments, engineered immune effector cells provided herein are derived from immune cells obtained from a subject that is not a patient. In some embodiments, engineered T cells for use in a therapeutic method are syngeneic (the donor and the recipients are different but are identical twins). In some embodiments, engineered T cells for use in a therapeutic method are allogenic (from the same species but different donor) as the recipient subject. In some embodiments, engineered T cells provided herein are derived from autologous stem cells (for autologous stem cell therapy or ASCT). In some embodiments, engineered immune cells provided herein are derived from non-autologous T cells. In some embodiments, engineered immune cells provided herein are derived from immune cells obtained from a healthy donor. In some embodiments, engineered immune cells provided herein are derived from immune cells obtained from a patient afflicted with a disease.

[0095] Immunoglobulin Variable Heavy Chain (IGHV) Gene-Encoded Protein

[0096] B cells are immunopathogenic drivers of systemic lupus erythematosus (SLE), an autoimmune disease characterized by a large repertoire of autoantibodies targeting self-protein and nucleic acids. While B cell-directed T-cell therapies have curative potential in the treatment of B-cell cancers and can achieve complete disease remission in refractory lupus, therapies that deplete all B cells are associated with excess morbidity and mortality from infection, precluding their use beyond life-threatening disease. Precision therapies that selectively target autoreactive B cells that drive SLE, while preserving normal B cell populations, are therefore critically needed, but targeting the plethora of autoreactive B cells in SLE poses practical challenges.

[0097] As used herein, the term “B cell receptors (BCRs)” refer to membrane-anchored immunoglobulins expressed on the surface of B cells, plasmablasts, and plasma cells that are encoded by one of many functional heavy-chain variable region (IGHV) genes, heavy-chain diversity region (1GHD) genes, heavy-chain joining region (IGHJ) genes, and heavy chain constant (CH) genes, together encoding the immunoglobulin heavy chain, as well as equivalent genes encoding the immunoglobulin light chain (i.e., IGKV or IGLV, IGKJ or IGLJ, and IGKC Attorney Docket No.: 44807-0494WO1 or IGLC, respectively). Each mature B cell expresses a single IGHV gene that is chosen during the process of genetic rearrangement of the IGH locus in the process of V(D)J recombination; consequently, all daughter cells derived from a specific B cell clone share the same IGHV and IGKV / IGLV gene usage. This shared and clone-specific usage offers unique opportunities for the precision targeting of B cells in cancer and autoimmune diseases.

[0098] The autoreactive B cell compartment in SLE is uniquely characterized by the expansion B cells bearing B-cell receptors (BCRs) that use the IGHV4-34 variable heavy chain. This IGHV4-34 gene encodes the 9G4 idiotype (9G4id B cells). 9G4id B cells contribute 10-45% of total IgG in patients with active SLE, including antibodies against canonical lupus autoantigens (dsDNA, DNAselL3, cardiolipin, and other nuclear antigens). 9G4id B cells are therefore promising targets for the selective depletion of the autoreactive B cell pool in SLE, opening opportunities to treat lupus without increasing the risk of infection. Moreover, 9G4id B cells play key pathogenic roles in other autoimmune diseases, including but not limited to cold agglutinin disease (comprising essentially all pathogenic clones), rheumatoid arthritis, Sjogren’s disease, and multiple sclerosis. Beyond their role in various autoimmune diseases, clonal expansion of 9G4id B cells underlies various forms of B cell lymphoma, including diffuse large B-cell lymphoma (DLBCL, -30%), chronic lymphocytic leukemia (CLL, -40%), follicular lymphoma (-5%), mantle cell lymphoma (-30%), Burkitt(-like) lymphoma (-10-20%), primary central nervous system lymphomas (-35%), vitreoretinal lymphomas (-65%), and hairy cell leukemia variant (-35%), and primary cold agglutinin-associated lymphoproliferative disease (-100%), making it an attractive precision target across a large group of B cell cancers.

[0099] In some embodiments, an engineered immune effector cell expresses a 9G4id (IGHV4-34 BCR)-targeted chimeric T cell receptor (cTCR), for the treatment of 9G4id B cell-driven autoimmune diseases or cancer. In some embodiments, an engineered immune effector cell expresses a 9G4id (IGHV4-34 BCR)-targeted chimeric antigen receptor (CAR), for the treatment of 9G4id B cell-driven autoimmune diseases or cancer.

[0100] In some embodiments, an engineered immune effector cell comprises a synthetic immune receptor that includes an antigen binding domain that binds specifically to an IGHV gene- encoded protein present on a surface of a B cell. In some embodiments, the IGHV gene-encoded protein is present on a surface of a plasmablast. In some embodiments, the IGHV gene-encoded protein is present on a surface of a plasma cell. In some embodiments, the IGHV gene-encoded Attorney Docket No.: 44807-0494WO1 protein is derived from a IGHV4-34 allele (e.g, IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene- encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

[0101] Antigen Binding Domain

[0102] In some embodiments, the antigen binding domain comprises an antibody fragment. In some embodiments, the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody (e.g, nanobody), a variable new antigen receptor (VNAR), or a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the antigen binding domain comprises a variable heavy (VH) chain and a variable light (VL) chain, wherein the VH chain and VL chain are not expressed as a single chain. In some embodiments, the antigen binding domain comprises a variable heavy (VH) chain and a variable light (VL) chain, wherein the VH chain and VL chain are expressed on different domains of a T cell receptor and assembled to form an scFv.

[0103] In some embodiments, the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences. In some embodiments, the antigen binding domain is humanized.

