IGHV-targeted bispecific immune effector cell engaging antibodies and uses thereof

Abstract

Provided herein are engineered polypeptides that include (a) 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, plasmablast, or plasma cell, and (b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

Description

[0001] Attorney Docket No.: 44807-0495WO1

[0002] IGHV-TARGETED BISPECIFIC IMMUNE EFFECTOR CELL ENGAGING

[0003] ANTIBODIES AND USES THEREOF

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Patent Application No. 63 / 693,408, 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 “44807-0495WOl_SL_ST26.xml.” The XML file, created on August 26, 2025, is 122,395 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 bispecific immune effector cell engaging antibodies and uses thereof. In some embodiments, it relates to the use of bispecific immune effector cell engaging antibodies to redirect T cells (or other immune cells) to bind and kill autoreactive immune cells in a subject that has an autoimmune or rheumatic disease.

[0010] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0011] This invention was made with government support under grant All 76764 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0012] BACKGROUND

[0013] CD19- and BCMA-targeted CAR-T-cell therapies hold promise for systemic lupus erythematosus (SLE) and lupus nephritis, but are critically limited by scalability, toxicity, and long-term safety. Therapies that indiscriminately deplete B cells or require cytotoxic conditioning are limited by infection risk, while integration of viral vectors into the T cell genome carries a risk of transformation and secondary T cell cancers. The autoreactive B cell compartment in active SLE is characterized by the expansion of B cells bearing the 9G4 idiotope Attorney Docket No.: 44807-0495WO1

[0014] (9G4id, IGHV4-34), a major source of disease-relevant autoantibodies (e g., anti-dsDNA, anti- DNAselL3, anticardiolipin, ANA). Provided herein are precision immunotherapies that selectively eliminate autoreactive B cells to treat patients with SLE, other autoimmune diseases, or IGHV4-34+ B cell cancers without causing immunosuppression.

[0015] SUMMARY

[0016] Provided herein are engineered polypeptides that include (a) 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, plasmablast, or plasma cell, and (b) an effector cellbinding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

[0017] 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.

[0018] In some embodiments, the engineered polypeptide comprises a single-chain polypeptide or multi-chain polypeptide. In some embodiments, the engineered polypeptide further comprises a linker sequence, a hinge, a dimerization domain, a bioconjugation domain, or any combination thereof. In some embodiments, the linker sequence, the hinge, the dimerization domain, the bioconjugation domain, or any combination thereof is between the antigen binding domain and the effector cell-binding domain.

[0019] In some embodiments, the antigen binding domain comprises an antibody, an antibody fragment, or an immunoligand. In some embodiments, the antigen binding domain comprises a single-chain antibody. In some embodiments, the antigen binding domain comprises a singlechain variable fragment (scFv), a single domain antibody, or a variable new antigen receptor (VNAR). In some embodiments, the single-chain variable fragment (scFv) comprises an immunoglobulin variable light chain and an immunoglobulin variable heavy chain. In some embodiments, the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences. In some embodiments, the antibody sequence is humanized. In some Attorney Docket No.: 44807-0495WO1 embodiments, the effector cell-binding domain comprises an antibody, antibody fragment, or immunoligand. In some embodiments, the effector cell-binding domain comprises a single-chain antibody. In some embodiments, the effector cell-binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, or a variable new antigen receptor (VNAR). In some embodiments, the single-chain variable fragment (scFv) of the effector cell-binding domain comprises an immunoglobulin variable light chain and an immunoglobulin variable heavy chain. In some embodiments, the effector cell-binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences. In some embodiments, the effector cell-binding antibody sequence is humanized.

[0020] In some embodiments, the engineered polypeptide comprises a bispecific antibody. In some embodiments, the engineered polypeptide comprises a single-chain diabody (scDb). In some embodiments, the engineered polypeptide comprises a bispecific T cell engager (BiTE).

[0021] In some embodiments, the effector cell-binding domain binds to a protein or epitope of a T cell receptor (TCR)-CD3 complex. In some embodiments, the effector cell-binding domain binds to CD3s, CD3y, CD38, Cot (TRAC), any C0 alleles (TRBC1, TRBC2), Va alleles (TRAV1-TRAV41), any V alleles (TRBV1-TRBV30), any Cy alleles (TRGC1, TRGC2), C8 (TRDC), any Vy alleles (TRGV1-9), any V8 alleles (TRDV1-3), or TCR Vy9V82 of the T cell receptor (TCR)-CD3 complex.

[0022] In some embodiments, the engineered polypeptide further comprises a fusion protein domain. In some embodiments, the fusion protein domain comprises a polypeptide sequence that extends half-life of the engineered polypeptide in vivo. In some embodiments, the polypeptide sequence is derived from an immunoglobulin constant heavy chain 1 (CHI), constant heavy chain 2 (CH2), constant heavy chain 3 (CH3), an Fc domain, an Ig constant light chain, a human plasma protein, or from peptides that extend half-life by binding to other plasma proteins. In some embodiments, the fusion protein domain further comprises an additional linker sequence. In some embodiments, the fusion protein domain is positioned at the N-terminus, at the C- terminus, or between the antigen binding domain and the effector cell-binding domain. In some embodiments, the fusion protein domain comprises a polypeptide sequence that comprises a payload or toxin. In some embodiments, the payload comprises MMAE, a glucocorticoid, a glucocorticoid receptor agonist, or a BTK inhibitor. Attorney Docket No.: 44807-0495WO1

[0023] In some embodiments, the antigen binding domain is N-terminal to the effector cellbinding domain. In some embodiments, the antigen binding domain is C-terminal to the effector cell-binding domain.

[0024] In some embodiments, the immune effector cell is a human immune cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell.

[0025] Also provided herein are nucleic acid molecules encoding any of the engineered polypeptides described herein.

[0026] Also provided herein are recombinant vectors comprising any of the nucleic acid molecules described herein.

[0027] Also provided herein are cells comprising any of the nucleic acid molecules or the recombinant vectors described herein.

[0028] Also provided herein are pharmaceutical compositions that include any of the engineered polypeptides, the nucleic acid molecules, the recombinant vectors, or the cells described herein, and a pharmaceutically acceptable carrier.

[0029] Also provided herein are methods of treating an autoimmune disease in a subject that include administering to the subject any of the recombinant vectors, the cells, or the pharmaceutical compositions described herein. In some embodiments, 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 subject is at risk of the autoimmune disease. In some embodiments, the subject has preclinical autoimmunity or a disease-associated autoantibody. In some embodiments, the disease 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).

[0030] 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 Attorney Docket No.: 44807-0495WO1 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.

[0031] 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.

[0032] BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 is an exemplary schematic showing 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, but increase the risk of infection and impair vaccine responses. C. Precise depletion of 9G4id B cells may aid in restoring immune homeostasis in SLE and other autoimmune diseases without increasing the risk of infection, while also avoiding the depletion of regulatory B cell (Breg) populations.

