EP4630459A1 - Bispecific autoantigen-immune effector cell engaging antibodies and uses thereof - Google Patents

Abstract

Provided herein are chimeric polypeptides comprising: (a) an antigenic domain comprising an (auto)antigenic moiety, wherein the (auto)antigenic moiety is recognized by a B cell receptor (BCR); and (b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell, leading to effector cell-mediated therapeutic effects (e.g., cytotoxic killing) towards the target cell.

Description

BISPECIFIC AUTOANTIGEN-IMMUNE EFFECTOR CELL ENGAGING ANTIBODIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/431,162, filed on December 8, 2022, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named 44807-0432WOl_ST26_SL.xml. The XML file, created on December 8, 2023, is 294,798 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to bispecific (auto)antigen-immune effector cell engaging antibodies and uses thereof. In some embodiments, it relates to the use of bispecific (auto)antigen-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 disease. In particular, it relates to compositions of “bispecific (auto)antigen T-cell engagers” (BaiTEs) binding T cells as effector cells, or “bispecific (auto)antigen therapeutic effector cell engagers” (BaitEs) binding other immune cells (e.g., NK cells) as effector cells, for the selective targeting of cognate B cell receptors (BCRs) expressed on autoreactive or pathogenic B cells, plasmablasts, or plasma cells (hereafter “B cells”). In some embodiments, it relates to compositions of “bispecific (auto)antigen T-cell engagers” or “bispecific (auto)antigen therapeutic effector cell engagers” for the selective targeting of cognate T cell receptors (TCRs) expressed on autoreactive or pathogenic T cells. The disclosure also includes methods of generating and using (auto)antigen-immune effector cell engaging antibodies to treat autoimmune diseases and other B cell-mediated diseases (e.g., allergy). BACKGROUND

Organ-specific and systemic autoimmune diseases are characterized by the development of abnormal immune responses against self-antigens (“autoantigens”). In patients with autoimmune diseases, target tissue damage is mediated by the concerted activity of B cells and T cells which carry autoantigen-specific cell surface receptors (e.g., autoreactive B cell receptors [BCRs] and T cell receptors [TCRs], respectively). This surface receptor antigen specificity, which is shared between an individual’s pathogenic B cells and among patients with a given autoimmune disease, provides an opportunity to target disease-causing B cells therapeutically despite their structural diversity.

Preclinical and clinical human studies have demonstrated the potency of B cell-targeted therapies in the treatment of organ-specific and systemic autoimmunity. However, the use of any therapy which indiscriminately depletes the entire B cell pool is limited by toxicity (e.g., risk of severe infection, lack of protective vaccine responses, and secondary immunodeficiency). Such pan-B cell-targeted therapies can be effective means to treat life-threatening autoimmune diseases in the acute setting but are not viable therapeutic strategies for primary prevention or life-long therapy. Indeed, infection is the most common cause of excess mortality in patients treated with CD20-targeted B cell-depleting antibodies for maintenance immunosuppressive therapy. In contrast, precision immunotherapies that selectively eliminate autoreactive B cells while preserving the diversity and function of normal B cells could overcome these limitations, and effectively treat or prevent autoimmune diseases without the excess morbidity and mortality related to infectious complications.

Antiphospholipid antibody syndrome (APS) is an autoimmune disease mediated by autoantibodies against phospholipid-binding proteins that promote complement and immune activation, coagulopathy, and various forms of end-organ damage. APS can occur in isolation (primary APS) or in the context of another rheumatic disease (secondary APS), variably presenting as venous or arterial thrombosis, fetal loss, pregnancy morbidity, or disseminated coagulation with multiorgan failure. In patients with systemic lupus erythematosus (SLE), thrombosis from APS is a leading cause of mortality, accounting for 27% of deaths during a 10-year period. Healthy individuals can harbor pathogenic antiphospholipid antibodies without fulfilling clinical criteria for APS but are at risk of thrombotic events (e.g., stroke, myocardial infarction, deep venous thrombosis) and pregnancy complications.

Immunologically, APS is characterized by loss of B cell tolerance against self and the emergence of specific autoantibodies. To date, autoantibody systems targeting three phospholipidbinding protein/phospholipid complexes have been identified, collectively referred to as antiphospholipid antibodies: (i) anti-beta-2-glycoprotein I (£2GPI)/ cardiolipin; (ii) anti- endothelial protein C receptor (EPCR)/ lysobisphosphatidic acid (LBPA); and (iii) antiprothrombin (PT)/ phosphatidylserine (PS). These autoantibodies are directly pathogenic in vitro and in vivo. Strategies that eliminate the sources of these antibodies (e.g., B cells, plasmablasts, and plasma cells) may prevent or cure APS. Autoreactive B cells and plasma cells that express surface B cell receptors (BCRs) specific for phospholipid-binding proteins are therefore ideal therapeutic targets for antigen-specific depletion strategies in patients with APS.

Therapies that can selectively eliminate autoreactive B cells targeting phospholipidbinding proteins (or their phospholipid complexes), and thereby eliminate the sources of pathogenic antibodies in APS, are advantageous to treat and prevent APS without increasing the risk of bleeding or infection. Furthermore, the herein described precision therapies provide methods and compositions to prevent and treat other specified autoimmune diseases and B cell- mediated diseases (e.g., allergy) with curative intent, while sparing protective immune and vaccine responses.

SUMMARY

Provided herein are chimeric polypeptides comprising: (a) an antigenic domain comprising an (auto)antigenic moiety, wherein the (auto)antigenic moiety is recognized by a B cell receptor (BCR); and (b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

In some embodiments, the chimeric polypeptide comprises a single-chain polypeptide or multi-chain polypeptide. In some embodiments, the chimeric polypeptide further comprises a linker sequence, hinge, dimerization domain, bioconjugation domain, or any combination thereof. In some embodiments, a first linker sequence, hinge, dimerization domain, bioconjugation domain, or any combination thereof is between the antigenic domain and the effector cell-binding domain.

In some embodiments, the (auto)antigenic moiety is derived from a phospholipid-binding protein. In some embodiments, the phospholipid-binding protein is beta-2 glycoprotein I (P2GPI), endothelial protein C receptor (EPCR), prothrombin (PT). In some embodiments, the (auto)antigenic moiety comprises at least part of a domain of 02GPI, wherein 02GPI comprises domain I (DI), domain II (DII), domain in (Dill), domain IV (DIV), or domain V (DV); an amino acid epitope comprising a sequence of “RGGMR”; any of their orthologs; or any combination thereof. In some embodiments, the (auto)antigenic moiety comprises at least part of an endothelial protein C receptor (EPCR); a full or part of an extracellular (EC) domain of EPCR; a modified extracellular (EC) domain of EPCR; or an EPCR in complex with a phospholipid. In some embodiments, the (auto)antigenic moiety comprises at least part of prothrombin (PT) or posttranslationally modified prothrombin; or a PT in complex with a phospholipid.

In some embodiments, the (auto)antigenic moiety further comprises a second linker sequence or hinge within the antigenic domain.

In some embodiments, the (auto)antigenic moiety is derived from proteinase 3 (PR3), myeloperoxidase (MPO), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG), coagulation factor VIII (FVIII), muscle skeletal receptor tyrosine-protein kinase (MuSK), phospholipase A2 receptor (PLA2R), disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), E3 ubiquitin-protein ligase TRIM21 (TRIM21), signal recognition particles (SRP), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), protein-arginine deiminase type-4 (PAD4), desmoglein 1 (DSG1), or desmoglein 3 (DSG3). In some embodiments, the (auto)antigenic moiety is a post-translationally modified peptide. In some embodiments, the posttranslational modification is citrullination/deimination, carbamylation, acetylation, glycosylation, deamination, phosphorylation, oxidation, or y-carboxylation.

In some embodiments, the (auto)antigenic moiety is mutated or otherwise modified to inhibit or abrogate specific or unspecific binding to tissues, cells, membranes, receptors, or ligands other than their cognate B cell receptors, to inhibit or abrogate enzymatic activity, or to alter other undesired biological function of the antigen.

In some embodiments, the (auto)antigenic moiety comprises a lipid or phospholipid. In some embodiments, the (auto)antigenic moiety comprises a single-stranded or double-stranded nucleic acid or an oligonucleotide. In some embodiments, the (auto)antigenic moiety comprises more than one (auto)antigen.

In some embodiments, the effector cell-binding domain is derived from an antibody, antibody fragment, or immunoligand. In some embodiments, the effector cell-binding domain comprises an immunoglobulin variable light chain, variable heavy chain, or both. 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). In some embodiments, the scFv comprises a variable light chain and a variable heavy chain.

In some embodiments, the effector cell-binding domain binds to a protein or epitope of the T cell receptor (TCR)-CD3 complex. In some embodiments, the effector cell-binding domain binds to CD3e, CD3y, CD35, Ca (TRAC), any Cp alleles (TRBC1, TRBC2), Va alleles (TRAV1- TRAV41), any Vp 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. In some embodiments, the effector cell-binding domain binds to a surface activating receptor or co-receptor on an immune effector cell.

In some embodiments, the effector cell-binding domain further comprises a third linker sequence. In some embodiments, the third linker sequence is between the variable light chain and the variable heavy chain.

In some embodiments, the effector cell-binding domain comprises more than one effector cell-binding domain. In some embodiments, the additional immune effector cell-binding domain comprises an antibody, antibody fragment, or immunoligand which provides a co-stimulatory signal to the immune effector cell.

In some embodiments, the chimeric polypeptide further comprises a fusion protein domain. In some embodiments, the fusion protein domain comprises a protein molecule that extends halflife of the chimeric polypeptide in vivo. In some embodiments, the protein molecule 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 comprises a protein molecule that comprises a payload or toxin.

In some embodiments, the fusion protein domain further comprises a fourth linker sequence. In some embodiments, the fusion protein domain is expressed N-terminal to both the antigenic domain and the effector cell-binding domain. In some embodiments, the fusion protein domain is expressed C-terminal to both the antigenic domain and the effector cell-binding domain. In some embodiments, the fusion protein domain is expressed in-between the antigenic domain and the effector cell-binding domain. In some embodiments, the antigenic domain is expressed N- terminal to the effector cell-binding domain. In some embodiments, the antigenic domain is expressed C-terminal to the effector cell-binding domain.

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.

Also provided herein are nucleic acid molecule encoding any one of the chimeric polypeptide described herein.

Also provided herein are recombinant vectors comprising any one of the nucleic acid molecules described herein.

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

Also provided herein are pharmaceutical compositions comprising: any of the chimeric polypeptides, the nucleic acid molecules, the recombinant vectors, or the cells described herein; and a pharmaceutically acceptable carrier.

Also provided herein are methods of treating a B cell-mediated disorder in a subject, the method comprising: administering to the subject any one of the recombinant vectors, the cells, or the pharmaceutical compositions described herein. In some embodiments, the B cell-mediated disease is an allergy.

In some embodiments, the B cell-mediated disease is an autoimmune disease. In some embodiments, the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease.

In some embodiments, the organ-specific autoimmune disease is acquired haemophilia, autoimmune encephalitis, anti-N-methyl-d-aspartate (NMDA) receptor encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune and paraneoplastic encephalitis, Addison’s disease, celiac disease, Graves’ disease, Hashimoto thyroiditis, immune thrombocytopenia purpura, multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody-related diseases, neuromyelitis optica (NMO) spectrum disorders, myasthenia gravis, Lambert-Eaton myasthenia syndrome, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, other autoimmune blistering diseases, autoimmune membranous nephropathy, primary membranous nephropathy, primary biliary cirrhosis, thrombotic thrombocytopenic purpura, or type I diabetes mellitus.

In some embodiments, the systemic autoimmune disease is rheumatoid arthritis, a spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, lupus nephritis, Sjogren’s disease, scleroderma/ systemic sclerosis, an idiopathic inflammatory myopathy, myositis, dermatomyositis, anti synthetase syndrome, an immune-mediated necrotizing myopathy, IgG4-related disease, vasculitis, granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, anti- glomerular basement membrane disease, Henoch-Schbnlein purpura, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, or giant cell arteritis.

In some embodiments, the autoimmune disease is antiphospholipid syndrome (APS) or catastrophic antiphospholipid syndrome.

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

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1C show exemplary schematic drawings that depicts how (auto)antigen-immune effector cell engaging antibodies function for the treatment of autoimmune disease and B-cell disorders. FIG. 1A shows model representation of a surface T cell receptor (TCR, bottom) of a bystander T cell in proximity to a surface B cell receptor (BCR, top) of an autoreactive B cell in the presence of a bispecific (auto)antigen-T cell engager (BaiTE) therapeutic antibody which can cross-link both cells by binding to the TCR with one end (e.g., using an anti-CD3 epsilon [CD3e] scFv) and to the BCR with the other end using cognate (auto)antigen as the binding moiety. An anti-CD3s scFv-BaiTE is shown as a non-limiting example.

FIG. IB shows how bispecific (auto)antigen T-cell engagers (BaiTEs) mediate binding and killing of autoreactive B cells expressing cognate B cell receptors (BCRs) by bystander T cells in patients with APS and other B cell-mediated autoimmune diseases. The exemplary schematic shows a T cell (with TCR) that is bound to the BCR on the surface of an autoreactive B cell through a BaiTE, resulting in immune synapse formation, T cell activation, and selective cytotoxic killing of the autoreactive B cell via the perforin/granzyme pathway.