[0104] In some embodiments, the antigen binding domain (e.g., scFV, VHH) is linked to the TCR alpha chain. In some embodiments, the antigen binding domain (e.g., scFV, VHH) is linked to the TCR beta chain. In some embodiments, the antigen binding domain (e.g., scFV, VHH) is linked to the TCR delta chain. In some embodiments, the antigen binding domain (e.g., scFV, VHH) is linked to the TCR gamma chain. In some embodiments, the VH is linked to the TCR beta chain and the VL is linked to the TCR alpha chain. In some embodiments, the VL is linked to the TCR beta chain and the VH is linked to the TCR alpha chain. In some embodiments, the VH is linked to the TCR delta chain and the VL is linked to the TCR gamma chain. In some embodiments, the VH is linked to the TCR gamma chain and the VL is linked to the TCR delta chain. In some embodiments, the antigen binding domain (e.g., scFV, VHH) is linked to the CD3 gamma (CD3y) chain; a CD3 epsilon (CD3E) chain; a CD3 delta (CD38) chain.

[0105] Chimeric T-cell receptor Attorney Docket No.: 44807-0494WO1

[0106] In some embodiments, an engineered immune effector cell comprises a 9G4id (IGHV4- 34 BCR)-targeted chimeric T cell receptor (cTCR). As used herein, a “chimeric T cell receptor” refers to a T-cell receptor that has been genetically engineered to produce an artificial antigen- directed T-cell receptor for use in immunotherapy. A T-cell receptor (TCR) is a protein complex found on the surface of T cells or T lymphocytes, wherein the TCR is responsible for recognizing presented antigen, immune synapse formation, inducing intracellular signaling, and initiating target cell killing. The TCR can include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the TCR includes a CD3 gamma (CD3y) chain, a CD3 epsilon (CD3s) chain, a CD3 delta (CD35) chain, a T cell receptor (TCR) alpha chain (with variable [V] and / or constant [C] regions), a T cell receptor (TCR) beta chain (with variable [V] and / or constant [C] regions), a T cell receptor (TCR) gamma chain (with variable [V] and / or constant [C] regions), and a T cell receptor (TCR) delta chain (with variable [V] and / or constant [C] regions). In some embodiments, the TCR can further include a CD3 zeta (CD3Q chain.

[0107] In some embodiments, the chimeric T cell receptor comprises at least one native or modified peptide of a T cell receptor-CD3 complex protein (e.g., a T cell receptor alpha chain, beta chain, gamma chain, delta chain, a CD3 gamma subunit, a CD3 delta subunit, or a CD3 epsilon subunit, or any part thereof), and may include a linker / hinge peptide(s) or sequence(s), and / or additional intracellular domains (e.g., co-stimulatory domains, immune regulatory domains). In some embodiments, the antigen binding domain comprises a modified CD3-TCR protein complex that is expressed on the surface of the engineered immune effector cell. In some embodiments, the antigen binding domain comprises an engineered TCR alpha chain and an engineered TCR beta chain. In some embodiments, the antigen binding domain comprises an engineered TCR delta chain and an engineered TCR gamma chain.

[0108] In some embodiments, the chimeric T cell receptor further includes an intracellular costimulatory, immunomodulatory, or signaling domain that is covalently linked or independently expressed of the synthetic immune receptor. In some embodiments, the intracellular costimulatory or immunomodulatory domain is derived from full or partial sequences of CD28, 4- 1BB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRT AM, CTLA-4, DAP 10, DNAM-1, DAP12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, Attorney Docket No.: 44807-0494WO1

[0109] FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, IT AM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof. In some embodiments, the intracellular co-stimulatory domain is derived from MyD88 and CD40.

[0110] For example, in some embodiments, the chimeric T cell receptor comprises a peptide of a TCR-CD3 complex protein including a sequence from SEQ ID NOs: 1-24 (Table 1).

[0111] [Table 1] Examples of TCR-CD3 complex domains incorporated into CATCRs Attorney Docket No.: 44807-0494WO1 Attorney Docket No.: 44807-0494WO1 Attorney Docket No.: 44807-0494WO1 Attorney Docket No.: 44807-0494WO1

[0112] Chimeric Antigen Receptor (CAR)

[0113] In some embodiments, an engineered immune effector cell comprises a 9G4id (IGHV4- 34 BCR)-targeted chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain, a linker, a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0114] As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably and refer to engineered immune receptors capable of triggering or inhibiting the activation of an immune cell. With a CAR, a receptor can be programmed to recognize an antigen (e.g., 9G4id idiotope), which when bound, activates immune cells (e.g., effector cells) to kill the cell expressing that antigen (e.g., 9G4id idiotope). Therefore, immune cells expressing CAR(s) for a 9G4id idiotope expressed on a B cell can target and kill the autoreactive lupus B cell. For example, recent clinical trials of a CD19-targeted CAR-transduced T cell (CD19-CAR T cell) against hematologic malignancies showed a strong effect of CAR T technology. (Kochenderfer, J. N. et al. (2010) Blood 116: 4099-4102; Porter, D. L., et al. (2011) N. Engl. J. Med. 365: 725-733; Grupp, S. A. et al. (2013) N. Engl. J. Med. 368: 1509-1518; Kochenderfer, J. N. et al. (2015) J. Clin. Oncol. 33: 540-549; Brown, C. E. et al. (2016) N. Engl. J. Med. 375: 2561-2569). The clinical success of CAR-T therapy is attributed, at least in part, to the fusion structure of the CAR, which is made by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus, M. V. et al. (2014) Blood 123: 2625-2635; van der Stegen, S. J. et al. (2015) Nat. Rev. Drug Discov. 14: 499-509).