[0034] FIG. 2 is an exemplary schematic drawing that depicts how bystander immune cells (e.g., cytotoxic T cells) are redirected by anti-9G4id bispecific immune cell engaging antibodies to bind and kill autoreactive 9G4id (IGHV4-34+) B cells in SLE and other B cell-mediated autoimmune diseases. Bispecific antibody -induced binding of T cells induces immune synapse formation, T cell activation, and the selective killing of autoreactive 9G4id B cells via the perforin / granzyme cytotoxicity pathway.

[0035] FIG. 3 shows exemplary compositions of anti-9G4id bispecific T cell engaging antibodies (9G4id-BsAbs) for the selective depletion of autoreactive 9G4id (IGHV4-34+) B cells, regardless of isotype or subclass, in patients with SLE and other B cell-mediated autoimmune or neoplastic diseases. These 9G4id-targeted immune cell engagers can be expressed in various therapeutic antibody formats but include i) at least one binding domain that engages immune effector cells (e.g., T cells or NK cells), for example a single-chain variable fragment (scFv) Attorney Docket No.: 44807-0495WO1 targeting CD3s or other parts of the TCR-CD3 protein complex, and ii) at least one domain that binds a 9G4id (IGHV4-34+) BCR of targeted B cells. 9G4id-targeted immune cell engagers can include various linker sequences, hinges, dimerization / bioconjugation sequences, protein tags, and fusion protein domains (e.g., to extend half-life or to add a payload). These domains can be incorporated in various parts of the 9G4id-targeted immune cell engager construct, depending on the therapeutic format chosen. A bispecific antibody strategy and product using the single chain diabody (scDb) format is shown here as an example which can redirect T cells to kill autoreactive B cells carrying IGHV4-34+ B cell receptors (BCRs). BCRs can be of any isotype (IgG, IgM, IgA, IgE, or IgD) or immunoglobulin subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2). UCHTlv9 and humanized OKT3 (hOKT3) are shown as exemplary T cell engaging scFvs. LHLH or HLHL denotes the order of variable light (L) or variable heavy (H) chains. In some compositions, the immune effector cell binding domain is an scFv. In some compositions, the immune effector cell binding domain is a single-domain antibody fragment (e.g., a camelid VHH fragment). Non-limiting compositions include heterodimerized heavy chain (hetero H), asymmetric antibody-based BsAb formats with a CHI-linked domain, a Fragment crystallizable region (Fc)-linked domain, a CL-linked domain; BsAb formats comprising a F(ab’)2 variants; Fab-based BsAbs; scFv-knobs-into-holes (KIH)-based BsAb, scFv-CH3 KIH-based BsAb, minibody scFv BsAb, miniantibody scFv BsAb. In some compositions, the BsAb can bind to activating receptors as expressed on the surface of NK cells and related cells, including CD16(a), NKp30, NKp44, NKp46, NKG2D, natural killer cell receptor 2B4, DNAM1.

[0036] FIG. 4 shows exemplary compositions of anti-9G4id bispecific T cell engaging antibodies (9G4id-BsAbs) for the selective depletion of autoreactive 9G4id (IGHV4-34+) B cells, regardless of isotype or subclass, in patients with SLE and other B cell-mediated autoimmune or neoplastic diseases. These 9G4id-targeted immune cell engagers can be expressed in various therapeutic antibody formats but include i) at least one binding domain that engages immune effector cells (e.g., T cells or NK cells), for example a single-chain variable fragment (scFv) targeting CD3s or other parts of the TCR-CD3 protein complex, and ii) at least one domain that binds a 9G4id (IGHV4-34+) BCR of targeted B cells. 9G4id-targeted immune cell engagers can include various linker sequences, hinges, dimerization / bioconjugation sequences, protein tags, and fusion protein domains (e.g., to extend half-life or to add a payload). These domains can be incorporated in various parts of the 9G4id-targeted immune cell engager construct, depending on Attorney Docket No.: 44807-0495WO1 the therapeutic format chosen. A bispecific antibody strategy and product using the bispecific T cell engager (BiTE) format is shown here as an example which can redirect T cells to kill autoreactive B cells carrying IGHV4-34+ B cell receptors (BCRs). UCHTlv9 and humanized OKT3 (hOKT3) are shown as exemplary T cell engaging scFvs. LHHL, LHLH, HLHL, and HLLH denote the order of variable light (L) or variable heavy (H) chains of the respective scFvs. BsAb can bind to activating receptors as expressed on the surface of NK cells and related cells, including CD16(a), NKp30, NKp44, NKp46, NKG2D, natural killer cell receptor 2B4, DNAM1. BCRs can be of any isotype (IgG, IgM, IgA, IgE, or IgD) or immunoglobulin subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2).

[0037] FIG. 5 shows exemplary compositions of anti-9G4id bispecific T cell engaging antibodies (9G4id-BsAbs) for the selective depletion of autoreactive 9G4id (IGHV4-34+) B cells, regardless of isotype or subclass, in patients with SLE and other B cell-mediated autoimmune or neoplastic diseases. An example for a half-life extended (HLE) BiTE or scDb format using an engineered Fc domain are shown. The HLE domain can comprise various polypeptides including Fc domains, human serum albumin, other serum proteins, other non-immunogenic proteins, or antibody or other synthetic fragments that facilitate binding to other proteins or circulating cells to extend half-life.

[0038] FIG. 6 shows a visualization of an exemplary structure of a 9G4id-targeted scDb in HLHL configuration as predicted by AlphaFold. Structures may or may not contain additional tags or purification domains. An example with a C-terminal His6 tag is shown.

[0039] FIG. 7 shows SDS-PAGE of four exemplary 9G4id-BsAbs. In-gel protein stain (PageBlue) showing 9G4id-BsAbs expressed in ExpiCHO cells after purification. From left to right, protein ladder, empty, anti-9G4 BiTE12, anti-9G4 scDb8, anti-9G4 scDb6, and anti-9G4 BiTE3 are shown.

[0040] FIG. 8 shows 9G4id-BsAbs bind autoreactive B cells expressing BCR 9G4id (IGHV4-34) and immune effector cells. Ramos B cells were modified by CRISPR-Cas9 to express SLE patient- derived, pathogenic 9G4id (IGHV4-34) BCRs or irrelevant, 9G4id-negative BCRs. A. The specificity of antibody binding to engineered 9G4id and 9G4id-negative Ramos B cells was demonstrated by flow cytometry using a rat anti-9G4id antibody and APC anti-rat IgG secondary antibody. The top histogram shows no binding of anti-9G4 to 9G4id-negative B cells (BCR10). The bottom histogram shows specific binding of rat anti-9G4 to 9G4id (IGHV4-34+) Ramos B Attorney Docket No.: 44807-0495WO1 cells (BCR19). B. 9G4id Ramos B cells and 9G4id-negative Ramos cells were incubated with (top histogram) or without (bottom histogram) 9G4id-BsAb. BCR-specific binding of 9G4id- BsAbs to B cells was detected using APC anti-His6 antibody. The top histogram shows selective binding of an exemplary 9G4id-BsAb (anti-9G4id BiTE3 is shown) to 9G4id Ramos B cells (BCR19, pink histogram), no binding to 9G4id-negative Ramos B cells (BCR10, green histogram), and no binding with secondary antibody only as a control (signal overlaps fully with BCR10). C. Binding of 9G4id-BsAb (here using an anti-human CD3 scFv as a T cell engaging domain) to polyclonal human T cells is dependent on surface expression of the TCR-CD3 protein complex. Primary human T cells (unedited) or TCR-knock out (TCR KO) T cells were incubated with (top histogram) or without (bottom histogram) anti-9G4 BiTE3. Binding of anti-9G4 BiTE3 to T cells was detected using APC anti-His6 antibody. Binding of anti-9G4 BiTE3 to T cells is dependent on surface expression of the TCR (red histogram, top panel) and not observed in TCR-KO T cells (grey histogram, top panel).