FIG. 1C shows how bispecific (auto)antigen therapeutic effector cell engagers (BaitEs) mediate binding and killing of autoreactive B cells expressing cognate B cell receptors (BCRs) by bystander NEC cells in patients with APS and other B cell-mediated autoimmune diseases. The exemplary schematic shows an NK cell (with activating surface receptor, e.g., CD16a) that is bound to the BCR on the surface of an autoreactive B cell through a BaitE, resulting in immune synapse formation, NK cell activation, and selective cytotoxic killing of the autoreactive B cell via the perforin/granzyme pathway.

FIGs. 2-4 show exemplary compositions of (auto)antigen-immune effector cell engaging antibodies for the selective depletion of autoreactive B cells in patients with APS and other B cell- mediated diseases. BaiTEs/ BaitEs 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 incorporates antigenic sequences recognized by the BCR of targeted B cells (e.g., an autoantigen, antigen, epitope, their mimotopes, or any parts thereof). BaiTEs/ BaitEs 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 BaiTE/ BaitE construct, depending on the therapeutic format chosen. FIG. 2 shows model representations of BaiTE variants to target B cells carrying autoreactive BCRs (e.g., BCRs that bind the autoantigen beta-2-glycoprotein I [02GPI]). Native T cell receptors (TCRs) with their structural subunits/domains CD3 gamma (y), CD3 delta (8), CD3 epsilon (s), TCR alpha constant region (Ca), TCR beta constant region (C0), TCR alpha variable region (Voc), TCR beta variable region (V0), TCR gamma constant region (Cy), TCR delta constant region (C8), TCR gamma variable region (Vy), TCR delta variable region (V8), and CD3 zeta (Q, respectively, are shown on the bottom. Autoreactive BCRs as expressed on the cell surface of autoreactive B cells are shown on the top, including their structural subunits/domains constant heavy chains (CH), constant light chain (CL), variable heavy chain (VH), and variable light chain (VL). These BCRs can be of any isotype (IgG, IgM, IgA, IgE, or IgD) or immunoglobulin subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2). Exemplary BaiTE variants using effector cell-binding domains engaging CD3 proteins (i.e., CD3e, CD3y, or CD36), or Ca (TRAC), or any C(3 alleles (TRBC1, TRBC2), or any Va alleles (TRAV), or any V0 alleles (TRBV), or any Cy alleles (TRGC1, TRGC2), or C8 (TRDC), or any Vy alleles (TRGV), or any V8 alleles (TRDV), or TCR Vy9V82, are shown. scFv BaiTEs are shown as an exemplary BaiTE format for these variants. “(Auto)antigen” denotes the antigenic domain (i.e., cognate B cell receptor-binding domain) of the BaiTE (e.g., beta-2-glycoprotein I or any parts thereof).

FIG. 3 shows model representations of bispecific (auto)antigen therapeutic effector cell engager (BaitE) variants to target B cells carrying autoreactive BCRs (e.g., BCRs that bind the autoantigen beta-2-glycoprotein I [02GPI]). Exemplary activating receptors as expressed on the surface ofNK cells and related cells, including CD16(a), NKp30, NKp44, NKp46, NKG2D, natural killer cell receptor 2B4, DNAM1, are shown on the bottom. Autoreactive BCRs as expressed on the cell surface of autoreactive B cells are shown on the top row, including their structural subunits/domains constant heavy chains (CH), constant light chain (CL), variable heavy chain (VH), and variable light chain (VL). These BCRs can be of any isotype (IgG, IgM, IgA, IgE, or IgD) or immunoglobulin subclass (IgGl, IgG2, IgG3, Ig4; IgAl, IgA2). Exemplary BaitE variants using effector cell-binding domains engaging CD 16(a), or NKp30, or NKp44, or NKp46, or NKG2D, or 2B4, or DNAM1 are shown. scFv BaitEs are shown as an exemplary BaitE format. “(Auto)antigen” denotes the antigenic domain (cognate B cell receptor-binding domain) of the BaiTE (e.g., beta-2 -glycoprotein I or parts thereof). FIG. 4A-B shows non-limiting antibody format variations for bispecific (auto)antigen-immune effector cell engaging antibodies (i.e., BaiTEs or BaitEs) described in FIGs. 2-3. Antibody format variations are shown for construct designs comprising an effector cell-binding domain that engages CD3s (yellow) but equally apply to BaiTE/ BaitE constructs incorporating any other effector cellbinding domains as described in FIGs. 2-3.

FIG. 4A shows compositions of six alternative BaiTE antibody formats as compared to an scFv BaiTE (first row, left), which uses an scFv as the effector cell-binding domain. Non-limiting alternative compositions shown include (single-chain) diabody BaiTEs, which introduce the antigenic domain between the variable heavy and variable light chains of the effector cell-binding domain (second from left), nanobody/VnH BaiTEs, which use a single-domain antibody fragment as the effector cell-binding domain (third from left), Fab BaiTEs, which use a Fab-derived effector cell-binding domain (second from right), half-life extended BaiTEs, which add at least one halflife extending fusion protein domain to the BaiTE (right), and BaiTE designs based on modified full or partial asymmetric antibodies (second row). Native T cell receptors (TCRs) with their structural subunits/domains CD3 gamma (y), CD3 delta (8), CD3 epsilon (s), TCR alpha constant region (Coe), TCR beta constant region (C0), TCR alpha variable region (Va), TCR beta variable region (V ), and CD3 zeta (Q are shown for context. Autoreactive BCRs of any isotype or subclass as expressed on the cell surface of autoreactive B cells are also shown for context, including their structural subunits/domains: constant heavy chains (CH), constant light chain (CL), variable heavy chain (VH), and variable light chain (VL).

FIG. 4B shows compositions of additional BaiTE/ BaitE antibody formats as compared to the structure of a full human antibody (top row, left) with its structural subunits/domains: constant heavy chain (CH) domain 1 (CHI), constant heavy chain (CH) domain 2 (CH2), constant heavy chain (CH) domain 3 (CH3), constant light chain (CL), variable heavy chain (VH), and variable light chain (VL). Effector cell-binding domains for each format are shown in yellow, including the relevant effector-engaging variable heavy chain (VH) and variable light chain (VL). In some compositions, the effector cell-binding domains is an scFv. In some compositions, the effector cell-binding domains is a single-domain antibody fragment (e.g., a camelid VHH fragment). Nonlimiting compositions shown include heterodimerized heavy chain (hetero H), asymmetric antibody-based BaiTE formats with a CHI -linked antigenic domain, a Fragment crystallizable region (Fc)-linked antigenic domain, a CL-linked antigenic domain (top row); BaiTEs formats comprising a F(ab’)2 variants (middle row); Fab-based BaiTEs linking the antigenic domains to CHI, or CL, or VL, or VH (middle row); scFv-knobs-into-holes (KIH)-based BaiTEs, scFv-CH3 KIH-based BaiTEs, minibody scFv BaiTEs, miniantibody scFv BaiTEs (bottom row); BaiTEs comprising their variants using single-domain antibody fragment instead of scFvs (bottom row); scFv-CH-CL-antigen-based BaiTEs and antigen-CH-CL-scFv-based BaiTE designs (bottom row).

FIGs. 5A-5B show schematic representations of exemplary BaiTE/ BaitE construct designs (i.e., scFv BaiTEs/ BaitEs) to selectively target autoreactive B cells expressing B cell receptors (BCRs) for a specific autoantigen. “Linker” denotes any linker sequence; "VH” denotes the variable heavy chain of the scFv of the effector cell-binding domain; “VL” denotes the variable light chain of the scFv of the effector cell-binding domain; “autoantigen(s)” denotes the antigenic domain (cognate B cell receptor-binding domain) of the BaiTE (e.g., the autoantigen beta-2-glycoprotein I [f32GPI] as recognized by disease-causing autoreactive B cells in patients with antiphospholipid syndrome); “+/-“ denotes absence or presence of a domain; “fusion protein” denotes any fusion protein domain (e.g., half-life extending protein domain, payload, toxin).

FIG. 5A shows exemplary construct designs for scFv BaiTEs/ BaitEs without a fusion protein domain. These scFv BaiTEs/ BaitEs comprise i) an effector cell-binding domain (e.g., an scFv specific for human CD3s such as scFvs derived from clones 0KT3, UCHT1, UCHTlv9, L2K-07, hXR32, 26II6, SP34, among many others; or an scFv specific for a surface activating receptor expressed on NK cells), and ii) an antigenic domain (i.e., the BCR-binding domain) that incorporates sequences of cognate autoantigen(s) (including full or partial sequences, epitopes, mimotopes, or highly homologous protein sequences) recognized by the targeted BCR. The antigenic domain of the BaiTE/ BaitE can be N-terminal or C-terminal to its effector cell-binding domain. The variable heavy chain (VH) of the effector cell-binding domain can be N-terminal or C-terminal to the variable light chain (VL) of the effector cell-binding domain. The antigenic domain and the effector cell-binding domain of the scFv BaiTE/ BaitE can be directly linked or indirectly linked via addition of a linker sequence (e.g., linker, hinge, dimerization domain, bioconjugation domain). Additional construct requirements are dependent on the mode of expression and may include, but are not limited to, promoter sequence, Kozak sequence, signal peptide(s), protein tag(s), stop codon, mammalian termination and polyadenylation signal sequence(s) (e.g., SV40, hGH, BGH, rbGlob poly(A) sequences). FIG. 5B shows exemplary construct designs as in FIG. 5A but for scFv BaiTEs/ BaitEs with a fusion protein domain. The fusion protein domain (e.g., a half-life extending protein, payload, toxin) can be directly linked to the antigenic domain and/or effector cell-binding domain of the scFv BaiTE/ BaitE. Alternatively, the fusion protein domain (e.g., a half-life extending protein, payload, toxin) can also be indirectly linked to the antigenic domain and/or effector cell-binding domain of the scFv BaiTE/ BaitE via addition of a linker sequence (e.g., a linker, hinge, dimerization domain, bioconjugation domain). The fusion protein domain can be N-terminal to the antigenic and effector cell-binding domains. The fusion protein domain can be C-terminal to the antigenic and effector cell-binding domains. The fusion protein domain can be in-between the antigenic domain and effector cell-binding domain. The fusion protein domain can be N-terminal to the scFv BaiTE/ BaitE. The fusion protein domain can be C-terminal to the scFv BaiTE/ BaitE.

FIGs. 6A-6B show schematic representations of exemplary BaiTE/ BaitE construct designs (i.e., nanobody BaiTEs/ BaitEs) to selectively target autoreactive B cells expressing B cell receptors (BCRs) for a specific autoantigen. “Linker” denotes any linker sequence; "VHH” denotes the single-domain antibody fragment (e.g., camelid VHH fragments) of the effector cell-binding domain; “autoantigen(s)” denotes the antigenic domain (cognate B cell receptor-binding domain) of the Bai TE/ BaitEs (e.g., the autoantigen beta-2-glycoprotein I [02GPI] as recognized by diseasecausing autoreactive B cells in patients with antiphospholipid syndrome); “+/-“ denotes absence or presence of a domain; “fusion protein” denotes any fusion protein domain (e.g., half-life extending protein domain, payload, toxin).

FIG. 6A shows exemplary construct designs for nanobody BaiTEs/ BaitEs without a fusion protein domain. These nanobody BaiTEs/ BaitEs comprise i) an effector cell-binding domain (e.g., a VHH specific for human CD3s; or a VHH specific for a surface activating receptor expressed on NK cells), and ii) an antigenic domain (i.e., the BCR-binding domain) that incorporates sequences of cognate autoantigen(s) (including full or partial sequences, epitopes, mimotopes, or highly homologous protein sequences) recognized by the targeted BCR. The antigenic domain of the BaiTE/ BaitE can be N-terminal or C-terminal to its effector cell-binding domain. The antigenic domain and the effector cell-binding domain of the nanobody BaiTE/ BaitE can be directly linked or indirectly linked via addition of a linker sequence (e.g., linker, hinge, dimerization domain, bioconjugation domain). Additional construct requirements are dependent on the mode of expression and may include, but are not limited to, promoter sequence, Kozak sequence, signal peptide(s), protein tag(s), stop codon, mammalian termination and polyadenylation signal sequence(s) (e.g., SV40, hGH, BGH, rbGlob poly(A) sequences).

FIG. 6B shows exemplary construct designs as in FIG. 6A but for nanobody BaiTEs/ BaitEs with a fusion protein domain. The fusion protein domain (e.g., a half-life extending protein, payload, toxin) can be directly linked to the antigenic domain and/or effector cell-binding domain of the nanobody BaiTEZ BaitE. Alternatively, the fusion protein domain (e.g., a half-life extending protein, payload, toxin) can also be indirectly linked to the antigenic domain and/or effector cellbinding domain of the nanobody BaiTE/ BaitE via addition of a linker sequence (e.g., a linker, hinge, dimerization domain, bioconjugation domain). The fusion protein domain can be N- terminal to the antigenic and effector cell-binding domains. The fusion protein domain can be C- terminal to the antigenic and effector cell-binding domains. The fusion protein domain can be inbetween the antigenic domain and effector cell-binding domain. The fusion protein domain can be N-terminal to the nanobody BaiTE/ BaitE. The fusion protein domain can be C-terminal to the nanobody BaiTE/ BaitE.