[0115] In some embodiments, a CAR comprises an extracellular antigen binding domain (e.g., a ligand / antigen-binding domain), a transmembrane domain and one or more intracellular signaling domains. As is known in the art, a CAR can typically comprise at least an extracellular antigen-binding domain, a transmembrane domain, a hinge region, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) that is capable of recognizing a 9G4id idiotope antigen. In some embodiments, a CAR comprises an extracellular antigen-binding domain, a hinge domain or a spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a Attorney Docket No.: 44807-0494WO1

[0116] CAR further comprises a co-stimulatory domain. In some embodiments, a CAR further comprises two co-stimulatory domains. In some embodiments, a CAR further comprises two or more co-stimulatory domains. In some embodiments, the transmembrane domain includes a CD8-alpha transmembrane domain. In some embodiments, the hinge region includes a CD28 hinge region. In some embodiments, the intracellular signaling domain includes a CD28 intracellular signaling domain or a CD3-zeta intracellular signaling domain. See, e.g., Abate- Daga et al., Molecular Therapy Oncolytics (2016) 3, 16014, the disclosure of which is incorporated herein by reference in its entirety.

[0117] In some embodiments, the transmembrane domain for use in a CAR provided herein includes a transmembrane domain from a endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD137, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CDl la,CDl lb, CDl lc, CDl ld, CDS, CEACAM1, CTLA-4, CRT AM, cytokine receptor, DAP-10,DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l,Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM,ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), an MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death- 1 (PD-1), PSGL1, SELPLG (CD 162), a Signaling Lymphocytic Activation Molecule (a SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0118] In some embodiments, a transmembrane domain for use in a CAR provided herein comprises portions of transmembrane domains present in two or more endogenous proteins, such that the chimeric transmembrane domain retains the ability to fold correctly and span the cell membrane. In some embodiments, a CAR provided herein can include a transmembrane domain that differs from a transmembrane domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR Attorney Docket No.: 44807-0494WO1 provided herein can include a transmembrane domain that shares a degree of amino acid sequence identity to a transmembrane domain present in an endogenous protein. For example, a transmembrane domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a transmembrane domain present in an endogenous protein.

[0119] In some embodiments, the intracellular signaling domain for use in a CAR provided herein includes an intracellular signaling domain from an endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD137, CD160 (BY55), CD18, CD19, CD19a,CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8,CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAM1, CTLA-4, CRT AM, a cytokine receptor, DAP- 10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2Rbeta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function- associated antigen- 1(LF A- 1), a MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), a Signaling Lymphocytic Activation Molecules (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0120] In some embodiments, an intracellular signaling domain for use in a CAR provided herein comprises portions of intracellular signaling domains present in two or more endogenous proteins, such that the chimeric intracellular signaling domain retains the ability to fold correctly and mediate signaling. In some embodiments, a CAR provided herein include an intracellular signaling domain that differs from an intracellular signaling domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein include an intracellular signaling domain that shares Attorney Docket No.: 44807-0494WO1 a degree of amino acid sequence identity to an intracellular signaling domain present in an endogenous protein. For example, an intracellular signaling domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a an intracellular signaling domain present in an endogenous protein.

[0121] In some embodiments, a CAR provided herein can include a co-stimulatory domain. In some embodiments, a CAR provided herein can include two or more co-stimulatory domains. For example, a CAR can include a co-stimulatory domain that is present in an endogenous polypeptide. Non-limiting examples of polypeptides having co-stimulatory domains that are suitable to include in engineered immune receptors provided herein include 4-1BB (CD137), CD28, CD2, CD4, 0X40, ICOS, BTLA, CD27, CD30, GITR, and HVEM, and CD8. In some embodiments, a co-stimulatory domain for use in a CAR provided herein comprises portions of co-stimulatory domains present in two or more endogenous proteins, such that the chimeric co- stimulatory domain retains the ability to fold correctly and enhance signaling. In some embodiments, a CAR provided herein include a co-stimulatory domain that differs from a co- stimulatory domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein include a co-stimulatory domain that shares a degree of amino acid sequence identity to a co-stimulatory domain present in an endogenous protein. For example, a co-stimulatory domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a co-stimulatory domain present in an endogenous protein.

[0122] Exemplary CARs, exemplary domains within CARs, and derivatives thereof (e.g., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ral72; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(l):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Attorney Docket No.: 44807-0494WO1

[0123] In some embodiments, the chimeric antigen receptor (CAR) comprises a hinge and / or linker domain (e.g., G4S, G3S, G2S, GS, EAAAK, CD8, CD28, IgGl, IgG2, IgG4), a transmembrane domain (e.g., CD8a, CD3^, CD4, CD28), at least one intracellular co-stimulatory or immunomodulatory domain (e.g., full or partial sequences of CD28, 4- IBB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP 10, DNAM-1, DAP 12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, IT AM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof), and at least one intracellular signaling / signal transducer domain (e.g., CD3(^, ZAP70, LCK, FYN, PLCG1, LCP2) in addition to the extracellular antibody fragment. In some embodiments, the CAR construct can include a sequence from SEQ ID NOs: 38-48 (Table 2).

[0124] [Table 2] Examples of CAR domains Attorney Docket No.: 44807-0494WO1

[0125] Engineered Polypeptides Attorney Docket No.: 44807-0494WO1

[0126] Provided herein are engineered polypeptides that include an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell), and a signaling domain. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV4-34 allele (e.g., IGHV4- 34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13). In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele. In some embodiments, the antigen binding domain binds specifically to a linear epitope on the IGHV gene-encoded protein on a surface of a B cell, plasmablast, or plasma cell. In some embodiments, the antigen binding domain binds specifically to a conformational epitope on the IGHV gene-encoded protein on a surface of a B cell, plasmablast, or plasma cell.