[0041] FIG. 9 shows a table that summarizes equilibrium dissociation constants (KD) for four exemplary 9G4id-BsAbs against three SLE patient-derived 9G4id monoclonals (BCR17, BCR18, BCR19) obtained by surface plasmon resonance (SPR).

[0042] FIG. 10 shows SPR sensorgrams for the 9G4id-BsAb BiTE3 against three patient-derived 9G4id monoclonals (BCR17, BCR18, BCR19) and data derived from shown binding models.

[0043] FIG. 11 shows SPR sensorgrams for the 9G4id-BsAb scDb6 against three patient-derived 9G4id monoclonals (BCR17, BCR18, BCR19) and data derived from shown binding models.

[0044] FIG. 12 shows SPR sensorgrams for the 9G4id-BsAb scDb8 against three patient-derived 9G4id monoclonals (BCR17, BCR18, BCR19) and data derived from shown binding models.

[0045] FIG. 13. shows SPR sensorgrams for the 9G4id-BsAb BiTE12 against three patient-derived 9G4id monoclonals (BCR17, BCR18, BCR19) and data derived from shown binding models. FIGs. 14A-14B show Ramos B cells were modified by CRISPR-Cas9 homology-directed repair to express one irrelevant, 9G4id-negative antibody / BCR (non-9G4 B cell) and three SLE patient- derived 9G4id (IGHV4-34+) autoantibody / BCRs. FIG. 14A shows 9G4id-negative (top panel) and 9G4id Ramos B cells (bottom three panels) were incubated with rat anti-9G4 antibody and APC anti-rat IgG or anti-rat IgG alone. Histograms demonstrate no binding of rat anti-9G4 to 9G4id-negative Ramos B cells (BCR10) but binding of rat anti-9G4 to 9G4id Ramos B cells (BCR17, BCR18, BCR19, respectively). FIG. 14B shows representative results of co-culture Attorney Docket No.: 44807-0495WO1 experiments incubating either 9G4id-negative Ramos cells or three different 9G4id Ramos B cells with primary human T cells in presence of 5 ng / mL anti-9G4id scDb. Flow graphs show live, single cells stained with AF488 anti-CD20 (detecting B cells) and StrepTactin XT (detecting engineered BCRs using a Strep-tag 2) after 48 hours of co-culture.

[0046] FIGs. 15A-15B show selective and dose-dependent elimination of 9G4id Ramos B cells by anti- 9G4 BiTE12. FIG. 15A shows representative co-culture experiment of human 9G4id Ramos B cells (top panels) or 9G4id-negative Ramos B cells (bottom panels) with primary human polyclonal T cells, in the presence (right panels) or absence (left panels) of anti-9G4 BiTE12. Dot plots of flow cytometric analysis showing GFP on the X-axis (all B cells) and StrepTactin XT staining (engineered BCRs) on the Y-axis at the end of co-culture (48 hours). Anti-9G4 BiTE12 did not deplete 9G4id-negative Ramos B cells (bottom panels, Q2). In contrast, anti-9G4 BiTE12 eliminated 9G4id Ramos B cells (top panels, Q2). FIG. 15B shows specific cytotoxicity in Ramos 9G4id B cells or 9G4id-negative B cells in the presence of increasing concentrations of anti-9G4 BiTE12 and primary human polyclonal T cells. BsAb treatment achieved complete depletion of 9G4id Ramos B cells while maintaining 9G4id-negative (irrelevant) B cells.

[0047] FIG. 16 shows representative flow plots showing the selective elimination of 9G4id Ramos B cells by anti-9G4 BiTE12 at 250 ng / mL. Co-culture experiments of different human 9G4id Ramos B cell clones (SLE-BCR17, SLE-BCR18, SLE-BCR19, Ramos wild-type) or engineered, 9G4id-negative Ramos B cells (APS-BCR1, APS-BCR10) with primary human polyclonal T cells. Top panels show treatment with anti-9G4id BiTE12; bottom panel show untreated coculture controls. Dot plots of flow cytometric analysis showing GFP on the X-axis (all B cells) and StrepTactin XT staining (engineered BCRs) on the Y-axis at the end of co-culture (48 hours). Anti-9G4 BiTE12 preserved 9G4id-negative Ramos B cells (panels shown on left: "other idiotypes", Q2). In contrast, anti-9G4 BiTE12 killed 9G4id Ramos B cells, as shown in the right four panels (9G4 idiotype positive; Q2 for engineered BCR17, BCR18, BCR19; Q3 for wildtype Ramos cells).

[0048] FIG. 17 shows half maximal effective concentration (EC50) of exemplary anti-9G4 BsAbs for cytotoxicity against 9G4id Ramos B cell clones (BCR17, BCR18, BCR19) and control, irrelevant B cell clones (BCR10). Ramos 9G4id B cells or 9G4id-negative Ramos B cells were co-cultured with primary human polyclonal T cells, in the presence of increasing concentrations of the anti-9G4 BsAbs BiTE12 (top left), BiTE3 (top right), scDb8 (bottom left), or scDb6 Attorney Docket No.: 44807-0495WO1

[0049] (bottom right). The percentage of viable B cells (%) was assessed by flow cytometric analysis at the end of co-culture, normalized to corresponding no-treatment conditions for each clone. Nonlinear regression analyses for 3 replicates (dots) are shown.

[0050] FIG. 18 shows anti-9G4 BsAbs eliminate primary human 9G4id B cells in a dose-dependent manner. The frequency of 9G4id human B cells as a percentage of all primary human B cells was quantified by flow cytometry at the end of the experiment (48 hours) by staining with anti-CD19 and anti-9G4id. Left panel shows the result of a co-culture experiment of primary human B cells with either activated (purple line) or freshly isolated, unactivated (green line) primary human T cells (matched donor) in the presence of increasing concentrations of anti-9G4 BiTE12. CD3+ T cells were isolated by negative selective and activated using anti-CD3 / anti-CD28 Dynabeads. Right panel shows direct co-incubation of unactivated, primary human PBMCs with increasing concentrations of anti-9G4 BiTE12.