FIGs. 7A-7C show schematic representations of exemplary BaiTE/ BaitE construct designs (i.e., asymmetric antibody-based BaiTEs/ BaitEs) to selectively target autoreactive B cells expressing B cell receptors (BCRs) for a specific autoantigen. "VH” denotes the variable heavy chain of the effector cell-binding domain; “VL” denotes the variable light chain of the effector cell-binding domain; “CHI” denotes constant heavy chain 1; “CH2” denotes constant heavy chain 2; “CH3” denotes constant heavy chain 3; “CL” denotes constant light chain; “Hinge” denotes hinge region of immunoglobulin; “CHS” denotes C-terminal end of a secreted immunoglobulin heavy chain; “autoantigen(s)” denotes the antigenic domain (cognate B cell receptor-binding domain) of the BaiTEZ BaitEs (e.g., the autoantigen beta-2-glycoprotein I [P2GPI] as recognized by diseasecausing autoreactive B cells in patients with antiphospholipid syndrome).

FIG. 7A shows an exemplary construct design for an asymmetric antibody -based BaiTEs/ BaitEs with a CHI -linked antigenic domain. The heavy and light chains (yellow) comprising the effector cell-binding domain are shown on the left. The heavy chain comprising the CHI -linked antigenic domain is shown on the right. Both can be combined to form an asymmetric antibody BaiTEs/ BaitEs. FIG. 7B shows an exemplary construct design for an asymmetric antibody -based BaiTEs/ BaitEs as in FIG. 7A, but with a CH2 -linked antigenic domain.

FIG. 7C shows an exemplary construct design for an asymmetric antibody -based BaiTEs/ BaitEs as in FIG. 7A, but with a CL-linked antigenic domain. The heavy chain and the light chain comprising the CL-linked antigenic domain are shown on the right.

FIGs. 8A-8B show schematic representations of scFv BaiTE/ BaitE construct designs that have been developed for the selective depletion of autoreactive B cells that bind the autoantigen beta- 2-glycoprotein I (02GPI), as found in patients with APS. These 02GPI-directed BaiTEs/ BaitEs (“P2GPI-BaiTEs/ BaitEs”) comprise an effector cell-binding domain (e.g., a T cell-engaging scFv specific for human CD3e or an NK cell-engaging domain specific for an activating surface receptor expressed on NK cells) and an antigenic domain (i.e., a BCR-binding domain) that incorporates autoantigenic sequences of 02GPI (e.g., including full or partial P2GPI, its domains, any epitopes, any mimotopes, any highly homologous protein sequences, or any combination thereof).

FIG. 8A shows different scFv p2GPI-BaiTE/ p2GPI-BaitE construct designs that have been developed. “Linker” denotes any linker sequence; "VH” denotes the variable heavy chain of the scFv of the effector cell-binding domain; “VL” denotes the variable light chain of the scFv of the effector cell-binding domain; “P2GPI” denotes beta-2-gly coprotein I; DI = P2GPI domain I; DII = domain II; Dill = domain III; DIV = domain IV; DV = domain V (including mutated sequences); “+/-“ denotes absence or presence of a domain.

FIG. 8B shows exemplary SDS-PAGE and in-gel protein stain of BaiTEs expressed in ExpiCHO mammalian cells after purification from culture supernatant. A single-chain diabody (scDb) of known concentration is shown as a standard.

FIG. 9 shows schematic representations of exemplary scFv BaiTE/ BaitE construct designs incorporating a fusion protein domain that have been developed for the selective depletion of autoreactive B cells that bind the autoantigen beta-2-glycoprotein I (P2GPI), as found in patients with APS. These p2GPI-BaiTEs/ BaitEs comprise an effector cell-binding domain (e.g., a T cellengaging scFv specific for human CD3s or an NK cell-engaging domain specific for an activating surface receptor expressed on NK cells), an antigenic domain (i.e., a BCR-binding domain) that incorporates autoantigenic sequences of [32GP I (e.g., including full or partial |32GPI, its domains, any epitopes, any mimotopes, any highly homologous protein sequences, or any combination thereof), and a fusion protein domain (e.g., a half-life extending single-chain Fc domain [scFc HLE], another half-life extending protein, a plasma protein-binding domain). “Linker” denotes any linker sequence; "VH” denotes the variable heavy chain of the scFv of the effector cell-binding domain; “VL” denotes the variable light chain of the scFv of the effector cell-binding domain; “P2GPI” denotes beta-2-glycoprotein I; DI = P2GPI domain I; DII = domain II; Dill = domain III; DIV = domain IV; DV = domain V (including mutated sequences); “Fc CH2” denotes constant heavy chain 2 of the Fc domain, “Fc CH3” denotes constant heavy chain 3 of the Fc domain; “+/- “ denotes absence or presence of a domain. Plasma protein binding domain refers to any domain that facilitates binding to human plasma proteins (e g., human serum albumin [HSA]) for the purpose of half-life extension (e.g., a scFv, a natural ligand, a synthetic ligand). Immunoligands refers to any natural or synthetic ligands that binds to one or more of the immune effector cell proteins otherwise targeted by an antibody or an antibody fragment.

FIG. 10 shows schematic representations of scFv BaiTE/ BaitE construct designs for targeting other autoreactive B cells in patients with APS and other autoimmune diseases. The non-limiting examples shown include scFv BaiTE/ BaitE construct designs with antigenic domains incorporating autoantigenic sequences of the endothelial protein C receptor (EPCR) (+/- lysobisphosphatidic acid [LBPA]), prothrombin (PT) (+/- phosphatidyl serine [PS]), proteinase 3 (PR3), myeloperoxidase (MPO), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG), coagulation factor VIII (FVIII), muscle skeletal receptor tyrosine-protein kinase (MuSK), phospholipase A2 receptor (PLA2R), disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), E3 ubiquitin-protein ligase TRIM21 (TRIM21), signal recognition particles (SRP), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), protein-arginine deiminase type-4 (PAD4), or desmoglein 1/3 (DSG1/3), respectively, or any parts thereof. The T cell- or NK cell-engaging domains as shown in FIG. 8A apply.

FIG. 11 shows binding of BaiTEs with different antigenic domains to polyclonal primary human T cells. Representative flow cytometric analysis of live, single CD3+ primary human T cells that were incubated in the presence of scFv p2GPI-BaiTE (left, top row) as compared to live, single CD3+ primary human T cells that incubated without BaiTE (left, bottom row). Binding of BaiTE to the T cells was detected using a fluorophore conjugated anti-Hise antibody. Histogram shows binding of different scFv p2GPI-BaiTEs to all CD3+ primary human T cells in fluid phase, as detected by flow cytometry using an AF 488-conjugated anti-Hise antibody. No staining above baseline is observed for CD3+ primary human T cells incubated with AF 488-conjugated anti-Hise antibody alone.

FIG. 12 shows that BaiTEs bind to polyclonal human CD8+ (CD4-) and CD4+ T cells: Flow cytometry analysis of human T cells incubated with or without p2GPI-BaiTE incorporating UCHTlv9 scFv as the effector cell-binding domain. Top row panels show the gating strategy used for live, single CD4+ and CD8+ (CD4-) T cells. Middle row histograms show exemplary staining of live, single CD8+ (CD4-) human T cells incubated in the presence or absence of a scFv [32GPI- BaiTE. p2GPI-BaiTE bound to the surface of CD8+ human T cells in fluid phase is detected using a monoclonal antibody (P2-6) against P2GPI, the autoantigen comprising the antigenic domain of this BaiTE, and DyLight 649-conjugated StrepTactin XT (middle panel & green population in the right panel). No binding above background is observed for CD8+ human T cells incubated with monoclonal antibody (P2-6) and DyLight 649-conjugated StrepTactin XT alone (left panel and grey population in the right panel). Bottom row histograms show exemplary staining of live, single CD4+ human T cells incubated in the presence or absence of a scFv p2GPI-BaiTE. p2GPI-BaiTE bound to the surface of CD4+ human T cells in fluid phase.

FIG. 13 shows p2GPI-BaiTEs bind autoreactive B cells expressing p2GPI-specific BCRs. Ramos B cells, a human B cell line, were modified by CRISPR/Cas9 to express a patient-derived autoreactive BCR specific for domain I/II of P2GPI (Pl-117). Anti-P2GPI B cells and unedited B cells (expressing irrelevant BCR) were incubated in the presence of different scFv p2GPI-BaiTEs (BaiTEs 1-7), EPCR-BaiTE (“irrelevant BaiTE”), or no BaiTE. Binding of BaiTEs to B cells was detected using a DyLight 488-conjugated anti-Hise antibody by flow cytometry. The histogram on the left shows binding of p2GPLBaiTEs (BaiTEs 3-6) to human anti-p2GPI B cells. No binding is observed for scFv BaiTE specific for other B cell receptors (irrelevant BaiTE). The histogram on the right shows analogous staining for unedited B cells (expressing irrelevant BCRs). FIG. 14 shows that p2GPI-BaiTEs redirect T cells to deplete autoreactive anti-P2GPI B cells in APS, but preserve other B cells. Human T cells were co-cultured with Ramos B cells expressing a P2GPI-specific BCR or wild-type Ramos B cells in the presence or absence of scFv p2GPI-BaiTE. In the absence of BaiTE, neither unedited Ramos B cells (top row, right panel, Q3) nor autoreactive anti-p2GPI Ramos B cells (third row, right panel, Q2) were killed by human T cells (green). In the presence of p2GPI-BaiTE, human primary T cells killed autoreactive anti-P2GPI Ramos B cells (bottom row, right panel, Q2) but not unedited Ramos B cells (second row, right panel, Q3). Flow cytometry gating on single, live cells: B cells are stained with FITC anti-CD20; autoreactive anti- P2GPI B cells are detected using DyLight 649-conjugated StrepTactin XT.

FIGs. 15-16 show p2GPI-BaiTEs selectively deplete pathogenic anti-P2GPI B cells of various B cell receptor surface antigen densities.

FIG. 15 shows generation of anti-P2GPI B cell clones expressing various densities of autoreactive B cell receptor (Pl-117) on their cell surface, as quantified by flow cytometry. The histogram (left) shows anti-p2GPI BCR surface densities for different Ramos B cell clones as detected by flow cytometry using DyLight 649-conjugated StrepTactin XT (binding a Strep tag 2 in the engineered BCR). The bar graphs (right) show the results of co-culture experiments of primary human T cells with Ramos B cell clones in the presence or absence of different concentrations of p2GPI-BaiTE (0-1000 ng/mL) for 40 hours. The percentage of viable CD20+ B cells at the end of the experiment, normalized to conditions incubated without p2GPI-BaiTE, is shown. B cell clones with high surface autoreactive BCR density (top row, left), medium-high surface autoreactive BCR density (top row, right), medium surface autoreactive BCR density (middle row, left), low surface autoreactive BCR density (middle row, right), absent autoreactive BCR density (bottom row, left), or irrelevant BCR expression (bottom row, right) are shown. p2GPI-BaiTE retained selectivity at highest protein concentrations.

FIG. 16 shows a Heat map showing the percentage (%) of remaining viable Ramos B cell clones after co-culture with human primary T cells in the presence of increasing concentrations of (32GPI- BaiTE (BaiTE3). Autoreactive Ramos B cells are efficiently killed at low BaiTE concentrations, while B cells expressing no anti-02GPI B cell receptors (bottom three rows) are not depleted even at highest BaiTE concentrations. “Ramos Pl-117 BCR+” denotes pool of edited anti-|32GPI B cells (Pl-117); “Ramos wt” denotes unedited Ramos B cells expressing irrelevant B cell receptors. Single cell clones of high (clone 90, clone 74), medium-high (clone C12.2, clone 2), medium (clone 83, clone 77), low (clone B09.2, H09.2), and negative surface anti-p2GPI B cell receptor density (clone E06.2, clone 71) are shown.

FIG. 17 shows p2GPI-BaiTE depletes anti-02GPI B cells expressing different patient-derived monoclonal B cell receptors (Pl-117, Pl-190, and P2-6, respectively). Human T cells were cocultured with Ramos B cells expressing one of three different anti-02GPI B cell receptors (P 1-117, Pl-190, or P2-6) in the presence or absence of scFv p2GPI-BaiTE. Remaining B cells (expressed as the percentage of total live cells in co-culture) at the end of the experiment are shown. Each data point represents a unique single cell clone.

FIG. 18 shows treatment with p2GPI-BaiTEs abrogates autoantibody production. Human T cells were co-cultured with Ramos B cells expressing one of three different anti-P2GPI B cell receptors (Pl-117, Pl-190, or P2-6) in the presence or absence of two different scFv P2GPI-BaiTEs. At the end of the experiment, anti-p2GPI autoantibody levels were measured in cell culture supernatants using an in-house ELISA assay. BaiTE3 effectively abrogated anti-P2GPI autoantibody production for all target B cell lines. BaiTEl was more potent at lower concentration in stopping anti-P2GPI autoantibody production for B cells expressing B cell receptor P2-6 but expectedly did not deplete B cells expressing B cell receptors Pl-117 and Pl-190, respectively.