[0127] In some embodiments, engineered polypeptides can comprise a single polypeptide chain or multiple polypeptide chains. As used herein, the term “single-chain engineered polypeptide” refers to a single protein chain that includes amino acid sequences (e.g., domains) derived from at least two different sources (e.g., two different naturally-occurring proteins). In some embodiments, a single-chain engineered polypeptide includes domains from at least two different naturally-occurring animal proteins. In some embodiments, a single-chain engineered polypeptide includes domains from at least two different naturally-occurring mammalian proteins. In some embodiments, a single-chain engineered polypeptide includes domains from at least two different naturally-occurring human proteins. In some embodiments, a single-chain engineered polypeptide includes a domain that is a synthetic sequence (e.g., a scFv) and a domain that is derived from a naturally-occurring protein (e.g., a naturally-occurring animal or human protein). In some embodiments, a single-chain engineered polypeptide includes at least two different domains that are synthetic sequences (e.g., two different scFvs; one scFv and another synthetic sequence). In some embodiments, a single-chain engineered polypeptide includes a domain that is not a protein / peptide (e.g., a nucleic acid, a lipid, a phospholipid, a glycan).

[0128] As used herein, the term “multi-chain engineered polypeptide” refers to a therapeutic that comprises more than one polypeptide chain, of which at least one chain is a engineered polypeptide chain. A multi-chain engineered polypeptide, in its assembled form, includes amino Attorney Docket No.: 44807-0494WO1 acid sequences (e g., domains) derived from at least two different sources (e.g., two different naturally-occurring proteins). In some embodiments, a multi-chain engineered polypeptide includes domains from at least two different naturally-occurring animal proteins. In some embodiments, a multi-chain engineered polypeptide includes domains from at least two different naturally-occurring mammalian proteins. In some embodiments, a multi-chain engineered polypeptide includes domains from at least two different naturally-occurring human proteins. In some embodiments, a multi -chain engineered polypeptide includes a domain that is a synthetic sequence (e.g., a scFv) and a domain that is derived from a naturally-occurring protein (e.g., a naturally-occurring animal or human protein). In some embodiments, a multi-chain engineered polypeptide includes at least two different domains that are synthetic sequences (e.g., two different scFvs; one scFv and another synthetic sequence). In some embodiments, a multi-chain engineered polypeptide includes a domain that is not a protein / peptide (e.g., a nucleic acid, a lipid, a phospholipid, a glycan). In some embodiments, the chains of the multi -chain engineered polypeptide are reversibly linked. In some embodiments, the chains of the multi-chain engineered polypeptide are irreversibly linked. In some embodiments, the chains of the multichain engineered polypeptide are covalently linked. In some embodiments, the chains of the multi-chain engineered polypeptide are non-covalently linked. In some embodiments, the chains of the multi-chain engineered polypeptide are linked by dimerization or bioconjugation domains.

[0129] Nucleic acids and vectors

[0130] Provided herein are nucleic acids encoding any one of the engineered polypeptides described herein. As used herein, “nucleic acid” is used to include any compound and / or substance that comprise a polymer of nucleotides. In some embodiments, a polymer of nucleotides is referred to as polynucleotides. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs) and / or deoxyribonucleic acids (DNAs).

[0131] In some embodiments, nucleic acid constructs may be inserted into a recombinant vector or viral vector by methods known to the art, and nucleic acid molecules may be operably linked to an expression control sequence. Non-limiting examples of recombinant vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral vectors (e.g., any adenoviral vectors, cytomegaloviral [CMV] vectors, simian viral [SV40] vectors, adeno-associated virus Attorney Docket No.: 44807-0494WO1 vectors, lentiviral vectors, and retroviral vectors). In some embodiments, the recombinant vector is a viral vector.

[0132] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, recombinant vectors, adapters, and linkers is well known in the art.

[0133] Also provided herein are recombinant vectors comprising any one the nucleic acid molecules or comprising nucleic acids encoding for any of the amino acid sequences described herein. In some embodiments, nucleic acid constructs include regions that encode a 9G4id (IGHV4-34 BCR)-targeted chimeric T cell receptor (cTCR). In some embodiments, nucleic acid constructs include regions that encode a 9G4id (IGHV4-34 BCR)-targeted chimeric antigen receptor (CAR).

[0134] Also provided herein are cells comprising any one of the nucleic acid molecules or the vectors described herein. In some embodiments, nucleic acid molecules are inserted into a vector that is able to express a single-chain engineered polypeptide or a multi-chain engineered polypeptide of the present disclosure when introduced into an appropriate cell. In some embodiments, the cell can be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as chordate, mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include any human primary cell, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), or their derivatives.

[0135] Methods of introducing nucleic acids and expression vectors into a cell (e.g., an eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include electroporation, microinjection, any form of transfection, lipofection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, nanoparticle transfection, cell-penetrating peptides, or viral transduction. Attorney Docket No.: 44807-0494WO1

[0136] Pharmaceutical compositions

[0137] In some embodiments, the present disclosure provides pharmaceutical compositions that include any of the engineered immune effector cells, the engineered polypeptides, the nucleic acid molecules, or the recombinant vectors, and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition can include a buffer, a diluent, solubilizer, emulsifier, preservative, adjuvant, an excipient, or any combination thereof. In some embodiments, a pharmaceutical composition, if desired, can also contain one or more additional therapeutically active substances.

[0138] In some embodiments, pharmaceutical compositions are formulated for parenteral administration. For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection, intramuscular injection, or intravenous infusion). For example, in some embodiments, a pharmaceutical composition is provided in a liquid dosage form that is suitable for injection. In some embodiments, a pharmaceutical composition is provided as powders (e.g., lyophilized and / or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, buffer, salt solution, etc.) prior to injection. In some embodiments, a pharmaceutical composition is diluted and / or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, a powder should be mixed gently with the aqueous diluent (e.g., not shaken).