[0051] FIG. 19 shows anti-9G4 BsAbs eliminate primary human 9G4id B cells in patients with SLE a dose-dependent manner. The number of residual 9G4id B cells in lupus patient PBMCs was quantified by IgG FluoroSpot after co-culture in the presence of increasing concentrations of anti-9G4 BiTE12. Mean 9G4id B cell counts (+SEM) are shown for two different patients who fulfilled American College of Rheumatology classification criteria for SLE.

[0052] FIG. 20 shows B cell ELISpot results of B cells in lupus patient PBMCs. Anti-9G4 BsAbs eliminate primary human 9G4id B cells in patients with SLE a dose-dependent manner, while not impairing the total number of IgG+ antibody secreting cells (ASCs). In contrast, anti-CD19 BsAbs (blinatumomab) completely depleted both 9G4id B cells and IgG ASCs in patients with SLE. Representative data are shown for one patient.

[0053] DETAILED DESCRIPTION

[0054] This disclosure describes the use of 9G4id (IGHV4-34 BCR)-targeted bispecific immune cell engaging antibodies, including anti-9G4 bispecific T cell engaging antibodies, for the treatment of 9G4id B cell-driven autoimmune diseases and cancer.

[0055] Provided herein are engineered polypeptides that include (a) 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, plasmablast, or plasma cell; and (b) an effector cell- Attorney Docket No.: 44807-0495WO1 binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

[0056] This disclosure also provides nucleic acids, vectors, pharmaceutical compositions, and methods of treatment using the engineered polypeptides that comprise (a) 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, plasmablast, or plasma cell; and (b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

[0057] Various non-limiting aspects of these engineered polypeptides 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 polypeptides are known in the art.

[0058] 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.

[0059] 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.

[0060] 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, intraarterial, 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 Attorney Docket No.: 44807-0495WO1 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.

[0061] 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 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 Attorney Docket No.: 44807-0495WO1 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 having a binding domain which is homologous or largely homologous to an immunoglobulin- binding domain.

[0062] 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.

[0063] 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). 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 Attorney Docket No.: 44807-0495WO1 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.

[0064] As used herein, an “effector cell-binding domain” refers to an antigen binding domain comprising an antibody, an antibody fragment, or ligand, or portion thereof that specifically binds to a target moiety or entity on an immune effector cell. 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, a protein, or a small molecule. In some embodiments, an antigen binding domain may be or comprise a polypeptide (or complex thereof). In some embodiments, an effector cell-binding domain is part of a fusion polypeptide or one or more fusion polypeptide chains.

[0065] 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 moieties; 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).

[0066] 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, Attorney Docket No.: 44807-0495WO1 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.

[0067] 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.

[0068] 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 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.

[0069] 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.

[0070] 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 Attorney Docket No.: 44807-0495WO1 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.

[0071] Engineered Polypeptides

[0072] Provided herein are engineered polypeptides that include (a) 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, plasmablast, or plasma cell; and (b) an effector cellbinding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell. 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. 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 IGHV 1-46 allele. In some embodiments, the IGHV gene-encoded protein is derived from a IGHV1-2, IGHV4-59, IGHV3-7, or IGHV4-39 allele. Attorney Docket No.: 44807-0495WO1

[0073] 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 singlechain 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 singlechain 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).

[0074] As used herein, the term “multi-chain engineered polypeptide” refers to a polypeptide complex 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 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 multichain 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 Attorney Docket No.: 44807-0495WO1 engineered polypeptide are reversibly linked. In some embodiments, the chains of the multichain engineered polypeptide are irreversibly linked. In some embodiments, the chains of the multi-chain 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.

[0075] As used herein, the term “bispecific T cell engaging antibody” refers to a single-chain or multi-chain engineered polypeptide that binds T cells (e.g., a otP-T cell, a yb-T cell, a CD4+T cell, a CD8+T cell, a CD3+CD4-CD8- double-negative T cell, a Thl T cell, a Th2 T cell, a Thl7 T cell, a regulatory T cell, another T cell subset) via its effector cell-binding domain. In some embodiments, the engineered polypeptide comprises a single-chain diabody (scDb). In some embodiments, the engineered polypeptide comprises a bispecific T cell engager (BiTE).

[0076] Antigen Bindins Domain

[0077] In some embodiments, an antigen binding domain binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein sequence present on a surface of a B cell. In some embodiments, an antigen binding domain binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein sequence present on a surface of a plasmablast. In some embodiments, an antigen binding domain binds specifically to an immunoglobulin variable heavy chain (IGHV) gene-encoded protein sequence present on a surface of a plasma cell. In some embodiments, the IGHV 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). 2. 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.

[0078] In some embodiments, the antigen binding domain comprises an antibody, an antibody fragment, or an immunoligand. In some embodiments, the antigen binding domain comprises a single-chain antibody. In some embodiments, the antigen binding domain comprises a singlechain variable fragment (scFv), a single domain antibody (e.g., nanobody), or a variable new antigen receptor (VNAR). In some embodiments, the single-chain variable fragment (scFv) comprises an immunoglobulin variable light chain and an immunoglobulin variable heavy chain. Attorney Docket No.: 44807-0495WO1

[0079] 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.

[0080] In some embodiments, the engineered polypeptide comprises a bispecific or multispecific antibody. The bispecific antibody includes: a first binding domain that specifically binds to an IGHV gene-encoded protein on a B cell, plasmablast, or plasma cell; and a second binding domain that specifically binds to an immune effector cell, such as a T cell, NK cell, NKT cell, or other cytotoxic or regulatory immune cell.

[0081] The bispecific antibody may be constructed in any suitable format known in the field. For example, it may be a single-chain diabody, which is a polypeptide containing two single-chain variable fragments (scFvs) arranged to provide two distinct binding sites. In some embodiments, the bispecific antibody recognizes an IGHV epitope and another that binds to a marker on an immune effector cell, such as CD3. Another format is a bispecific T cell engager, which typically consists of a single polypeptide chain linking an IGHV-binding domain to a domain that engages T cells, thereby facilitating the redirection of T cell activity toward IGHV-expressing B cells. Other contemplated formats include stabilized bispecific molecules with two separate binding arms, tetravalent antibodies that provide two binding sites for each target, and immunoglobulin- based molecules engineered to contain two different variable domains per heavy and light chain, allowing for dual specificity. Additional formats include antibodies with domain exchanges to ensure correct pairing of binding sites, antibodies engineered to promote heterodimerization, and bispecifics constructed from antigen-binding fragments. Smaller antibody formats, such as those based on single-domain antibodies or nanobodies, may also be used to achieve bispecific or multispecific binding. Multispecific antibodies are contemplated, which are capable of binding to two or more different antigens, epitopes, or immune effector cell types.

[0082] In some embodiments, the bispecific or multispecific antibody further comprises a fusion protein domain, such as an Fc domain, albumin, or other half-life extending moiety, or a payload such as a toxin, cytokine, or other therapeutic agent.

[0083] Immunoglobulin Variable Heavy Chain (IGHV) Gene-Encoded Protein

[0084] B cells are immunopathogenic drivers of systemic lupus erythematosus (SLE), an autoimmune disease characterized by a large repertoire of autoantibodies targeting self-protein Attorney Docket No.: 44807-0495WO1 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.