FIG. 19 shows p2GPI-BaiTE depletes anti-P2GPI B cells expressing patient-derived monoclonal B cell receptor (Pl-117) of various immunoglobulin isotypes or subclasses. Human T cells were co-cultured with Ramos B cells expressing IgM, IgA, IgGl, or IgG3 anti-P2GPI B cell receptors in the presence or absence of scFv p2GPI-BaiTE. B cell viability (expressed as the percentage of untreated B cells) at the end of the experiment is shown. Ramos B cells expressing anti-P2GPI BCRs are eliminated regardless of isotype (IgM, IgA, IgG) or subclass (IgGl, IgG3).

DETAILED DESCRIPTION

Provided herein are bispecific (auto)antigen-immune effector cell engaging antibodies (e.g., bispecific (auto)antigen T-cell engagers [BaiTEs] or bispecific (auto)antigen therapeutic effector cell engagers [BaitEs]) that can selectively cross-link immune cells (e.g., T cells, NK cells, or other cytotoxic cells) with targeted B cells via their cognate B cell receptors (BCR), as well as methods of making and using the same.

Provided herein are (single-chain or multi-chain) chimeric polypeptides that include at least (a) an antigenic domain comprising an (auto)antigenic sequence, and (b) an effector cell-binding domain, wherein the antigenic domain binds specifically to a cognate B cell receptor (BCR) and the effector cell-binding domain binds specifically to an immune effector cell.

Also provided herein are nucleic acid molecules encoding any one of the (single-chain or multi-chain) chimeric polypeptides described herein. Also provided herein are recombinant vectors, compositions, and methods of treatment using any one of the (single-chain or multi-chain) chimeric polypeptides described herein.

Various non-limiting aspects of these single-chain or multi-chain chimeric 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 single-chain or multi-chain chimeric polypeptides are known in the art.

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.

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.

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. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. 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, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

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®; Transbodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR). In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

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 some embodiments, an (auto)antigen binds to a membrane-bound immunoglobulin (i.e., a B cell receptor [BCR] of any isotype or subclass) on a B-cell surface. In general, an (auto)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 (auto)antigen is or comprises a polypeptide. In some embodiments, an (auto)antigen is or comprises a glycan. In some embodiments, an (auto)antigen is or comprises a lipid or phospholipid. In some embodiments, an (auto)antigen is or comprises a phospholipidprotein complex. Those of ordinary skill in the art will appreciate that, in general, an (auto)antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some certain embodiments, an (auto)antigen is present in a cellular context (e.g., an antigen is expressed on the surface of a cell or expressed in a cell or is bound to a cell). In some embodiments, an (auto)antigen is a recombinant (auto)antigen.

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. 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. In some embodiments, an effector cell-binding domain is part of a bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) antibody.

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).

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, 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 (i.e., 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, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, derivatives and/or progeny of an engineered polypeptide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

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.

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.

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 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.

As used herein, a “vector” or “recombinant vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, 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 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference for any purpose.

Chimeric Polypeptides

Provided herein are chimeric polypeptides comprising bispecific (auto)antigen-immune effector cell engaging antibodies (i.e., bispecific (auto)antigen T-cell engagers [BaiTEs] or bispecific (auto)antigen therapeutic effector cell engagers [BaitEs]) that include (a) an antigenic domain comprising an (auto)antigenic sequence, and (b) an effector cell-binding domain, wherein the antigenic domain binds specifically to a cognate B cell receptor (BCR) and the effector cellbinding domain binds specifically to an immune effector cell. Chimeric polypeptides can comprise a single polypeptide chain or multiple polypeptide chains. As used herein, the term “single-chain chimeric 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 chimeric polypeptide includes domains from at least two different naturally-occurring animal proteins. In some embodiments, a single-chain chimeric polypeptide includes domains from at least two different naturally-occurring mammalian proteins. In some embodiments, a single-chain chimeric polypeptide includes domains from at least two different naturally-occurring human proteins. In some embodiments, a single-chain chimeric polypeptide includes a domain that is a synthetic sequence (e.g., a scFv) and a domain that is derived from a naturally-occurring protein (e.g., a naturally-occurring animal or human protein). In some embodiments, a single-chain chimeric 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 chimeric polypeptide includes a domain that is not a protein/peptide (e.g., a nucleic acid, a lipid, a phospholipid, a glycan).

As used herein, the term “multi-chain chimeric polypeptide” refers to a therapeutic that comprises more than one polypeptide chain, of which at least one chain is a chimeric polypeptide chain. A multi-chain chimeric 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 chimeric polypeptide includes domains from at least two different naturally-occurring animal proteins. In some embodiments, a multi-chain chimeric polypeptide includes domains from at least two different naturally-occurring mammalian proteins. In some embodiments, a multi-chain chimeric polypeptide includes domains from at least two different naturally-occurring human proteins. In some embodiments, a multi-chain chimeric 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 chimeric polypeptide includes at least two different domains that are synthetic sequences (e.g., two different scFvs; one scFv and another synthetic sequence). In some embodiments, a multi-chain chimeric 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 chimeric polypeptide are reversibly linked. In some embodiments, the chains of the multi-chain chimeric polypeptide are irreversibly linked. In some embodiments, the chains of the multi-chain chimeric polypeptide are covalently linked. In some embodiments, the chains of the multi-chain chimeric polypeptide are non-covalently linked. In some embodiments, the chains of the multi-chain chimeric polypeptide are linked by dimerization or bioconjugation domains.

As used herein, the term “bispecific (auto)antigen-immune effector cell engaging antibody” describes both bispecific (auto)antigen T-cell engagers (BaiTEs) and bispecific (auto)antigen therapeutic effector cell engagers (BaitEs). As used herein, the term “bispecific (auto)antigen T- cell engager” or “BaiTE” refers to a single-chain or multi-chain chimeric polypeptide that binds T cells (e.g., a a0-T cell, a y6-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. As used herein, the term “bispecific (auto)antigen therapeutic effector cell engager” or “BaitE” refers to a single-chain or multi-chain chimeric polypeptide that binds other immune effector cells (e.g., NK cells, NKT cells, innate lymphoid cells, other non-T cytotoxic cells, or other immune cells) via its effector cell-binding domain.

Antigenic domain (cognate B cell receptor-bindin domain)

In some embodiments, the antigenic domain of a BaiTE or BaitE includes one or more autoantigenic sequences. As used herein the term “autoantigenic sequence” is a sequence that is derived from an autoantigen. As used herein, the term “autoantigen” refers to an antigen that, despite being a normal tissue constituent, is the target of a humoral or cell-mediated immune response. In some embodiments, an autoantigen is an endogenous antigen that stimulates autoantibody production that evokes an immune response by the host.

In some embodiments, the autoantigenic sequence is recognized by a B cell receptor (BCR). As used herein, a “B cell receptor” or “BCR” refers to a transmembrane protein on the surface of a B cell, wherein the transmembrane protein comprises a membrane-bound immunoglobulin molecule (of any isotype or subclass) and a signal transduction moiety. In some embodiments, a BCR binds to (auto)antigens and controls the activation of the B cell. In some embodiments, after binding to an (auto)antigen, the BCR transmits an intracellular signal to initiate various immune responses or cell proliferation. In some embodiments, a B cell produces a type of BCR with a high level of specificity to a specific (auto)antigen. Diversity of amino acid sequences at an antigen-binding site is responsible for the specificity of a BCR. Sequences at an (auto)antigen-binding site greatly vary among BCR molecules and are called variable regions (V regions) or hypervariable regions. Meanwhile, a sequence of a constant region (C region) is highly conserved among BCR molecules or antibody molecules of a given isotype or subclass. Such a region has an effector function of an antibody or a signaling function of a receptor.

In some embodiments, the autoantigenic sequence is an epitope, peptide, polypeptide, or domain derived from an autoantigen, part of an autoantigen, the entire autoantigen, or the autoantigen in complex with another molecule. In some embodiments, the autoantigenic sequence is derived from a phospholipid-binding protein. In some embodiments, the phospholipid binding protein is beta-2 glycoprotein I (P2GPI), endothelial protein C receptor (EPCR), or prothrombin (PT), or their mammalian analogs. In some embodiments, the autoantigenic sequence comprises one or at least part of one domain of 02GPI, including domain I (DI), domain II (DII), domain III (Dill), domain IV (DIV), or domain V (DV). In some embodiments, the autoantigenic sequence comprises a combination of domain I (DI) with other domains of 02GPI, including - but not limited to - 02GPI DI-DII, 02GPI DI-DIII, 02GPI DI-DIV, or 02GPI DI-DV. In some embodiments, the autoantigenic sequence comprises two consecutive domains of (32GPI (or parts thereof), including 02GPI DI-DII, P2GPI DII-DIII, 02GPI DIII-DIV, or 2GPI DIV-DV. In some embodiments, the autoantigenic sequence comprises three consecutive domains of [32GPI (or parts thereof), including 02GPI DI-DIII, 02GPI DII-DIV, or p2GPI DIII-DV. In some embodiments, the autoantigenic sequence comprises four consecutive domains of [32GPI (or parts thereof), including 02GPI DI-DIV or 02GPI DII-DV. In some embodiments, the autoantigenic sequence comprises domains of 02GPI (or parts thereof) that are mutated to increase binding specificity. In some embodiments, the autoantigenic sequence comprises domains of P2GPI (or parts thereof) that are mutated to inhibit or abrogate specific or unspecific binding to tissues, cells, membranes, receptors, or ligands other than their cognate B cell receptors, and/or alter other biological function. In some embodiments, the autoantigenic sequence comprises domains of P2GPI (or parts thereof) that are mutated to increase binding affinity. In some embodiments, the autoantigenic sequence comprises any combination of the domains of P2GPI or their parts. In some embodiments, the autoantigenic sequence comprises the epitope R39-R43 “RGGMR”, either in isolation or embedded in another protein sequence. In some embodiments, the autoantigenic sequence comprises a bacterial or viral mimotope or orthologs of this epitope. In some embodiments, the autoantigenic sequence comprises at least part of the endothelial protein C receptor (EPCR). In some embodiments, the autoantigenic sequence comprises the extracellular (EC) domains of EPCR. In some embodiments, the extracellular domain of EPCR has been modified by introduction of a tag sequence. In some embodiments, EPCR has been complexed or “loaded” with the associated phospholipid LBPA to produce the phospholipidbinding protein/phospholipid complex.

In some embodiments, the autoantigenic sequence comprises at least part of a prothrombin. In some embodiments, the autoantigenic sequence comprises prothrombin expressed in the presence of its natural pro-peptide and vitamin K to facilitate processing and modification. In some embodiments, the autoantigenic sequence comprises partial sequences of 02GPI, EPCR, and/or PT

In some embodiments, the autoantigenic peptide is derived from or comprises at least part of native or mutant proteinase 3 (PR3), myeloperoxidase (MPO), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG), coagulation factor VIII (FVIII), muscle skeletal receptor tyrosine-protein kinase (MuSK), phospholipase A2 receptor (PLA2R), disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), E3 ubiquitin-protein ligase TRIM21 (TRIM21), signal recognition particles (SRP), 3 -hydroxy-3 -methylglutaryl-coenzyme A reductase (HMGCR), protein-arginine deiminase type-4 (PAD4), desmoglein 1 (DSG1), or desmoglein 3 (DSG3).