[0139] In some embodiments, a pharmaceutical composition of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer’s solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. A vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). In some embodiments, a formulation is sterilized by known or suitable techniques. A pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening, or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Attorney Docket No.: 44807-0494WO1

[0140] Practice of Pharmacy, 21 st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0141] Therapeutic Applications

[0142] Also provided herein are methods of treating an autoimmune disease in a subject that include administering to the subject any one of the engineered immune effector cells or the pharmaceutical compositions described herein. In some embodiments, the methods provided herein can be used to treat a cancer in a subject. In some embodiments, the methods provided herein can be used to treat a B-cell mediated disease.

[0143] Also provided herein are uses of the engineered immune effector cells in the manufacture of a medicament for treating an autoimmune disease in a subject in need thereof. In some embodiments, the engineered immune effector cells can be used in the manufacture of a medicament for treating a cancer in a subject in need thereof. In some embodiments, the engineered immune effector cells can be used in the manufacture of a medicament for treating a B-cell mediated disease in a subject in need thereof.

[0144] An “autoimmune disease” is a disease that arises from an abnormal immune response to a functioning body part, wherein a body’s immune system attacks and damages its own normal healthy cells or tissues. In some embodiments, in response to an unknown or known trigger, the immune system may begin producing antibodies (e.g., autoantibodies) and self-antigen-directed immune cells that, instead of fighting infections or cancer, attack the body’s own tissues. Systemic or organ-specific autoimmune diseases suitable for treatment by a method of the present disclosure can include, but are not limited to Addison’s disease, (adult-onset) Still’s disease, alopecia areata / autoimmune hair loss, antiphospholipid syndrome (APS), APS-related fetal loss, and catastrophic antiphospholipid syndrome (CAPS), autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalitis (including anti-NMDAR encephalitis), autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis or pericarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune Attorney Docket No.: 44807-0494WO1 paraneoplastic syndromes, autoimmune retinopathy, autoimmune urticaria, autoimmune uveitis, axonal & neuronal neuropathy (AMAN), Bal6 disease, Behcet’s disease, benign mucosal pemphigoid (mucous membrane pemphigoid), bullous pemphigoid, celiac disease, acute or chronic inflammatory demyelinating polyneuropathy, eosinophilic granulomatosis with polyangiitis (formerly Churg-Strauss syndrome), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease (CAD), autoantibody-mediated congenital heart block, Crohn’s disease, cryoglobulinemia / cryoglobulinemic vasculitis, dermatitis herpetiformis, various subtypes of dermatomyositis, neuromyelitis optica (NMO) spectrum disorders, discoid and other forms of cutaneous lupus, Dressier’s syndrome, eosinophilic fasciitis and eosinophilic myositis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, antibody-mediated forms of glomerulonephritis, Goodpasture’s syndrome (anti- glomerular basement membrane disease), granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schbnlein purpura (HSP), herpes gestationis or pemphigoid gestationis, hidradenitis suppurativa, IgA nephropathy, IgA vasculitis, IgG4-related disease, autoimmune interstitial lung disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), various forms of juvenile idiopathic arthritis, type 1 diabetes mellitus, juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia syndrome, lichen planus, lichen sclerosus, ligneous conjunctivitis, systemic lupus erythematosus (SLE), lupus nephritis, and drug-induced lupus, Meniere’s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myelin oligodendrocyte glycoprotein (MOG) antibody disease, idiopathic inflammatory myopathies, immune-mediated necrotizing myopathies, anti synthetase syndrome, narcolepsy, neonatal lupus, autoimmune neutropenia, autoimmune lymphopenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, paraneoplastic cerebellar degeneration, pars planitis (peripheral uveitis), pemphigus vulgaris, pemphigus vulgaris foliaceous, IgA pemphigus, paraneoplastic pemphigus, autoimmune peripheral neuropathies, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary membranous nephropathy, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, Raynaud’s phenomenon, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, scleroderma / limited cutaneous and diffuse cutaneous systemic sclerosis, Sjogren’s Attorney Docket No.: 44807-0494WO1 disease, Stiff person syndrome spectrum disorders, Susac’s syndrome, sympathetic ophthalmia, Takayasu's arteritis, thyroid eye disease, transverse myelitis, ulcerative colitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, or warm or cold autoimmune hemolytic anemia, and any autoantibody-mediated pathology.

[0145] In some embodiments, an autoimmune rheumatic disease refers to a systemic disease characterized by an abnormal immune response to normal cells and tissues, wherein the abnormal immune response is often directed against multiple tissues and organ systems of the body, such as the joints, muscles, kidneys, lungs, the skin, and / or other connective tissue. Some examples of autoimmune rheumatic diseases can include, but are not limited to rheumatoid arthritis (RA), spondyloarthropathies (e.g., ankylosing spondylitis and psoriatic arthritis), juvenile idiopathic arthritis, systemic lupus erythematosus, Sjogren’s disease, scleroderma / systemic sclerosis, idiopathic inflammatory myopathies (e.g., dermatomyositis, anti synthetase syndrome, immune-mediated necrotizing myopathies), vasculitis (e.g., granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, Henoch- Schbnlein purpura, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis).

[0146] In some embodiments, the autoimmune disease can be an autoimmune rheumatic disease. In some embodiments, the autoimmune disease is an organ-specific autoimmune disease such as Addison’s disease, celiac disease, Graves’ disease, Hashimoto thyroiditis, multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody-related diseases, neuromyelitis optica (NMO), myasthenia gravis, pernicious anemia, primary biliary cirrhosis, primary membranous nephropathy, or type I diabetes mellitus. In some embodiments, the autoimmune disease is antiphospholipid antibody syndrome (APS).