[0085] 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 (IGHD) genes, heavy-chain j oining 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 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.

[0086] 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 5-45% 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 Attorney Docket No.: 44807-0495WO1 variant (-35%), and primary cold agglutinin-associated lymphoproliferative disease (-100%), making it an attractive precision target across a large group of B cell cancers.

[0087] In some embodiments, an engineered polypeptide comprises a 9G4id (IGHV4-34 BCR)- targeted bispecific immune cell engaging antibody. In some embodiments, an engineered polypeptide comprises an anti-9G4 bispecific T cell engaging antibody for the treatment of 9G4id B cell-driven autoimmune diseases or cancer. In some embodiments, an engineered polypeptide comprises a bispecific immune cell engaging antibody targeting B cell receptor variable or light chains (IGHV, IGHJ, IGKV, IGLV, IGKJ, IGLJ alleles / genes) or B cell 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.

[0088] In some embodiments, an engineered polypeptide comprises 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 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.

[0089] Non-limiting examples of a IGHV4-34 allele that encodes the IGHV gene-encoded protein are given in Table 1.

[0090] [Table 1] Non-limiting examples of IGHV4-34 alleles Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0091] Effector cell-binding domain

[0092] In some embodiments, an engineered polypeptide comprises an effector cell-binding domain that binds to an immune effector cell. As used herein, “immune effector cells” refer to Attorney Docket No.: 44807-0495WO1 cells of the immune system which can be categorized as lymphocytes (e.g., T cells, B cells, natural killer [NK] cells, NKT cells), dendritic cells, monocytes / macrophages, granulocytes (e.g., neutrophils, eosinophils, basophils), mast cells, and their subsets.

[0093] In some embodiments, the immune effector cell is a cytotoxic cell, e.g., an immune cell capable of killing through cytotoxic immune effector pathways, including perforin / granzyme- mediated cellular cytotoxicity. In some embodiments, the immune effector cell is a T cell. In specific embodiments, the immune effector cell can be a subset of T cells, e.g., a aP-T cell, a y8- T cell, a CD4+T cell, a CD8+T cell, a CD3+CD4-CD8- double-negative (DN) T cell, a Thl T cell, a Th2 T cell, a Thl 7 T cell, a regulatory T cell (Treg), another subset of T cells, or a population of T cells that comprises a combination of any of the foregoing. In some embodiments, the immune effector cell is an NK cell, an NKT cell, or an innate lymphoid cell. In some embodiments, the immune effector cell is a monocyte or macrophage. In some embodiments, the immune effector cell is another immune cell. In some embodiments, “immune effector cells” includes immune effector 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 (e.g., an engineered T cell). In some embodiments, binding of the immune effector cell through the effector cell-binding domain activates the immune effector cell. In some embodiments, binding of the effector cell-binding domain redirects the immune effector cell to kill -or otherwise therapeutically impair- target cells that are bound through the antigen binding domain.

[0094] In some embodiments, the effector cell-binding domain comprises an antibody. In some embodiments, the effector cell-binding domain comprises one or more heavy chains and light chains. In some embodiments, the effector cell-binding domain comprises one or more antibody fragments. In some embodiments, the effector cell-binding domain comprises one or more Fab, F(ab'), or F(ab')2 immunoglobulin fragments. In some embodiments, the effector cell-binding domain comprises one or more single-chain variable fragments (scFvs). In some embodiments, the effector cell-binding domain comprises at least one immunoglobulin variable heavy (VH) chain and immunoglobulin variable light (VL) chain. In some embodiments, the effector cellbinding domain is derived from human or other mammalian (e.g., monkey, murine, rat, rabbit, goat, llama, camel, alpaca, vicuna, guanaco) antibodies. In certain embodiments, the effector cell-binding domain is derived from non-mammalian antibodies (e.g., shark or other cartilaginous fish). In some embodiments, the effector cell-binding domain is derived from one Attorney Docket No.: 44807-0495WO1 or more single-domain antibodies (e.g., camelid VHH fragments, cartilaginous fish VNAR fragments). In certain embodiments, the effector cell-binding domain is derived from naturally occurring antibodies. In certain embodiments, the effector cell-binding domain comprises synthetic antibody sequences. In some embodiments, the effector cell-binding domain is a natural immunoligand. In some embodiments, the effector cell-binding domain comprises an antibody, antibody fragment, or immunoligand. In some embodiments, the effector cell-binding domain comprises a single-chain antibody. In some embodiments, the effector cell-binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, or a variable new antigen receptor (VNAR). In some embodiments, the single-chain variable fragment (scFv) of the effector cell-binding domain comprises an immunoglobulin variable light chain and an immunoglobulin variable heavy chain. In some embodiments, the effector cellbinding antibody sequence is humanized.

[0095] In some embodiments, the antibody or antibody fragment (e g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) of the effector cell-binding domain binds to a T-cell receptor (TCR)- CD3 complex protein. The T-cell receptor (TCR)-CD3 complex is a protein complex found on the surface of T-cells, wherein the TCR is responsible for recognizing presented antigen, followed by immune synapse formation, intracellular signaling, and initiation of target cell killing. The TCR-CD3 complex can include extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains. In aP-T cells, the TCR-CD3 complex includes a CD3 gamma (CD3y) chain, a CD3 epsilon (CD3s) chain, a CD3 delta (CD35) chain, a T cell receptor variable and constant alpha (Va+Ca) chain, and a T cell receptor variable and constant beta (VP+CP) chain. In yb-T cells, the TCR-CD3 complex includes a CD3 gamma (CD3y) chain, a CD3 epsilon (CD3e) chain, a CD3 delta (CD3b) chain, a T cell receptor variable and constant gamma (Vy+Cy) chain, and a T cell receptor variable and constant delta (Vb +Cb) chain. In some embodiments, the TCR-CD3 complex includes a CD3 zeta (CD3Q chain.

[0096] In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds CD3 epsilon (CD3s). In some non-limiting embodiments, the effector cell-binding domain is derived from the anti-CD3s antibody clone OKT3 or humanized OKT3. In some non-limiting embodiments, the effector cell-binding domain comprises the anti-CD3s antibody clone UCHT1 Attorney Docket No.: 44807-0495WO1 or UCHTlv9. In some non-limiting embodiments, the effector cell-binding domain is derived from the anti-CD3s antibody clones L2K-07, hXR32, 26II6, SP34. In other embodiments, the effector cell-binding domain comprises or is derived from any other antibody that binds CD3s. In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds CD3 gamma (CD3y). In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds CD3 delta (CD38).