In some embodiments, the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) comprises autoantigenic sequences of and targets B cell receptors (BCRs) that bind one or more of the following: beta-2-glycoprotein 1/ B2GPI (Gene Name: APOH, UniProt ID: P02749), endothelial protein C receptor/ EPCR (PROCR, Q9UNN8), prothrombin (F2, P00734), cardiolipin, lysobisphosphatidic acid, phosphatidylserine, annexin A2 (ANXA2, P07355), Annexin A5 (ANXA5, P08758); histone Hl .0 (H10, P07305), histone Hl.l (Hl-1, Q02539), histone H1.2 (Hl-2, P16403), histone H1.3 (Hl-3, P16402), histone Hl.4 (Hl-4, P10412), histone Hl .5 (Hl-5, P16401), histone Hit (Hl-6, P22492), testis-specific Hl histone (Hl-7, Q75WM6), histone Hl.8 (Hl-8, Q8IZA3), histone Hl.10 (Hl- 10, Q92522), histone H2A proteins, histone H2B proteins, histone H3 proteins, histone H4 (H4C1, P62805), hnRNP-AO (HNRNPAO, Q13151), hnRNP-Al (HNRNPA1, P09651), hnRNP-Al-like 2 (HNRNPA1L2, Q32P51), hnRNP-A2/Bl (HNRNPA2B1, P22626), hnRNP-A3 (HNRNPA3, P51991), hnRNP-A/B (HNRNP AB, Q99729), hnRNP-Cl/C2 (HNRNPC, P07910), hnRNP-C- like 1 (HNRNPCL1, 060812), hnRNP-C-like 2 (HNRNPCL2, B2RXH8), hnRNP -DO (HNRNPD, Q 14103), hnRNP -DL (HNRNPDL, 014979), hnRNP-El, hnRNP -F (HNRNPF, P52597), hnRNP- H (HNRNPH1, P31943), hnRNP-H2 (HNRNPH2, P55795), hnRNP-H3 (HNRNPH3, P31942), hnRNP-I (PTBP1, P26599), hnRNP K (HNRNPK, P61978), hnRNP-L (HNRNPL, Pl 4866), hnRNP -L-like (HNRNPLL, Q8WVV9), hnRNP -M (HNRNPM, P52272), hnRNP-Q (HNRPQ, 060506), hnRNP -R (HNRNPR, 043390), hnRNP-U (HNRNPU, Q00839), hnRNP-U-like protein 1 (HNRNPUL1, Q9BUJ2), Protein-arginine deiminase type-1/ PAD1 (Q9ULC6), Protein-arginine deiminase type-2/ PAD2 (PADI2, Q9Y2J8), Protein-arginine deiminase type-3/ PAD3 (PADI3, 9ULW8), Protein-arginine deiminase type-4/ PAD4 (PADI4, Q9UM07), vimentin (VIM, P08670), filaggrin (FLG, P20930), filaggrin-2 (FLG2, Q5D862), fibrinogen alpha chain (FGA, P02671), fibrinogen beta chain (FGB, P02675), fibrinogen gamma chain (FGG, P02679), fibronectin (FN1, P02751), alpha-enolase (EN01, P06733), elongation factor 1-alpha 1 (EEF1A1, P68104), elongation factor 1-alpha 2 (EEF1A2, Q05639), beta-actin (ACTB, P60709), gamma-actin (ACTG1, P63261), alpha-1 type I collagen (COL1 Al, P02452), alpha-2 type I collagen (COL1 A2, P08123), alpha- 1 type II collagen (COL2A1, P02458), fructose-bisphosphate aldolase A (ALDOA, P04075), fructose-bisphosphate aldolase B (ALDOB, P05062), fructose-bisphosphate aldolase C (ALDOC, P09972), heat shock 60 kDa proteins (HSP60), heat shock 70 kDa proteins (HSP70), heat shock protein HSP 90 proteins (HSP90), immunoglobulin gamma-1 heavy chain (IGHG1, P01857), immunoglobulin heavy constant gamma 2 (IGHG2, P01859), immunoglobulin heavy constant gamma 3 (IGHG3, P01860), immunoglobulin heavy constant gamma 4 (IGHG4, P01861); myeloblastin/ proteinase 3 (PRTN3, P24158), myeloperoxidase (MPO, P05164), neutrophil elastase (ELANE, P08246), lysosome-associated membrane glycoprotein 2 (LAMP2, P13473), collagen alpha-3(IV) chain (COL4A3, Q01955), secretory phospholipase A2 receptor (PLA2R1, QI 3018), thrombospondin type-1 domain-containing protein 7A (THSD7A, Q9UPZ6); histone H3-like centromeric protein A/ CENP-A (CENPA, P49450), major centromere autoantigen B/ CENP-B (CENPB, P07199), centromere protein C/ CENP-C (CENPC, Q03188), DNA topoisomerase 1/ Scl-70 (TOPI, Pl 1387), exosome complex component RRP45/ PM/Scl- 75 (EXOSC9, Q06265), exosome component 10/ PM/Scl-100 (EXOSCIO, Q01780), DNA- directed RNA polymerase III subunit RPC 1 (POLR3A, 014802), DNA-directed RNA polymerase III subunit RPC2 (POLR3B, Q9NW08), DNA-directed RNA polymerase III subunit RPC3 (P0LR3C, P0LR3C), DNA-directed RNA polymerase III subunit RPC4 (POLR3D, P05423), DNA-directed RNA polymerase III subunit RPC5 (POLR3E, Q9NVU0), DNA-directed RNA polymerase III subunit RPC6 (POLR3F, Q9H1D9), DNA-directed RNA polymerase III subunit RPC7 (POLR3G, 015318), DNA-directed RNA polymerase III subunit RPC8 (P0LR3H, Q9Y535), DNA-directed RNA polymerase III subunit RPC9 (CROP, 075575), DNA-directed RNA polymerase III subunitRPCIO (P0LR3K, Q9Y2Y1), DNA-directed RNA polymerases I and III subunit RPAC1 (P0LR1C, 015160), DNA-directed RNA polymerases I and III subunit RPAC2 (P0LR1D, P0DPB6), DNA-directed RNA polymerases I, II, and III subunit RPABC1 (P0LR2E, Pl 9388), DNA-directed RNA polymerases I, II, and III subunit RPABC2 (P0LR2F, P61218), DNA-directed RNA polymerases I, II, and III subunit RPABC3 (P0LR2H, P52434), DNA-directed RNA polymerases I, II, and III subunit RPABC4 (P0LR2K, P53803), DNA- directed RNA polymerases I, II, and III subunit RPABC5 (POLR2L, P62875), RNA Binding Region Containing 3/ RNPC3 (RNPC3, Q96LT9), ribonuclease P protein subunit p25/ Th/To antigen (RPP25, Q9BUL9), translation initiation factor eIF-2B subunit alpha (EIF2B1, Q14232), translation initiation factor eIF-2B subunit beta (EIF2B2, P49770), translation initiation factor elF- 2B subunit gamma (EIF2B3, Q9NR50), translation initiation factor eIF-2B subunit delta (EIF2B4, Q9UI10), translation initiation factor eIF-2B subunit epsilon (EIF2B5, Q13144), gammainterferon-inducible protein 16 (IFI16, Q16666), protein bicaudal D homolog 2 (BICD2, Q8TD16), fibrillin-1 (FBN1, P35555), rRNA 2'-0-methyltransferase fibrillarin/ fibrillarin 34 kDa (FBL, P22087), X-ray repair cross-complementing protein 6/ 70 kDa subunit of Ku antigen (XRCC6, P12956), X-ray repair cross-complementing protein 5/ 86 kDa subunit of Ku antigen (XRCC5, P13010); interferon-induced helicase C domain-containing protein 1/ melanoma differentiation-associated protein 5 (IFIH1, Q9BYX4), chromodomain-helicase-DNA-binding protein 4/ Mi-2 antigen (CHD4, Q14839), chromodomain-helicase-DNA-binding protein 3/ Mi-2 antigen (CHD3, Q12873), histidine-tRNA ligase, cytoplasmic/ Jo-1 antigen (HARS1, P12081), histidine-tRNA ligase, mitochondrial (HARS2, P49590), threonine-tRNA ligase 1, cytoplasmic/ PL-7 antigen (TARSI, P26639), threonine-tRNA ligase, mitochondrial (TARS2, Q9BW92), threonine-tRNA ligase 2, cytoplasmic (TARS3, A2RTX5), alanine— tRNA ligase, cytoplasmic/ PL-12 antigen (AARS1, P49588), alanine-tRNA ligase, mitochondrial (AARS2, Q5JTZ9), glycine-tRNA ligase/ EJ antigen (GARS1, P41250), isoleucine— tRNA ligase, cytoplasmic/ OJ antigen (IARS1, P41252), isoleucine-tRNA ligase, mitochondrial (IARS2, Q9NSE4), asparagine- -tRNA ligase, cytoplasmic/ KS antigen (NARS1, 043776), probable asparagine-tRNA ligase, mitochondrial (NARS2, Q96I59), phenylalanine-tRNA ligase alpha subunit/ ZO antigen (FARSA, Q9Y285), phenylalanine-tRNA ligase beta subunit (FARSB, Q9NSD9), phenyl al anine- -tRNA ligase, mitochondrial (FARS2, 095363), tyrosine-tRNA ligase, cytoplasmic/ HA antigen (YARS1, P54577), tyrosine-tRNA ligase, mitochondrial (YARS2, Q9Y2Z4), 3-hydroxy-3- methylglutaryl-coenzyme A reductase (HMGCR, P04035), signal recognition particle subunit SRP72 (SRP72, 076094), signal recognition particle subunit SRP68 (SRP68, Q9UHB9), signal recognition particle 54 kDa protein (SRP54, P61011), signal recognition particle 14 kDa protein (SRP14, P37108), signal recognition particle 19 kDa protein (SRP19, SRP19), signal recognition particle 9 kDa protein (SRP9, P49458), E3 ubiquitin-protein ligase TRIM33/ Transcription intermediary factor 1-gamma (TRIM33, Q9UPN9), MORC family CW-type zinc finger protein 3/ nuclear matrix protein 2 (M0RC3, Q14149), SUMO-activating enzyme subunit 1/ SAE antigen (SAE1, Q9UBE0), SUMO-activating enzyme subunit 2/ SAE antigen (UBA2, Q9UBT2), cytosolic 5'-nucleotidase 1A (NT5C1A, Q9BXI3), cell division cycle and apoptosis regulator protein 1 (CCAR1, Q8IX12), transcription factor SOX-5 (SOX5, P35711); E3 ubiquitin-protein ligase TRIM21/ Ro-52 kDa (TRIM21, P19474), RNA-binding protein RO60/ Ro-60 kDa (RO60, Pl 0155), E3 ubiquitin-protein ligase TRIM68 (TRIM68, Q6AZZ1), Lupus La protein/ La (SSB, P05455); double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), or their protein complexes; small nuclear ribonucleoprotein Sm DI (SNRPD1, P62314), small nuclear ribonucleoprotein Sm D2 (SNRPD2, P62316), small nuclear ribonucleoprotein Sm D3 (SNRPD3, P62318); small nuclear ribonucleoprotein Sm E (SNRPE, P62304), small nuclear ribonucleoprotein Sm F (SNRPF, P62306), small nuclear ribonucleoprotein Sm G (SNRPG, P62308), small nuclear ribonucleoprotein-associated proteins B and B' (SNRPB, P14678), small nuclear ribonucleoprotein-associated protein N/ Sm-N (SNRPN, P63162), U1 small nuclear ribonucleoprotein 70 kDa (SNRNP70, P08621), U1 small nuclear ribonucleoprotein C (SNRPC, P09234), U1 small nuclear ribonucleoprotein A (SNRPA, P09012), U2 small nuclear ribonucleoprotein (SNRPA1, P09661), U2 small nuclear ribonucleoprotein B" (SNRPB2, P08579), 60S acidic ribosomal protein PO (RPLPO, P05388), 60S acidic ribosomal protein Pl (RPLP1, P05386), 60S acidic ribosomal protein P2 (RPLP2, P05387), plasma protease Cl inhibitor (SERPING1, PO5155), complement Clq subcomponent subunit A (C1QA, P02745), complement Clq subcomponent subunit B (Cl QB, P02746), complement Clq subcomponent subunit C (C1QC, P02747), glycosylphosphatidylinositol-anchored high density lipoproteinbinding protein 1 (GPIHBP1, Q8IV16); a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13, Q76LX8), complement factor H (CFH, P08603), complement factor I (CFI, P05156), von Willebrand factor (VWF, P04275), coagulation factor VIII (F8, P00451), integrin alpha-IIb (GPIIb) (ITGA2B, P08514), integrin beta-3 (GPIIIa) (ITGB3, P05106), platelet glycoprotein lb alpha chain (GPIb) (GP1BA, P07359), platelet glycoprotein lb beta chain (GP1BB, P13224), platelet glycoprotein IX (GP9, P14770), integrin alpha-2 (ITGA2, P17301), integrin beta-1 (ITGB1, P05556), platelet glycoprotein 4 (CD36, P16671), platelet glycoprotein V (GP5, P40197), P-selectin (SELP, QI 4242), platelet factor 4 (PF4, P02776); aquaporin-4 Ml (AQP4, P55087), aquaporin-4 M23 (AQP4, P55087), aquaporin-4 orthogonal arrays, myelin proteolipid protein (PLP1, P60201), myelin-oligodendrocyte glycoprotein (MOG, Q16653), myelin basic protein (MBP, P02686), myelin-associated oligodendrocyte basic protein (MOBP, QI 3875), myelin-associated glycoprotein (MAG, P20916), alpha-crystallin B chain (CRY AB, P02511), glial fibrillary acidic protein (GFAP, P14136), claudin-11 (Claudin-11, 075508), endoplasmic reticulum chaperone BiP (HSPA5, Pl 1021); acetylcholine receptor subunit alpha (CHRNA1, P02708), acetylcholine receptor subunit beta (CHRNB1, Pl 1230), acetylcholine receptor subunit gamma (CHRNG, P07510), acetylcholine receptor subunit delta (CHRND, Q07001), acetylcholine receptor subunit epsilon (CHRNE, Q04844), muscle skeletal receptor tyrosineprotein kinase (MUSK, 015146), low-density lipoprotein receptor-related protein 4 (LRP4, 075096), agrin (AGRN, 000468), acetylcholinesterase collagenic tail peptide (COLQ, Q9Y215), a-subunit of the voltage-gated potassium channel Kvl.4, titin (TTN, Q8WZ42), ryanodine receptor 1 (RYR1, P21817), ryanodine receptor 2 (RYR2, Q92736), ryanodine receptor 3 (RYR3, Q 15413), A-kinase anchor protein 12 (AKAP12, Q02952); glutamate receptor ionotropic, NMDA 1/ GluNl (GRIN1, Q05586), glutamate receptor ionotropic, NMDA 2A7 GluN2A (GRIN2A, Q12879), glutamate receptor ionotropic, NMDA 2B/ GluN2B (GRIN2B, Q13224), glutamate receptor ionotropic, NMDA 2C/ GluN2C (GRIN2C, Q14957), glutamate receptor ionotropic, NMDA 2D/ GluN2D (GRIN2D, 015399), cerebellar degeneration-related protein 2/ Yo antigen (CDR2, Q01850), ELAV-like protein 1 (ELAV-like protein 1, Q15717), ELAV-like protein 2 (ELAVL2, Q12926), ELAV-like protein 3 (ELAVL3, Q14576), ELAV-like protein 4 (ELAVL4, P26378), leucine-rich glioma-inactivated protein 1 (LGI1, 095970), contactin-associated protein- like 2/ CASPR2 (CNTNAP2, Q9UHC6), contactin-2 (CNTN2, Q02246), dihydropyrimidinase- related protein 5/ CRMP5 (DPYSL5, Q9BPU6), glutamate receptor 1/ GluAl (GRIA1, P42261), glutamate receptor 2/ GluA2 (GRIA2, P42262), glutamate receptor 3/ GluA3 (GRIA3, P42263), glutamate receptor 4/ GluA4 (GRIA4, P48058), gamma-aminobutyric acid receptor subunit alpha- 1 (GABRA1, P14867), gamma-aminobutyric acid receptor subunit beta-3 (GABRB3, P28472), gamma-aminobutyric acid type B receptor subunit 1 (GABBR1, Q9UBS5), gamma-aminobutyric acid type B receptor subunit 2 (GABBR2, 075899), gamma-aminobutyric acid receptor subunit gamma-2 (GABRG2, P18507), metabotropic glutamate receptor 1 (GRM1, Q13255), metabotropic glutamate receptor 5 (GRM5, P41594), amphiphysin (AMPH, P49418), adenylate kinase isoenzyme 5 / AK5 (AK5, AK5), dipeptidyl aminopeptidase-like protein 6 (DPP6, P42658), delta and Notch-like epidermal growth factor-related receptor (DNER, Q8NFT8), neurexin-3 (NRXN3, Q9Y4C0), RNA-binding protein Nova- 1/ Ri antigen (N0VA1, P51513), paraneoplastic antigen Mai (PNMA1, Q8ND90), paraneoplastic antigen Ma2 (PNMA2, Q9UL42), paraneoplastic antigen Ma3 (PNMA3, Q9UL41), modulator of apoptosis 1/ Ma4 (M0AP1, Q96BY2), microtubule-associated protein IB (MAP1B, P46821), dihydropyrimidinase-related protein 1 (CRMP1, Q14194); ganglioside GM1, ganglioside GMlb, ganglioside GDla, ganglioside GDlb, ganglioside GQlb, ganglioside GTla; protein-glutamine gammaglutamyltransferase 2 (TGM2, P21980), protein-glutamine gamma-glutamyltransferase E (TGM3, Q08188), protein-glutamine gamma-glutamyltransferase 6 (TGM6, 095932); thyroid peroxidase (TPO, P07202), thyrotropin receptor (TSHR, P16473), thyroglobulin (TG, P01266); islet cell autoantigen 1 (ICA1, Q05084); islet cell autoantigen 1-like protein (ICA1L, Q8NDH6); glutamate decarboxylase 1 (GAD1, Q99259), glutamate decarboxylase 2 (GAD-65) (GAD2, Q05329); receptor-type tyrosine-protein phosphatase-like N / IA2 (PTPRN, QI 6849); receptor-type tyrosine-protein phosphatase N2/ IAR (PTPRN2, Q92932), zinc transporter 8 (SLC30A8, Q8IWU4); steroid 21 -hydroxylase (CYP21A2, P08686), steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A1, P05093), 3 beta-hydroxysteroid dehydrogenase/Delta 5->4-isomerase type 2 (HSD3B2, P26439), adrenocorticotropic hormone receptor (MC2R, Q01718), NACHT, LRR and PYD domains-containing protein 5 (NLRP5, P59047), testis-specific gene 10 protein (TSGA10, Q9BZW7), hyaluronidase PH-20 (SPAM1, P38567), disintegrin and metalloproteinase domaincontaining protein 2 (ADAM2, Q99965), follicle-stimulating hormone receptor (FSHR, P23945), follitropin subunit beta (FSHB, P01225), glycoprotein hormones alpha chain (CGA, P01215), lutropin-choriogonadotropic hormone receptor (LHCGR, P22888), lutropin subunit beta (LHB, P01229); cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A1, P05108); cobalamin binding intrinsic factor (CBLIF, P27352), potassium-transporting ATPase alpha chain 1 (ATP4A, P20648), potassium-transporting ATPase subunit beta (ATP4B, P51164); desmoglein-1 (DSG1, Q02413), desmoglein-3 (DSG3, P32926), collagen alpha-l(XVII) chain/ 180 kDa bullous pemphigoid antigen (COL17A1, Q9UMD9), dystonin/ 230 kDa bullous pemphigoid antigen (DST, Q03001), collagen alpha-l(VII) chain (COL7A1, Q02388), envoplakin (EVPL, Q92817), epiplakin (EPPK1, P58107), periplakin (PPL, 060437), plectin (PLEC, Q15149), desmoplakin (DSP, P15924), desmocollin-1 (DSC1, Q08554), laminin gamma-1, laminin 5, laminin 6, ladinin- 1 (LAD1, 000515), integrin alpha-4 (ITGA4, P13612), integrin beta-6 (ITGB6, P18564), neuronal acetylcholine receptor subunit alpha-9 (CHRNA9, Q9UGM1); tumor necrosis factor (TNF, P01375), lymphotoxin-alpha (LTA, P01374), granulocyte-macrophage colony-stimulating factor (CSF2, P04141), granulocyte colony-stimulating factor (CSF3, P09919), interleukin-1 alpha (ILIA, P01583), interleukin-1 beta (IL1B, P01584), interleukin-6 (IL6, P05231), interleukin- 17A (IL17A, Q16552), interleukin- 17F (IL17F, Q96PD4), interferon alpha-1/13 (IFNA1, P01562), interferon alpha-2 (IFNA2, P01563), interferon alpha-4 (IFNA4, P05014), interferon alpha-5 (IFNA5, P01569), interferon alpha-6 (IFNA6, PO5O13), interferon alpha-7 (IFNA7, P01567), interferon alpha-8 (IFNA8, P32881), interferon alpha-10 (IFNA10, P01566), interferon alpha-14 (IFNA14, P01570), interferon alpha-16 (IFNA16, P48551), interferon alpha-17 (IFNA17, P01571), interferon alpha-21 (IFNA21, P01568), interferon omega-1 (IFNW1, P05000), interferon kappa (IFNK, Q9P0W0), interferon lambda- 1 (IFNL1, Q8IU54), interferon lambda-2 (IFNL2, Q8IZJ0), interferon lambda-3 (IFNL3, Q8IZI9), interferon lambda-4 (IFNL4, K9M1U5), interferon gamma (IFNG, P01579); cytochrome P450 2D6/ LKM-1 (CYP2D6, Pl 0635), cytochrome P450 2C9 (CYP2C9, Pl 1712), cytochrome P450 2A6 (CYP2A6, Pl 1509), UDP- glucuronosyltransferase 1A (UGT1A1, P22309), cytochrome P450 1A2 (CYP1A2, P05177), formimidoyltransferase-cyclodeaminase (FTCD, 095954), dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial/ PDC-E2 (DLAT, Pl 0515), dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial/ OGDC-E2 (DLST, P36957), lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial/ BCOADC- E2 (DBT, Pl 1182), pyruvate dehydrogenase complex protein X component, mitochondrial (PDX1, P16451), dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT, Pl 0515), nuclear autoantigen Sp-100 (SP100, P23497), coilin (COIL, P38432), nuclear pore membrane glycoprotein 210 (NUP210, Q8TEM1), lamin-B receptor (LBR, Q8MLV1), nuclear pore glycoprotein p62 (NUP62, P37198), o- phosphoseryl-tRNA(Sec) selenium transferase (SEPSECS, Q9HD40), or any parts thereof, any of their isoforms, any of their splicing variants, any autoantigenic complexes with other proteins, nucleic acids, lipids, or carbohydrates containing the same, in either their unmodified or in their post-translationally modified (e.g., citrullinated, carbamylated, acetylated, glycosylated, y- carboxylated, among many other), or proteolytically processed forms. In some embodiments, the autoantigenic sequences are mutated to increase binding specificity. In some embodiments, the autoantigenic sequences are mutated to inhibit or abrogate specific or unspecific binding to tissues, cells, membranes, receptors, or ligands other than their cognate B cell receptors (BCRs). In some embodiments, the autoantigenic sequences are mutated to inhibit or abrogate enzymatic activity, or to alter other undesired biological function. In some embodiments, the autoantigenic sequences are mutated or modified to increase binding affinity to cognate B cell receptors (BCRs). In some embodiments, the BaiTE/ BaitE targets B cell receptors (BCRs) that bind to other autoantigens. In some embodiments, the BaiTE/ BaitE targets B cell receptors (BCRs) that bind to allergens or haptens. In some embodiments, the BaiTE or BaitE targets B cell receptors (BCRs) that bind to protein drugs, therapeutic vectors, or natural or synthetic viral antigens.