[0147] As used herein, a “B cell-mediated disease” is a disease that arises from B cell subsets, wherein those B cells directly or indirectly (e.g., through antibodies) contribute to the disease and wherein targeting of B cells directly or indirectly ameliorates the disease, its symptoms, or its clinical course. In some embodiments, the methods can be applied, through introduction of an allergen or other antigen into the polypeptide chain, to the treatment of other B cell-mediated diseases including type I allergies (e.g., any IgE-dependent allergic reactions, allergic asthma, urticaria, angioedema, allergic rhinitis), type II allergies (e.g., immune cytopenias, chronic Attorney Docket No.: 44807-0494WO1 idiopathic urticaria), type III allergies (e.g., serum sickness and serum sickness-like reactions), B cell cancers, and B cell dyscrasias (for B cell clones expressing B -cell receptors).

[0148] In some embodiments, the disease is a B cell cancer. In some embodiments, the disease is a B cell cancer, wherein the B cell cancer is a B cell lymphoma, Diffuse Large B cell Lymphoma (DLBCL), Chronic Lymphocytic Leukemia (CLL) / Small Lymphocytic lymphoma, Follicular lymphoma, Primary CNS lymphoma, primary vitreoretinal lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Hairy cell lymphoma, or a B cell acute lymphoblastic leukemia (ALL).

[0149] EXAMPLES

[0150] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0151] Example 1 - Precision Targeting of Autoreactive 9G4id B Cells in Systemic Lupus Erythematosus Using Engineered Chimeric Antigen Receptor (CAR)- and Chimeric T Cell Receptor (cTCR)-T Cells

[0152] Synthetic immune receptor T-cell therapies were developed to selectively eliminate 9G4id B cells in SLE that are optimized for the treatment of patients with autoimmune disease (FIGs. 2-3). Ramos B cells were CRISPR-Cas9-edited to replace their endogenous BCR with monoclonal BCRs from patients with SLE, CAD, or APS. PBMCs were collected from healthy donors and patients with SLE. Homology -directed repair template encoding anti-9G4 CARs or anti-9G4 chimeric T cell receptors (cTCRs) and Casl2a-gRNA ribonucleoprotein targeting TCR genes were transfected into activated T cells from healthy and SLE donors. CAR / cTCR expression was quantified by flow cytometry, and edited T cells isolated by positive selection. The potency and specificity of engineered T cells against Ramos B cells was quantified by livecell imaging (IncuCyte), flow cytometry, CellTrace Violet dye dilution to measure T-cell proliferation, and interferon (IFN)-y ELISA. SLE PBMCs were treated with engineered T cells, and depletion of IgG 9G4id B cells visualized by FluoroSpot. Autoantibody depletion was quantified using custom ELISAs.

[0153] Anti-9G4 CAR-T cells and anti-9G4 cTCR-T cells eliminated autoreactive 9G4id Ramos B cells with similar potency, while sparing non-9G4id Ramos B cells (FIGs. 7-9). Despite equal Attorney Docket No.: 44807-0494WO1 cytotoxic potency in one-time and repeated target cell stimulation assays (p>0.99 for CAR vs cTCR), anti-9G4 cTCR-T cell designs demonstrated ~10-fold lower IFN-y release as compared to anti-9G4 CAR-T cells (465 vs 4679 pg / mL for cTCR2 vs CAR, p<0.0001). Increased cytokine release in CAR-T cells was mirrored by greater T-cell proliferation following target 9G4id B cell exposure (weighted mean cell proliferation 3.24 vs 1.67 for CAR vs cTCR2, p<0.0001). Notably, 9G4 B cell-independent proliferation was observed for CAR-T cells, but not with cTCR-T cells (37.7% vs 5.1% T cells divided; weighted mean cell proliferation 1.28 vs 0.14 for CAR vs cTCR2, p<0.0001). In co-culture with SLE PBMCs, donor-matched cTCR-T cells and CAR-T cells depleted 9G4id primary human B cells in a dose-dependent manner.

[0154] The present disclosure describes precision cellular immunotherapies for the depletion of autoreactive, 9G4id B cells in SLE, CAD, and other autoimmune diseases. Both 9G4-CAR-T cells and 9G4-cTCR-T cells completely and efficiently eliminated primary or cancerous 9G4id B cells. Anti-9G4 cTCR-T cells, as compared to CAR-T cells, have preferable safety characteristics for the treatment of patients with autoimmune diseases.

[0155] Example 2. Experimental validation of IGHV-targeted Synthetic Immune Receptors

[0156] Engineered immune effector cells were evaluated for functional performance using constructs and methods described herein. Exemplary CAR constructs comprise an EAAAK linker, a CD8a hinge, a CD28 transmembrane and co-stimulatory domain, and a CD3^ signaling domain (FIG. 17); alternative CAR designs such as IgG4 hinge and (EAAAK)3 linkers are shown in FIG. 25. Genome editing was performed by CRISPR-Cas9 or Casl2a using homology-directed repair templates targeted to TCR loci, and edited cells were enriched by tNGFR expression and confirmed to lack endogenous CD3 (FIG. 6).