[0097] In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds the T cell receptor variable chains. In some embodiments, the effector cell-binding domain binds at least one Vy allele (e.g., TRGV1-9), V8 allele (e.g., TRDV1-3), Va allele (e.g., TRAV1-TRAV41), or VP allele (e.g., TRBV1-TRBV30). In some embodiments, the effector cell-binding domain binds to CD3e, CD3y, CD38, Ca (TRAC), any Cp alleles (TRBC1, TRBC2), Voc alleles (TRAV1- TRAV41), any VP alleles (TRBV1-TRBV30), any Cy alleles (TRGC1, TRGC2), C8 (TRDC), any Vy alleles (TRGV1-9), any V8 alleles (TRDV1-3), or TCR Vy9V82 of the T cell receptor (TCR)-CD3 complex.

[0098] Non-limiting examples of amino acid sequences of TCR-CD3 complex proteins that are targeted by effector cell-binding domains are given in Table 2.

[0099] [TABLE 2] NON-LIMITING EXAMPLES OF TCR-CD3 COMPLEX PROTEINS TARGETED

[0100] BY THE EFFECTOR CELL-BINDING DOMAIN Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0101] Non-limiting examples of amino acid sequences encoding variable heavy (VH) chains and variable light (VH) chains, as used in effector cell-binding domains are given in Tables 3-4. [TABLE 3] NON-LIMITING EXAMPLES OF VARIABLE LIGHT CHAIN PEPTIDES FROM WHICH EFFECTOR CELL-BINDING DOMAIN IS DERIVED Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0102] [TABLE 4] NON-LIMITING EXAMPLES OF VARIABLE HEAVY CHAIN PEPTIDES FROM

[0103] WHICH THE EFFECTOR CELL-BINDING DOMAIN IS DERIVED Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0104] Non-limiting examples of amino acid sequences of NK cell activating receptors that are targeted by effector cell-binding domains are given in Table 5. [TABLE 5] NON-LIMITING EXAMPLES OF NK CELL ACTIVATING RECEPTORS

[0105] TARGETED BY THE EFFECTOR CELL-BINDING DOMAIN Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0106] In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment (e g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds the T cell receptor constant chains. In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant beta (CP) chain-1 (TRBC1). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant beta (C ) chain-2 (TRBC2). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant beta (Ca) chain (TRAC). In some embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant gamma (Cy) chain-1 (TRGC1). In some embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant gamma (Cy) chain-2 (TRGC2). In some embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant delta (C8) chain (TRDC). In some embodiments, the effector cell-binding domain comprises an Attorney Docket No.: 44807-0495WO1 antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) that binds the yb-T cell TCR Vy9V52.

[0107] In some non-limiting embodiments, the antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) of the effector cell-binding domain binds to an activating surface receptors or proteins on NK cells, NKT cells, or innate lymphoid cells. In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the low affinity immunoglobulin gamma Fc region receptor III-A (CD16A, P08637). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the natural cytotoxicity triggering receptor 3 (NKp30, 014931). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the natural cytotoxicity triggering receptor 2 (NKp44, 095944). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds the natural cytotoxicity triggering receptor 1 (NKp46, 076036). In some non-limiting embodiments, the effector cellbinding domain is derived from an antibody that binds NKG2-D type II integral membrane protein (NKG2D, P26718). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds natural killer cell receptor 2B4 (2B4, Q9BZW8). In some non-limiting embodiments, the effector cell-binding domain is derived from an antibody that binds CD226 antigen (DNAM1, Q15762). In some embodiments, the effector cell-binding domain comprises an antibody or antibody fragment that binds other activating immune cell surface receptors. In some embodiments, the effector cell-binding domain is derived from a natural or synthetic immunoligands of activating immune cell surface receptors (e.g., MHC class I polypeptide-related sequence A [MICA], MHC class I polypeptide-related sequence B [MICB], UL16-binding proteins (ULBP) 1-6, B7-H6, large proline-rich protein BAG6 / Bat3, CD48, poliovirus receptor).

[0108] In certain embodiments, the antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) of the effector cell-binding domain binds to introduced epitopes or polypeptides of engineered surface receptors of immune cells (e.g., engineered T cells or NK cells).

[0109] Linker Sequences and Fusion Protein Domains Attorney Docket No.: 44807-0495WO1

[0110] In some embodiments, the antigen binding domain is directly linked to its effector cellbinding domain. In some embodiments, the antigen binding domain is indirectly linked to its effector cell-binding domain via a linker sequence. In some embodiments, the linker sequence is a peptide linker. In some embodiments, the linker sequence is a natural peptide linker. In some embodiments, the linker sequence is a synthetic peptide linker. In some non-limiting embodiments, the linker sequence is a flexible peptide linker (e.g., [G4S]n, [SSGGGSSGGGS]n, [Gly]n,) or a rigid peptide linker (e.g., [EAAAK]n, A[EAAAK]nALEA[EAAAK]nA, AEAAAKEAAAKA, PAPAP, [Ala-Pro]n).

[0111] In some embodiments, the linker sequence comprises one or more peptide hinges (e.g., immunoglobulin [Ig] G1 CHI, CH2, CH3, CL, CD8A, CD28, or any parts thereof). In some embodiments, the linker sequence comprises reversible or irreversible dimerization domains or protein bioconjugation domains. Non-limiting examples of linkers or hinges incorporated into the engineered polypeptides are given in Table 6. [TABLE 6] NON-LIMITING EXAMPLES OF LINKERS OR HINGES INCORPORATED INTO BISPECIFIC IMMUNE EFFECTOR CELL ENGAGING ANTIBODIES Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0112] Non-limiting examples of amino acid sequences encoding fusion protein domains, as used in BiTEs, are given in Table 7. [TABLE 7] NON-LIMITING EXAMPLES OF FUSION PROTEIN DOMAINS (or any parts thereof) Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0113] In some embodiments, an engineered polypeptide further comprises at least one fusion protein domain. As used herein, the term “fusion protein domain” refers to a functional and / or structural domain within a polypeptide that can be joined to a separate protein domain so that the separate domains can be transcribed or translated as a single unit, producing a single polypeptide.

[0114] In some embodiments, a fusion protein domain can comprise a half-life extending protein domain. In certain non-limiting embodiments, the half-life extending protein domain can be derived from immunoglobulins (e.g., immunoglobulin constant heavy [CH] chain 1, CH2, CH3, Ig constant light chains), plasma proteins (e.g., human serum albumin), or be derived from peptides that extend half-life by binding to other plasma proteins. In some embodiments, the half-life extending protein domain is an immunoglobulin Fc domain.

[0115] In some embodiments, a fusion protein domain can comprise a payload. As used herein, the term “payload” refers to a peptide, polypeptide, protein, glycoprotein, enzyme, small Attorney Docket No.: 44807-0495WO1 molecule, or drug which can induce therapeutic effects (e.g., inhibition of protein synthesis, inhibition of mitosis, DNA damage, cytotoxicity, cell death) when taken up by target cells. In some embodiments, the payload can be a tubulin inhibitor. In some embodiments, the payload can be a tubulin inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, maytansinoids). In some embodiments, the payload can be a DNA damaging agent (e.g., calicheamicins, pyrrolobenzodiazepines, duocarymycins, camptothecin analogs). In some embodiments, the payload can be derived from a eukaryotic or prokaryotic protein toxin (e.g., Pseudomonas exotoxin A, diphtheria toxin). In some embodiments, the payload comprises MMAE, a glucocorticoid, a glucocorticoid receptor agonist, or a BTK inhibitor.