In some embodiments, the autoantigenic domain of the BaiTE/ BaitE is derived from and targets B cell receptors (BCRs) that bind to nucleic acids (e.g., natural or synthetic single-stranded DNA, double-stranded DNA, natural or synthetic RNA, oligonucleotides) or their mimotopes, as relevant for the treatment of lupus erythematosus and its organ manifestations.

In some embodiments, the autoantigenic domain of the BaiTE/ BaitE comprises post- translationally modified proteins, in which the post-translational modification can increase binding to or specificity for the targeted B cell receptor(s) (BCRs). In certain embodiments, the post- translational modification is citrullination/deimination, carb amyl ati on, acetylation, glycosylation, deamination, phosphorylation, or y-carboxylation. In certain embodiments, the autoantigenic domain contains at least one citrullinated residue, at least one carbamylated/ homocitrullinated residue, at least one acetylated residue, at least one glycosylated residue, at least one deaminated residue, at least one phosphorylated residue, or at least one y-carboxylated residue, a relevant for the treatment of autoimmune diseases.

In some embodiments, the antigenic domain may be an antigenic complex. In certain embodiments, the antigenic complex is derived from human leukocyte antigen (HLA)/ major histocompatibility complex (MHC) proteins bound to the relevant (auto)antigenic peptide. In some embodiments, the peptide-HLA autoantigenic domain can specifically bind cognate T cell receptors (TCRs) in patients with an autoimmune disease or allergy, or in individuals at risk of a disease.

Effector cell-binding domain

In some embodiments, the effector cell-binding domain of the bispecific (auto)antigen T- cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) binds to an immune cell. As used herein, “immune cells” refer to 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.

In some embodiments, the immune cell is a cytotoxic cell, i.e., an immune cell capable of killing through cytotoxic immune effector pathways, including perforin/granzyme-mediated cellular cytotoxicity. In some embodiments, the immune cell is a T cell. In specific embodiments, the immune cell can be a subset of T cells, e.g., a aP-T cell, a yb-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 Thl7 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 cell is an NK cell, an NKT cell, or an innate lymphoid cell. In some embodiments, the immune cell is a monocyte or macrophage. In some embodiments, the immune cell is another immune cell. In some embodiments, “immune cells” includes immune 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 cell through the effector cellbinding domain activates the immune cell. In some embodiments, binding of the effector cellbinding domain of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) redirects the immune cell to kill -or otherwise therapeutically impair- target cells that are bound through the antigenic domain. In some embodiments, binding of an additional effector cell-binding domain of the bi specific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) provides a co-stimulatory signal to the immune effector cell.

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 cell-binding 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 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 antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) of the bispecific (auto)antigen T-cell engager (BaiTE) 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 (CD36) chain, a T cell receptor variable and constant alpha (Va+Ca) chain, and a T cell receptor variable and constant beta (V +CP) chain. In yb-T cells, the TCR-CD3 complex includes a CD3 gamma (CD3y) chain, a CD3 epsilon (CD3e) chain, a CD3 delta (CD36) chain, a T cell receptor variable and constant gamma (Vy+Cy) chain, and a T cell receptor variable and constant delta (V5 +C6) chain. In some embodiments, the TCR-CD3 complex includes a CD3 zeta (CD3Q chain.

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 0KT3 or humanized 0KT3. In some non-limiting embodiments, the effector cellbinding domain comprises the anti-CD3e antibody clone UCHT1 or UCHTlv9. In some nonlimiting 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 (CD36).

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 antibody or antibody fragment binds at least one Vy allele (e.g., TRGV1-9), V6 allele (e.g., TRDV1-3), Va allele (e.g., TRAV1-TRAV41), or Vp allele (e g., TRBVI-TRBV30).