[0157] Functional assays were performed, including cytokine release, proliferation, cytotoxicity, and biomarker analysis. IFN-y secretion was quantified by ELISA after 62 hours at an effectortarget (E:T) ratio of 5: 1 (FIG. 10). Under these conditions, 9G4-cTCR-T cells secreted IFN-y at levels approximately ten-fold lower than 9G4-CAR-T cells (465 pg / mL vs 4,679 pg / mL; p < 0.0001), achieving >5-fold reduction. Antigen-independent proliferation was assessed by CellTrace Violet dilution after 108 hours, revealing markedly reduced spontaneous division in cTCR-T cells compared to CAR-T cells (weighted mean 0.14 vs 1.28; p < 0.0001) (FIG. 11A-B). Attorney Docket No.: 44807-0494WO1

[0158] Cytotoxicity was evaluated by live-cell imaging (IncuCyte) and flow cytometry across multiple E:T ratios, demonstrating dose-dependent elimination of 9G4id B cells with no measurable depletion of non-9G4 B cells (FIGs. 8-9, FIG. 24). In SLE PBMC co-cultures, 9G4-targeted products preserved total IgG+B-cell numbers, whereas CD19-CAR-T cells eliminated this portion (FIG. 20). Further, bulk BCR repertoire sequencing was performed, which showed depletion of VH4-34+clones with preservation of other IGHV families (FIGs. 21- 22).

[0159] Autoantibody analysis revealed significant reductions in both 9G4id and anti-dsDNA antibodies in co-culture supernatants (****p < 0.0001) (FIGs. 12-13). Finally, the compositions and methods here were evaluated in other disease states, including cold agglutinin disease (CAD) models using Ramos cells expressing CA+9G4id BCRs . Results showed 9G4-targeted cells selectively depleted pathogenic clones while sparing irrelevant B cells; cTCR-T cells again exhibited mitigated cytokine release relative to CAR-T cells (FIG. 28-30). Collectively, these data showed IGHV-targeted cTCR-T cells maintain potent, selective cytotoxicity while reducing cytokine release and antigen-independent proliferation, and preserved IgG+B-cells. The approach extends to other disease models, including CAD.

Claims

Attorney Docket No.: 44807-0494WO1WHAT IS CLAIMED IS:

1. An engineered immune effector cell comprising a synthetic immune receptor, wherein the synthetic immune receptor comprises: an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell.

2. The engineered immune effector cell of claim 1, wherein the IGHV gene-encoded protein is derived from a IGHV4-34 allele (IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13).

3. The engineered immune effector cell of claim 1, wherein the IGHV gene-encoded protein is derived from a IGHV1-46 allele.

4. The engineered immune effector cell of claim 1, wherein the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.

5. The engineered immune effector cell of any one of claims 1-4, wherein the antigen binding domain comprises an antibody fragment.

6. The engineered immune effector cell of any one of claims 1-5, wherein the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, a variable new antigen receptors (VNARs), or a variable heavy (VH) chain and a variable light (VL) chain.

7. The engineered immune effector cell of any one of claims 1-6, wherein the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences.

8. The engineered immune effector cell of any one of claims 1-7, wherein the antigen binding domain is humanized.Attorney Docket No.: 44807-0494WO19. The engineered immune effector cell of any one of claims 1-8, wherein the antigen binding domain comprises an engineered TCR alpha chain and / or an engineered TCR beta chain.

10. The engineered immune effector cell of any one of claims 1-9, wherein the antigen binding domain comprises an engineered TCR delta chain and / or an engineered TCR gamma chain.

11. The engineered immune effector cell of any one of claims 1-10, wherein the immune effector cell is a human immune cell.

12. The engineered immune effector cell of claim 11, wherein the immune effector cell is a human T cell, NK cell, or NK / T cell.

13. The engineered immune effector cell of claim 6, further comprising an engineered intracellular co-stimulatory, immunomodulatory, or signaling domain.

14. The engineered immune effector cell of claim 13, wherein the intracellular co-stimulatory or immunomodulatory domain is derived from full or partial sequences of CD28, 4- IBB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP10, DNAM-1, DAP12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, ITAM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof.Attorney Docket No.: 44807-0494WO115. The engineered immune effector cell of claim 13, wherein the intracellular co-stimulatory domain is derived from MyD88 and CD40.

16. The engineered immune effector cell of claim 1, wherein the antigen binding domain comprises a chimeric antigen receptor.

17. The engineered immune effector cell of claim 16, wherein the chimeric antigen receptor comprises an antibody fragment, a linker, a transmembrane domain, a co-stimulatory domain, and a signaling domain.

18. The engineered immune effector cell of claim 16, wherein the chimeric antigen receptor (CAR) comprises a hinge and / or linker domain, a transmembrane domain, at least one intracellular co-stimulatory or immunomodulatory domain, and at least one intracellular signaling / signal transducer domain.

19. An engineered polypeptide comprising: an antigen binding domain that binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein present on a surface of a B cell, plasmablast, or plasma cell; and a signaling domain.

20. The engineered polypeptide of claim 19, wherein the IGHV gene-encoded protein is derived from an IGHV4-34 allele (IGHV4-34*01, 34*02, 34*03, 34*04, 34*05, 34*06, 34*07, 34*08, 34*09, 34*10, 34*11, 34*12, or 34*13).

21. The engineered polypeptide of claim 19, wherein the IGHV gene-encoded protein is derived from an IGHV1-46 allele.

22. The engineered polypeptide of claim 19, wherein the IGHV gene-encoded protein is derived from an IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele.Attorney Docket No.: 44807-0494WO123. The engineered polypeptide of any one of claims 19-22, wherein the antigen binding domain comprises an antibody fragment.

24. The engineered polypeptide of any one of claims 19-23, wherein the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, a variable new antigen receptors (VNARs), or a variable heavy (VH) chain and a variable light (VL) chain.

25. The engineered polypeptide of any one of claims 19-24, wherein the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences.

26. The engineered polypeptide of any one of claims 19-25, wherein the antigen binding domain is humanized.

27. The engineered polypeptide of any one of claims 19-26, wherein the antigen binding domain comprises an engineered TCR alpha chain and / or an engineered TCR beta chain.