[0116] In some embodiments, the fusion protein domain is expressed at the N-terminus, at the C- terminus, or between the antigen binding domain and the effector cell-binding domain. In some embodiments, the fusion protein domain is expressed N-terminal to the engineered polypeptide (comprising at least the antigen binding domain and the effector cell-binding domain). In some embodiments, the fusion protein domain is expressed C-terminal to the engineered polypeptide (comprising at least the antigen binding domain and the effector cell-binding domain). In some embodiments, the fusion protein domain is expressed between the antigen binding domain and the effector cell-binding domain of the engineered polypeptide.

[0117] In some embodiments, the antigen binding domain is expressed N-terminal to the effector cell-binding domain. In some embodiments, the antigen binding domain is expressed C-terminal to the effector cell -binding domain.

[0118] In some embodiments, the engineered polypeptide comprises one or more linker sequences. In some embodiments, a linker sequence is present between the antigen binding domain and the effector cell-binding domain. In some embodiments, the effector cell-binding domain also comprises a linker sequence (e.g., between variable light [VL] chain and variable heavy [VH] chains for scFvs; between antigen binding domain and VH or VL, respectively, for diabodies). In some embodiments, the antigen binding domain further comprises a linker sequence. In some embodiments, a linker sequence is present between the fusion protein domain and the effector cell-binding domain and / or the antigen binding domain. In some embodiments, the fusion protein domain also comprises a linker sequence.

[0119] Nucleic acids and vectors Attorney Docket No.: 44807-0495WO1

[0120] 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).

[0121] 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 vectors, lentiviral vectors, and retroviral vectors). In some embodiments, the recombinant vector is a viral vector.

[0122] 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.

[0123] 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 bispecific T cell engager (BiTE) or scDb.

[0124] 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 Attorney Docket No.: 44807-0495WO1 cell, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), or their derivatives.

[0125] 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.

[0126] Pharmaceutical compositions

[0127] In some embodiments, the present disclosure provides pharmaceutical compositions that include any of the engineered polypeptides, the nucleic acid molecules, the cells, 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.

[0128] 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).

[0129] In some embodiments, a pharmaceutical composition of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are Attorney Docket No.: 44807-0495WO1 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 Practice of Pharmacy, 21st 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.

[0130] Therapeutic Applications

[0131] Also provided herein are methods of treating an autoimmune disease in a subject that include administering to the subject any one of 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.

[0132] Also provided herein are uses of the engineered polypeptides in the manufacture of a medicament for treating an autoimmune disease in a subject in need thereof. In some embodiments, the engineered polypeptides can be used in the manufacture of a medicament for treating a cancer in a subject in need thereof. In some embodiments, the engineered polypeptides can be used in the manufacture of a medicament for treating a B-cell mediated disease in a subject in need thereof.

[0133] 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 Attorney Docket No.: 44807-0495WO1 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 paraneoplastic syndromes, autoimmune retinopathy, autoimmune urticaria, autoimmune uveitis, axonal & neuronal neuropathy (AMAN), Balo 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 Attorney Docket No.: 44807-0495WO1 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 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.

[0134] 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).

[0135] 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 Attorney Docket No.: 44807-0495WO1 nephropathy, or type I diabetes mellitus. In some embodiments, the autoimmune disease is antiphospholipid antibody syndrome (APS).

[0136] 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, methods provided herein 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 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).

[0137] 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).

[0138] In some embodiments, the subject is at risk of an autoimmune disease. In some embodiments, the subject has preclinical autoimmunity or a disease-associated autoantibody.

[0139] In some embodiments, subjects undergo a selection process before treatment with a therapeutic composition disclosed herein. In some embodiments, subjects are selected based on the frequency or absolute count of B cells expressing the 9G4 idiotope (IGHV4-34+) in peripheral blood or tissue. For example, subjects may be eligible if 9G4id B cells constitute >0.5%, >1%, >2%, or >5% of total CD19+ B cells, as determined by flow cytometry using an anti-9G4 monoclonal antibody.

[0140] EXAMPLES

[0141] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims. Attorney Docket No.: 44807-0495WO1

[0142] Example 1 - Bispecific T Cell Engaging Antibodies To Selectively Target Autoreactive 9G4id B Cells in Systemic Lupus Erythematosus (SLE)

[0143] Ramos B cells were CRISPR-Cas9-edited to introduce monoclonal BCRs cloned from patients with SLE or APS. Anti-9G4 and anti-CD3 single-chain variable fragment (scFv) sequences were synthesized, cloned, and 9G4xCD3 BsAbs expressed in single-chain diabody (scDb) or bispecific T cell engager (BiTE) formats in ExpiCHO. Binding of BsAbs to 9G4id B cells, and T cells was determined by flow cytometry. Binding affinities of BsAbs were quantified by surface plasmon resonance (SPR). The potency and specificity of 9G4xCD3 BsAbs against autoreactive Ramos cells expressing SLE-derived, 9G4id BCRs or non-9G4id BCRs was interrogated in co-culture, and cytotoxicity quantified by live-cell imaging, flow cytometry, and cytokine assays. PBMCs from patients with SLE were treated with 9G4xCD3 BsAbs, and depletion of 9G4id B cells quantified by FluoroSpot.

[0144] It was hypothesized that BsAbs redirect T cells to selectively kill autoreactive 9G4id B cells (FIGs. 2 and 6). Binding of BsAbs to human T cells was dependent on CD3e surface expression (FIG. 8). 9G4xCD3 BsAbs, expressed as scDbs and BiTEs, selectively bound IGHV4-34 BCRs, but not non-9G4id B cells (FIG. 8). Equilibrium dissociation constants (KD) for 9G4xCD3 BsAbs against SLE-9G4 BCRs were 0.3-4.6 nM by SPR (FIGs. 9-13). In coculture of T cells with autoreactive 9G4id Ramos B cells expressing anti-dsDNA / anti- DNAselL3 BCRs or Ramos cells expressing non-9G4id BCRs, 9G4xCD3 BsAbs selectively eliminated 9G4id B cells, while sparing other B cells (FIG. 16). Target cell killing was observed at low BsAb concentrations (EC50 0.08-1.56 ng / mL) but maintained specificity across a wide range of titration (60 ng / mL) (FIG. 17). In co-culture with SLE PBMCs, 9G4xCD3 BiTE eliminated 9G4 B cells in a dose-dependent manner (FIG. 19).

[0145] Non-limiting examples of compositions of scFv IGHV4-34-targeted bispecific antibodies are given in Tables 8-9.