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 (C0) chain- 1 (TRBC1). In some nonlimiting embodiments, the effector cell-binding domain is derived from an antibody that binds the T cell receptor constant beta (CP) 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 (C5) chain (TRDC). 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 yo-T cell TCR Vy9V52.

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 bispecific (auto)antigen therapeutic effector cell engager (BaitE) 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 nonlimiting 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 cell-binding domain is derived from an antibody that binds NKG2-D type II integral membrane protein (NKG2D, P26718). In some non-limiting embodiments, the effector cellbinding 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, QI 5762). 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).

In certain embodiments, the antibody or antibody fragment (e.g., scFv, Fab, [sc]diabody, F(ab'), F(ab')2, VHH, VNAR) of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) binds to introduced epitopes or polypeptides of engineered surface receptors of immune cells (e.g., engineered T cells or NK cells). In some embodiments, an additional 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 a surface protein or epitope on the effector cell membrane whose engagement provides a co-stimulatory signal. In some non-limiting embodiments, the additional effector cell-binding domain is derived from an antibody or antibody fragment that binds CD27, CD28, CD40L, CD137, 0X40, or ICOS.

In some embodiments, an additional effector cell binding domain provides a co-stimulatory signal to the immune effector cell. In some embodiments, the additional co-stimulatory signal to the immune effector cell is provided by a single-chain antibody. In some embodiments, the additional co-stimulatory signal to the immune effector cell is provided by a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a variable light chain and a variable heavy chain. In some embodiments, the scFv binds to a surface protein or epitope on an immune effector cell membrane. In some embodiments, the scFv binds to CD27, CD28, CD40L, CD 137, 0X40, or ICOS. In some embodiments, the effector cell binding domain further comprises a linker sequence. In some embodiments, wherein the linker sequence is between the variable light chain and the variable heavy chain of the scFv. In some embodiments, a linker sequence is between a first effector cell-binding domain and a second effector cell-binding domain.

Linker Sequences and Fusion Protein Domains

In some embodiments, the antigenic domain (e.g., the autoantigen) of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) is directly linked to its effector cell-binding domain.

In some embodiments, the antigenic domain (e.g., the autoantigen) of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) is indirectly linked to its effector cell-binding domain via a linker sequence.

In some embodiments, the linker sequence of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) 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). In some embodiments, the linker sequence of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) 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 of the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) comprises reversible or irreversible dimerization domains or protein bioconjugation domains.

In some embodiments, a bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) further comprises at least one fusion protein domain. As used herein, the term “fusion protein domain” can refer 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. 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.

In some embodiments, a fusion protein domain can comprise a payload. As used herein, the term “payload” can refer to a peptide, polypeptide, protein, glycoprotein, enzyme, small 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 auristatinE, monomethyl auristatinF, 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 antigenic domain is expressed N-terminal to the effector cellbinding domain. In some embodiments, the antigenic domain is expressed C-terminal to the effector cell-binding domain.

In some embodiments, the fusion protein domain is expressed N-terminal to the chimeric polypeptide (comprising at least the antigenic domain and the effector cell-binding domain). In some embodiments, the fusion protein domain is expressed C-terminal to the chimeric polypeptide (comprising at least the antigenic domain and the effector cell-binding domain). In some embodiments, the fusion protein domain is expressed between the antigenic domain and the effector cell-binding domain of the chimeric polypeptide.

In some embodiments, the chimeric polypeptide that constitutes the bispecific (auto)antigen T-cell engager (BaiTE) or bispecific (auto)antigen therapeutic effector cell engager (BaitE) comprises one or more linker sequences. In some embodiments, a linker sequence is present between the antigenic 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 antigenic domain and VH or VL, respectively, for diabodies). In some embodiments, one or more effector cell-binding domains also comprise a linker sequence (e.g., between variable light [VL] chain and variable heavy [VH] chains for scFvs). In some embodiments, the antigenic 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 antigenic domain. In some embodiments, a linker sequence is present between different effector cell-binding domains. In some embodiments, the fusion protein domain also comprises a linker sequence.

Phospholipid-binding proteins and antiphospholipid syndrome

Phospholipid-binding proteins are proteins that form complexes with phospholipids and play a regulatory function in controlling biological functions. Examples of phospholipid-binding proteins can include, but are not limited to, beta-2-glycoprotein I (02GPI), endothelial protein C receptor (EPCR), prothrombin (PT), annexin V, and annexin II.

Antiphospholipid antibody syndrome (APS) is a multisystem autoimmune disease associated with disease-causing autoantibodies directed against phospholipid-binding proteins and their complexes with specific phospholipids. Immunologically, APS is characterized by loss of B cell tolerance against self and the emergence of autoantibodies. In some embodiments, autoantibody systems that target phospholipid-binding protein/phospholipid complexes can be referred to as antiphospholipid antibodies. In some embodiments, these antiphosphoplipid antibodies can include (i) anti-beta-2-glycoprotein I (02GPI)/ cardiolipin, (ii) anti-endothelial protein C receptor (EPCR)/ lysobisphosphatidic acid (LBPA), and (iii) anti-prothrombin (PT)/ phosphatidyl serine (PS). In some embodiments, these autoantibodies can be directly pathogenic in vitro and in vivo. Therefore, strategies that eliminate the sources of these antibodies (e.g., B cells, plasmablasts, or plasma cells) may be used to prevent or cure APS. Autoreactive B cells, plasmablasts, and plasma cells that express surface B cell receptors (BCRs) specific for phospholipid-binding proteins are therefore ideal therapeutic targets for antigen-specific depletion strategies.

Nucleic acids and vectors

Provided herein are nucleic acids encoding any one of the chimeric 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).

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.

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.

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 (auto)antigen T-cell engager (BaiTE) or a bispecific (auto)antigen therapeutic effector cell engager (BaitE).

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 chimeric polypeptide or a multi-chain chimeric 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, nonhuman primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as chordate, mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include any human primary cell, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), or their derivatives.

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.

Therapeutic applications

Provided herein are pharmaceutical compositions that include any of the single-chain or multi-chain chimeric polypeptides, the nucleic acid molecules, the recombinant vectors, or the cells described herein. In some embodiments, pharmaceutical compositions provided herein include a pharmaceutically acceptable carrier.

Also provided herein are methods of treating an autoimmune disease or B cell-mediated disease in a subject that include administering to the subject any one of the chimeric polypeptides, the nucleic acid molecules, the recombinant vectors, or the cells described herein, or the pharmaceutical compositions described herein. In some embodiments, the therapeutic is administered as a polypeptide in its active form. In some embodiments, the therapeutic is administered as a polypeptide in an inactive form (e.g., proform). In some embodiments, the therapeutic is administered as a vector for expression in vivo.

An “autoimmune disease” is a disease that arises from an abnormal immune response to a functioning body part, wherein a body’s immune system attacks and damages its own normal healthy cells or tissues. In some embodiments, in response to an unknown or known trigger, the immune system may begin producing antibodies (e.g., autoantibodies) and self-antigen-directed immune cells that, instead of fighting infections or cancer, attack the body’s own tissues. Systemic or organ-specific autoimmune diseases suitable for treatment by a method of the present disclosure can include, but are not limited to Addison’s disease, (adult-onset) Still’s disease, alopecia areata/ autoimmune hair loss, antiphospholipid syndrome (APS), APS-related fetal loss, and catastrophic antiphospholipid syndrome (CAPS), autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalitis (including anti-NMDAR encephalitis), autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis or pericarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune 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 (EGPA, formerly Churg-Strauss syndrome), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, 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 (GPA), Graves’ disease, Guillain- Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch- Schbnlein purpura (HSP), herpes gestationis or pemphigoid gestationis, hidradenitis suppurativa, IgA nephropathy and 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 (MPA), mixed connective tissue disease (MCTD), multifocal motor neuropathy (MMN), multiple sclerosis, myasthenia gravis, myelin oligodendrocyte glycoprotein (MOG) antibody-related diseases, idiopathic inflammatory myopathies, immune-mediated necrotizing myopathies, anti synthetase syndrome, narcolepsy, neonatal lupus, autoimmune neutropenia, autoimmune lymphopenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, paraneoplastic cerebellar degeneration, pars planitis (peripheral uveitis), pemphigus vulgaris, pemphigus vulgaris foliaceous, IgA pemphigus, paraneoplastic pemphigus, autoimmune peripheral neuropathies, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary membranous nephropathy, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, Raynaud’s phenomenon, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, scleroderma/ limited cutaneous and diffuse cutaneous systemic sclerosis, Sjogren’s 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.

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). 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) spectrum disorders, myasthenia gravis, pernicious anemia, primary biliary cirrhosis, primary membranous nephropathy, or type I diabetes mellitus. In some embodiments, the autoimmune disease is antiphospholipid antibody syndrome (APS).

A “B cell-mediated disease” is a disease that arises from B cell subsets, wherein those B cells directly or indirectly (e.g., through antibodies) contribute to the disease and wherein targeting of B cells directly or indirectly ameliorates the disease, its symptoms, or its clinical course. In some embodiments, the methods can be applied - through introduction of an allergen or other antigen into the polypeptide chain - to the treatment of other B cell-mediated diseases including type I allergies (e.g., any IgE-dependent allergic reactions, allergic asthma, urticaria, angioedema, allergic rhinitis), type II allergies (e.g., immune cytopenias, chronic 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).

Pharmaceutical compositions

In some embodiments, the present disclosure provides pharmaceutical compositions that include any of the chimeric polypeptides, the nucleic acid molecules, the recombinant vectors, or the cells described herein, 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 composition, if desired, can also contain one or more additional therapeutically active substances.

In some embodiments, 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).

In some embodiments, a pharmaceutical composition of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer’s solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. A vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). In some embodiments, a formulation is sterilized by known or suitable techniques. A pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening, or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21^st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

EXAMPLES

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

Example 1 - Bispecific (auto)antigen immune effector cell-engaging antibodies for the treatment of autoimmune disease and B-cell disorders

The present disclosure describes an antigen-specific immunotherapeutic strategy for the treatment of autoimmune diseases and other B-cell disorders. Specifically, the disclosure describes bispecific (auto)antigen T-cell engager (BaiTE) and bispecific (auto)antigen therapeutic effector cell engager (BaitE) that can cross-link T cells or other immune effector cells, respectively, with selectively targeted B cells via their cognate B cell receptors. By cross-linking any bystander T cells with disease-relevant B cells, BaiTE therapeutics facilitate immune synapse formation, T cell activation, and the killing of targeted B cells via the cytotoxic effector pathways of the T cell (FIG. 1A-1B). Analogously, by cross-linking bystander NK cells (or other cytotoxic immune effector cells) with disease-relevant B cells, BaitE therapeutics facilitate immune synapse formation, NK cell activation (or other cytotoxic immune effector cell activation), and the killing of targeted B cells via cytotoxic effector pathways (FIG. 1C).

BaiTE proteins encompass various therapeutic formats but in all compositions are characterized by at least two binding moi eties: i) an effector cell-binding domain that binds to and activates T cells (e.g., an scFv targeting CD3s; an scFv targeting any part of the TCR-CD3 complex), and ii) an antigenic (i.e., BCR-binding) domain that binds to the intended B cell target by exploiting the antigen-specificity of its BCR (FIGs. 2, 4-10).

BaitE proteins encompass various therapeutic formats but in all compositions are characterized by at least two binding moi eties: i) an effector cell-binding domain that binds to and activates NK cells or other immune effector cells (e.g., an scFv targeting an activating surface NK cell receptor), and ii) an antigenic (i.e., BCR-binding) domain that binds to the intended B cell target by exploiting the antigen-specificity of its BCR (FIGs. 3-10).

For the treatment of autoimmune diseases, exploiting the antigen-specificity of autoreactive B cells is achieved by incorporating antigenic sequences of their BCRs’ cognate autoantigen (or any parts thereof), any combination of epitopes derived from their cognate autoantigen, mimotopes of their cognate autoantigen, or orthologs of their cognate autoantigen into the antigenic (BCR-binding) domain of the BaiTE or BaitE construct.

For other B cell-mediated diseases, BaiTE/ BaitE constructs are designed to incorporate cognate antigen (or any parts thereof) into the antigenic domain to target B cells expressing a BCR of interest. BaiTEs/ BaitEs can be expressed in various formats which may include linker sequences, hinges, or fusion protein domains (e.g., payloads, protein domains to increase half-life) that confer other desirable characteristics to the BaiTE therapeutic (FIGs. 4-10).

In one example, BaiTE construct designs have been developed to target autoreactive B cells in patients with APS, expressing BCRs that bind the autoantigen beta-2-glycoprotein I (02GPI) (FIGs. 5-9). B2GPI-BaiTEs comprise an effector cell-binding domain that binds T cells (e.g., an scFv specific for CD3e, CD3y, CD35, Ca (TRAC), any Cp alleles (TRBC1, TRBC2), Voc alleles (TRAV), any VP alleles (TRBV), any Cy alleles (TRGC1, TRGC2), C6 (TRDC), any Vy alleles (TRGV), any V6 alleles (TRDV), TCR Vy9V52) and an antigenic domain that incorporates sequences of 02GPI (e.g., full or partial sequences, one or more of its domains, epitopes, mimotopes, or highly homologous protein sequences). (FIGs. 8-9). Examples of BaiTEs expressed in ExpiCHO cells and resolved after affinity tag purification are shown in FIG. 8B. P2GPI-BaitEs comprise an effector cell-binding domain that binds NK cells or other immune effector cells (e.g., an scFv specific for activating surface receptors on NK cells, such as CD16(a), NKp30, NKp44, NKp46, NKG2D, natural killer cell receptor 2B4, or DNAM1) and an antigenic domain that incorporates sequences of P2GPI (e.g., full or partial sequences, one or more of its domains, epitopes, mimotopes, or highly homologous protein sequences). (FIGs. 8-9).