28. The engineered polypeptide of any one of claims 19-27, wherein the antigen binding domain comprises an engineered TCR delta chain and / or an engineered TCR gamma chain.

29. The engineered polypeptide of any one of claims 19-28, wherein the immune effector cell is a human immune cell.

30. The engineered polypeptide of claim 29, wherein the immune effector cell is a human T cell, NK cell, or NK / T cell.

31. The engineered polypeptide of claim 24, further comprising an intracellular costimulatory, immunomodulatory, or signaling domain.Attorney Docket No.: 44807-0494WO132. The engineered polypeptide of claim 31 , wherein the intracellular co-stimulatory domain or immunomodulatory domain is derived from full or partial sequences of CD28, 4- IBB, 0X40, ICOS, MYD88, 2B4, BTLA, CD2, CD22, CD27, CD30, CD3d, CD3e, CD3g, CD3z, CD4, CD40, CD79a, CD79b, CD84, CD8a, CD8b, CRACC, CRTAM, CTLA-4, DAP10, DNAM-1, DAP12, DR3, FCER1G, FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, GITR, HVEM, ITAM, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, LAT, LILRB1, LILRB2, Ly9, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, PD-1, PILRB, SIRPa, SLAMF1, SLAMF6, SLAMF7, TIGIT, TIM1, TIM3, TNFL6, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or any combination thereof.

33. The engineered polypeptide of claim 31, wherein the intracellular co-stimulatory domain is derived from MyD88 and CD40.

34. The engineered polypeptide of claim 19, wherein the antigen binding domain comprises a scFv.

35. The engineered polypeptide of claim 34, wherein the engineered polypeptide further comprises an antibody fragment, a linker, a transmembrane domain, a co-stimulatory domain, and a signaling domain.

36. The engineered polypeptide of claim 34, wherein the engineered polypeptide further comprises a hinge and / or linker domain, a transmembrane domain, at least one intracellular co-stimulatory or immunomodulatory domain, and at least one intracellular signaling / signal transducer domain.

37. A nucleic acid molecule encoding the engineered polypeptide of any one of claims 19-36.

38. A recombinant vector comprising the nucleic acid of claim 37.Attorney Docket No.: 44807-0494WO139. The engineered immune effector cell of any one of claims 1-18, wherein the synthetic immune receptor is a chimeric T-cell receptor (cTCR) comprising a VH domain linked to a TCR beta chain and a VL domain linked to a TCR alpha chain.

40. The engineered immune effector cell of claim 39, wherein the engineered immune effector cell, when contacted by 9G4id B cells, secretes a cytokine at a lower level than a 9G4-targeted CAR-T cell comparator under similar conditions.

41. The engineered immune effector cell of claim 40, wherein the cytokine is interferongamma.

42. The engineered immune effector cell of any one of claims 39-41, wherein the cell exhibits antigen-independent proliferation that is at least two-fold, at least three-fold, at least four-fold, or at least five-fold lower than a 9G4-targeted CAR-T comparator under similar conditions.

43. The engineered immune effector cell of any one of claims 39-42, wherein the cell increases preservation of IgG+ B-cells compared to a CD19-CAR-T treatment.

44. A chimeric antigen receptor (CAR) comprising:(a) an extracellular antigen-binding domain that specifically binds a 9G4 idiotope; and(b) a linker comprising an EAAAK motif.

45. The CAR of claim 44, comprising a CD8a hinge or IgG4 hinge.

46. The CAR of claim 44 or claim 45, comprising a CD28 transmembrane domain or a CD3(^ transmembrane domain.

47. The CAR of any one of claims 44-46, comprising a CD28 intracellular co- stimulatory domain.Attorney Docket No.: 44807-0494WO148. The CAR of any one of claims 44-47, comprising a CD3(j intracellular signaling domain.

49. An engineered T cell comprising the CAR of any one of claims 44-48.

50. A pharmaceutical composition comprising: any one of the engineered immune effector cells of claims 1-18 and 39-43, engineered polypeptides of claims 19-30, CARs of claims 44-48, the nucleic acid molecules of claim 37, the engineered T cell of claim 49, or the recombinant vectors of claim 38; and a pharmaceutically acceptable carrier.

51. A method of treating a disease in a subject, the method comprising: administering to the subject any one of the engineered immune effector cells of claims 1-18 and 39-43 or the pharmaceutical compositions of claim 50.

52. The method of claim 51, wherein the disease is an autoimmune disease, wherein the autoimmune disease is systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren's disease, scleroderma, rheumatoid arthritis, cold agglutinin disease, pemphigus vulgaris, or multiple sclerosis.

53. The method of claim 52, wherein the autoimmune disease is SLE.

54. The method of claim 53, wherein the method reduces one or more autoantibodies selected from 9G4id antibodies and anti-dsDNA antibodies.

55. The method of claim 52, wherein the autoimmune disease is cold agglutinin disease.

56. The method of claim 53, wherein the method depletes or significantly reduces 9G4id B cells expressing cold agglutinin B-cell receptors without significantly depleting irrelevant B cells.Attorney Docket No.: 44807-0494WO157. The method of any one of claims 52-56, wherein total IgG+ B-cell numbers are preserved relative to a CD19-CAR-T treatment.

58. The method of claim 51, wherein the disease is a B cell cancer, wherein the B cell cancer is a B cell lymphoma, Diffuse Large B cell Lymphoma (DLBCL), Chronic LymphocyticLeukemia (CLL) / Small Lymphocytic lymphoma, Follicular lymphoma, Primary CNS lymphoma, primary vitreoretinal lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Hairy cell lymphoma, or a B cell acute lymphoblastic leukemia (ALL).

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