[0146] [Table 8] Non-limiting compositions for a IGHV-targeted BiTE (VH-VL-VH-VL BiTE shown) Attorney Docket No.: 44807-0495WO1 Attorney Docket No.: 44807-0495WO1

[0147] [Table 9] Non-limiting compositions for a IGHV-targeted scDb (VL-VH-VL-VH scDb shown)

[0148] The present disclosure describes an off-the-shelf, precision immunotherapy approach for the selectively depletion of autoreactive, 9G4id B cells in SLE. 9G4xCD3 T cell engaging antibodies eliminated 9G4id B cells, while sparing other B cells— an opportunity to treat SLE without increasing the risk of infection. Different to ex-vivo engineered CAR-T cells, 9G4xCD3 BsAbs can be produced and administered at scale, without increasing the risk of T-cell cancer. Attorney Docket No.: 44807-0495WO1

[0149] Beyond autoimmune diseases, the herein developed BsAbs have utility in the treatment of B cell lymphomas.

Claims

Attorney Docket No.: 44807-0495WO1WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising:(a) 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, plasmablast, or plasma cell; and(b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

2. The engineered polypeptide 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 polypeptide of claim 1, wherein the IGHV gene-encoded protein is derived from a IGHV1-46 allele.

4. The engineered polypeptide 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 polypeptide of any one of claims 1-4, wherein the engineered polypeptide comprises a single-chain polypeptide or multi-chain polypeptide.

6. The engineered polypeptide of any one of claims 1-5, wherein the engineered polypeptide further comprises a linker sequence, a hinge, a dimerization domain, a bioconjugation domain, or any combination thereof.

7. The engineered polypeptide of claim 6, wherein the linker sequence, the hinge, the dimerization domain, the bioconjugation domain, or any combination thereof is between the antigen binding domain and the effector cell-binding domain.Attorney Docket No.: 44807-0495WO18. The engineered polypeptide of any one of claims 1 -7, wherein the antigen binding domain comprises an antibody, an antibody fragment, or an immunoligand.

9. The engineered polypeptide of any one of claims 1-8, wherein the antigen binding domain comprises a single-chain antibody.

10. The engineered polypeptide of claim 9, wherein the antigen binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, or a variable new antigen receptor (VNAR).

11. The engineered polypeptide of claim 10, wherein the single-chain variable fragment (scFv) comprises an immunoglobulin variable light chain and an immunoglobulin variable heavy chain.

12. The engineered polypeptide of any one of claims 1-11, wherein the antigen binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences.

13. The engineered polypeptide of claim 12, wherein the antibody sequence is humanized.

14. The engineered polypeptide of any one of claims 1-13, wherein the effector cell-binding domain comprises an antibody, antibody fragment, or immunoligand.

15. The engineered polypeptide of any one of claims 14, wherein the effector cell-binding domain comprises a single-chain antibody.

16. The engineered polypeptide of claim 13, wherein the effector cell-binding domain comprises a single-chain variable fragment (scFv), a single domain antibody, or a variable new antigen receptor (VNAR).

17. The engineered polypeptide of claim 16, wherein the single-chain variable fragment (scFv) of the effector cell-binding domain comprises an immunoglobulin variable lightAttorney Docket No.: 44807-0495WO1 chain and an immunoglobulin variable heavy chain.

18. The engineered polypeptide of any one of claims 1-17, wherein the effector cell-binding domain is derived from human, murine, rabbit, rat, camelid, or shark antibody sequences.

19. The engineered polypeptide of claim 18, wherein the effector cell-binding antibody sequence is humanized.

20. The engineered polypeptide of any one of claims 1-20, wherein the engineered polypeptide comprises a bispecific antibody.

21. The engineered polypeptide of claim 20, wherein the engineered polypeptide comprises a single-chain diabody (scDb).

22. The engineered polypeptide of claim 20, wherein the engineered polypeptide comprises a bispecific T cell engager (BiTE).

23. The engineered polypeptide of any of the claims 1-22, wherein the effector cell-binding domain binds to a protein or epitope of a T cell receptor (TCR)-CD3 complex.

24. The engineered polypeptide of claim 23, wherein the effector cell-binding domain binds to CD3e, CD3y, CD38, Ca (TRAC), any C alleles (TRBC1, TRBC2), Voc alleles (TRAV1-TRAV41), any V0 alleles (TRBV1-TRBV30), any Cy alleles (TRGC1, TRGC2), C8 (TRDC), any Vy alleles (TRGV1-9), any V5 alleles (TRDV1-3), or TCR Vy9V82 of the T cell receptor (TCR)-CD3 complex.

25. The engineered polypeptide of any one of claims 1-24, further comprising a fusion protein domain.Attorney Docket No.: 44807-0495WO126. The engineered polypeptide of claim 25, wherein the fusion protein domain comprises a polypeptide sequence that extends half-life of the engineered polypeptide in vivo.

27. The engineered polypeptide of claim 26, wherein the polypeptide sequence is derived from an immunoglobulin constant heavy chain 1 (CHI), constant heavy chain 2 (CH2), constant heavy chain 3 (CH3), an Fc domain, an Ig constant light chain, a human plasma protein, or from peptides that extend half-life by binding to other plasma proteins.

28. The engineered polypeptide of claim 25, wherein the fusion protein domain further comprises an additional linker sequence.

29. The engineered polypeptide of claim 25, wherein the fusion protein domain is positioned at the N-terminus, at the C-terminus, or between the antigen binding domain and the effector cell-binding domain.

30. The engineered polypeptide of claim 22, wherein the fusion protein domain comprises a polypeptide sequence that comprises a payload or toxin.

31. The engineered polypeptide of claim 30, wherein the payload comprises MMAE, a glucocorticoid, a glucocorticoid receptor agonist, or a BTK inhibitor.

32. The engineered polypeptide of any one of claims 1-31, wherein the antigen binding domain is N-terminal to the effector cell-binding domain.

33. The engineered polypeptide of any one of claims 1-31, wherein the antigen binding domain is C-terminal to the effector cell-binding domain.

34. The engineered polypeptide of any one of claims 1-33, wherein the immune effector cell is a human immune cell.Attorney Docket No.: 44807-0495WO135. The engineered polypeptide of any one of claims 1-34, wherein the immune effector cell is a T cell.

36. The engineered polypeptide of any one of claims 1-34, wherein the immune effector cell is an NK cell.

37. A nucleic acid molecule encoding the engineered polypeptide of any one of claims 1-36.

38. A recombinant vector comprising the nucleic acid molecule of claim 37.

39. A cell comprising the nucleic acid molecule of claim 37 or the recombinant vector of claim 38.

40. A pharmaceutical composition comprising: any of the engineered polypeptides of claims 1-36, the nucleic acid molecules of claim 37, the recombinant vector of claim 38, or the cell of claim 39; and a pharmaceutically acceptable carrier.

41. A method of treating an autoimmune disease in a subject, the method comprising: administering to the subject the recombinant vector of claim 38, the cell of claim 39, or the pharmaceutical composition of claim 40.

42. The method of claim 41, 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.

43. The method of claim 41 or 42, wherein the subject is at risk of the autoimmune disease.

44. The method of claim of 43, wherein the subject has preclinical autoimmunity or a disease-associated autoantibody.Attorney Docket No.: 44807-0495WO145. The method of any one of claim 41-44, wherein the disease 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).

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