In other examples, BaiTEs were developed comprising an antigenic domain derived from the autoantigens endothelial protein C receptor (EPCR), prothrombin (PT), proteinase 3 (PR3), myeloperoxidase (MPO), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG), signal recognition particles (SRP), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), protein-arginine deiminase type-4 (PAD4), or desmoglein 1/3 (DSG1/3), among others (FIG. 10).

Non-limiting examples of amino acid sequences of other autoantigens from which antigenic domains used in BaiTEs or BaitEs were derived are given in Table 1. TABLE 1. NON-LIMITING EXAMPLES OF AUTOANTIGENIC PEPTIDES FROM WHICH THE ANTIGENIC DOMAIN OF BISPECIFIC (AUTO) ANTIGEN-IMMUNE EFFECTOR CELL ENGAGING ANTIBODIES IS DERIVED

Table 1 Additional Sequences: In some embodiments, the antigenic domain may further be derived from the amino acid or nucleotide sequences encoding for one of the following proteins provided, with their full sequences listed under the UniProtKB accession numbers provided: annexin A2 (ANXA2, P07355), Annexin A5 (ANXA5, P08758); histone H1.0 (H10, P07305), histone Hl.1 (Hl-1, Q02539), histone Hl.2 (Hl-2, P16403), histone Hl.3 (Hl -3, Pl 6402), histone H1.4 (Hl-4, P10412), histone Hl.5 (Hl-5, P16401), histone Hit (Hl-6, P22492), testis-specific Hl histone (Hl-7, Q75WM6), histone Hl.8 (Hl-8, Q8IZA3), histone Hl.10 (Hl-10, Q92522), histone H2A proteins, histone H2B proteins, histone H3 proteins, histone H4 (H4C1, P62805), hnRNP-AO (HNRNPAO, Q13151), hnRNP-Al (HNRNPA1, P09651), hnRNP-Al-like 2 (HNRNPA1L2, Q32P51), hnRNP-A2/Bl (HNRNP A2B1, P22626), hnRNP-A3 (HNRNPA3, P51991), hnRNP-A/B (HNRNP AB, Q99729), hnRNP-Cl/C2 (HNRNPC, P07910), hnRNP-C- like 1 (HNRNPCL1, 060812), hnRNP-C-like 2 (HNRNPCL2, B2RXH8), hnRNP -DO (HNRNPD, Q 14103), hnRNP -DL (HNRNPDL, 014979), hnRNP-El, hnRNP -F (HNRNPF, P52597), hnRNP- H (HNRNPH1, P31943), hnRNP -H2 (HNRNPH2, P55795), hnRNP -H3 (HNRNPH3, P31942), hnRNP-I (PTBP1, P26599), hnRNP K (HNRNPK, P61978), hnRNP-L (HNRNPL, Pl 4866), hnRNP -L-like (HNRNPLL, Q8WVV9), hnRNP -M (HNRNPM, P52272), hnRNP-Q (HNRPQ, 060506), hnRNP -R (HNRNPR, 043390), hnRNP-U (HNRNPU, Q00839), hnRNP-U-like protein 1 (HNRNPUL1, Q9BUJ2), Protein-arginine deiminase type-1/ PAD1 (Q9ULC6), Protein-arginine deiminase type-2/ PAD2 (PADI2, Q9Y2J8), Protein-arginine deiminase type-3/ PAD3 (PADI3, 9ULW8), Protein-arginine deiminase type-4/ PAD4 (PADI4, Q9UM07), vimentin (VIM, P08670), filaggrin (FLG, P20930), filaggrin-2 (FLG2, Q5D862), fibrinogen alpha chain (FGA, P02671), fibrinogen beta chain (FGB, P02675), fibrinogen gamma chain (FGG, P02679), fibronectin (FN1, P02751), alpha-enolase (EN01, P06733), elongation factor 1-alpha 1 (EEF1A1, P68104), elongation factor 1-alpha 2 (EEF1A2, Q05639), beta-actin (ACTB, P60709), gamma-actin (ACTG1, P63261), alpha-1 type I collagen (COL1 Al, P02452), alpha-2 type I collagen (COL1 A2, P08123), alpha-1 type II collagen (C0L2A1, P02458), fructose-bisphosphate aldolase A (ALDOA, P04075), fructose-bisphosphate aldolase B (ALDOB, P05062), fructose-bisphosphate aldolase C (ALDOC, P09972), heat shock 60 EDa proteins (HSP60), heat shock 70 kDa proteins (HSP70), heat shock protein HSP 90 proteins (HSP90), immunoglobulin gamma- 1 heavy chain (IGHG1, P01857), immunoglobulin heavy constant gamma 2 (IGHG2, P01859), immunoglobulin heavy constant gamma 3 (IGHG3, P01860), immunoglobulin heavy constant gamma 4 (IGHG4, P01861); myeloblastin/ proteinase 3 (PRTN3, P24158), myeloperoxidase (MPO, P05164), neutrophil elastase (ELANE, P08246), lysosome-associated membrane glycoprotein 2 (LAMP2, Pl 3473), collagen alpha-3(IV) chain (COL4A3, Q01955), secretory phospholipase A2 receptor (PLA2R1, QI 3018), thrombospondin type-1 domain-containing protein 7A (THSD7A, Q9UPZ6); histone H3-like centromeric protein A/ CENP-A (CENPA, P49450), major centromere autoantigen B/ CENP-B (CENPB, P07199), centromere protein C/ CENP-C (CENPC, Q03188), DNA topoisomerase 1/ Scl-70 (TOPI, Pl 1387), exosome complex component RRP45/ PM/Scl-75 (EXOSC9, Q06265), exosome component 10/ PM/Scl-100 (EXOSCIO, Q01780), DNA-directed RNA polymerase III subunit RPC1 (POLR3A, 014802), DNA-directed RNA polymerase III subunit RPC2 (POLR3B, Q9NW08), DNA-directed RNA polymerase III subunit RPC3 (POLR3C, POLR3C), DNA-directed RNA polymerase III subunit RPC4 (POLR3D, P05423), DNA-directed RNA polymerase III subunit RPC5 (POLR3E, Q9NVU0), DNA-directed RNA polymerase III subunit RPC6 (POLR3F, Q9H1D9), DNA-directed RNA polymerase III subunit RPC7 (POLR3G, 015318), DNA-directed RNA polymerase III subunit RPC8 (POLR3H, Q9Y535), DNA-directed RNA polymerase III subunit RPC9 (CRCP, 075575), DNA-directed RNA polymerase III subunit RPC10 (POLR3K, Q9Y2Y1), DNA-directed RNA polymerases I and III subunit RPAC1 (POLR1C, 015160), DNA-directed RNA polymerases I and III subunit RPAC2 (POLR1D, P0DPB6), DNA-directed RNA polymerases I, II, and III subunit RPABC1 (POLR2E, Pl 9388), DNA-directed RNA polymerases I, II, and III subunit RPABC2 (POLR2F, P61218), DNA- directed RNA polymerases I, II, and III subunit RPABC3 (P0LR2H, P52434), DNA-directed RNA polymerases I, II, and III subunit RPABC4 (P0LR2K, P53803), DNA-directed RNA polymerases I, II, and III subunit RPABC5 (POLR2L, P62875), RNA Binding Region Containing 3/ RNPC3 (RNPC3, Q96LT9), ribonuclease P protein subunit p25/ Th/To antigen (RPP25, Q9BUL9), translation initiation factor eIF-2B subunit alpha (EIF2B1, Q14232), translation initiation factor eIF-2B subunit beta (EIF2B2, P49770), translation initiation factor eIF-2B subunit gamma (EIF2B3, Q9NR50), translation initiation factor eIF-2B subunit delta (EIF2B4, Q9UI10), translation initiation factor eIF-2B subunit epsilon (EIF2B5, Q 13144), gamma-interferon- inducible protein 16 (IFI16, QI 6666), protein bicaudal D homolog 2 (BICD2, Q8TD16), fibrillin- 1 (FBN1, P35555), rRNA 2 ’-O-m ethyltransferase fibrillarin/ fibrillarin 34 kDa (FBL, P22087), X- ray repair cross-complementing protein 6/ 70 kDa subunit of Ku antigen (XRCC6, P12956), X- ray repair cross-complementing protein 5/ 86 kDa subunit of Ku antigen (XRCC5, P13010); interferon-induced helicase C domain-containing protein 1/ melanoma differentiation-associated protein 5 (IFIH1, Q9BYX4), chromodomain-helicase-DNA-binding protein 4/ Mi-2 antigen (CHD4, QI 4839), chromodomain-helicase-DNA-binding protein 3/ Mi -2 antigen (CHD3, Q12873), histidine-tRNA ligase, cytoplasmic/ Jo-1 antigen (HARS1, P12081), histidine-tRNA ligase, mitochondrial (HARS2, P49590), threonine-tRNA ligase 1, cytoplasmic/ PL-7 antigen (TARSI, P26639), threonine-tRNA ligase, mitochondrial (TARS2, Q9BW92), threonine-tRNA ligase 2, cytoplasmic (TARS3, A2RTX5), alanine-tRNA ligase, cytoplasmic/ PL-12 antigen (AARS1, P49588), alanine-tRNA ligase, mitochondrial (AARS2, Q5JTZ9), glycine-tRNA ligase/ EJ antigen (GARS1, P41250), isoleucine-tRNA ligase, cytoplasmic/ OJ antigen (IARS1, P41252), isoleucine-tRNA ligase, mitochondrial (IARS2, Q9NSE4), asparagine-tRNA ligase, cytoplasmic/ KS antigen (NARS1, 043776), probable asparagine-tRNA ligase, mitochondrial (NARS2, Q96I59), phenylalanine-tRNA ligase alpha subunit/ ZO antigen (FARSA, Q9Y285), phenylalanine-tRNA ligase beta subunit (FARSB, Q9NSD9), phenylalanine-tRNA ligase, mitochondrial (FARS2, 095363), tyrosine-tRNA ligase, cytoplasmic/ HA antigen (YARS1, P54577), tyrosine-tRNA ligase, mitochondrial (YARS2, Q9Y2Z4), 3 -hydroxy-3 -methylglutaryl- coenzyme A reductase (HMGCR, P04035), signal recognition particle subunit SRP72 (SRP72, 076094), signal recognition particle subunit SRP68 (SRP68, Q9UHB9), signal recognition particle 54 kDa protein (SRP54, P61011), signal recognition particle 14 kDa protein (SRP14, P37108), signal recognition particle 19 kDa protein (SRP19, SRP19), signal recognition particle 9 kDa protein (SRP9, P49458), E3 ubiquitin-protein ligase TRIM33/ Transcription intermediary factor 1-gamma (TRIM33, Q9UPN9), MORC family CW-type zinc finger protein 3/ nuclear matrix protein 2 (M0RC3, Q14149), SUMO-activating enzyme subunit 1/ SAE antigen (SAE1, Q9UBE0), SUMO-activating enzyme subunit 2/ SAE antigen (UBA2, Q9UBT2), cytosolic 5’- nucleotidase 1A (NT5C1A, Q9BXI3), cell division cycle and apoptosis regulator protein 1 (CCAR1, Q8IX12), transcription factor SOX-5 (SOX5, P35711); E3 ubiquitin-protein ligase TRIM21/ Ro-52 kDa (TRIM21, Pl 9474), RNA-binding protein RO60/ Ro-60 kDa (RO60, Pl 0155), E3 ubiquitin-protein ligase TRIM68 (TRIM68, Q6AZZ1), Lupus La protein/ La (SSB, P05455); double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), or their protein complexes; small nuclear ribonucleoprotein Sm DI (SNRPD1, P62314), small nuclear ribonucleoprotein Sm D2 (SNRPD2, P62316), small nuclear ribonucleoprotein Sm D3 (SNRPD3, P62318); small nuclear ribonucleoprotein Sm E (SNRPE, P62304), small nuclear ribonucleoprotein Sm F (SNRPF, P62306), small nuclear ribonucleoprotein Sm G (SNRPG, P62308), small nuclear ribonucleoprotein-associated proteins B and B’ (SNRPB, P14678), small nuclear ribonucleoprotein-associated protein N/ Sm-N (SNRPN, P63162), U1 small nuclear ribonucleoprotein 70 kDa (SNRNP70, P08621), U1 small nuclear ribonucleoprotein C (SNRPC, P09234), U1 small nuclear ribonucleoprotein A (SNRPA, P09012), U2 small nuclear ribonucleoprotein A’ (SNRPA1, P09661), U2 small nuclear ribonucleoprotein B” (SNRPB2, P08579), 60S acidic ribosomal protein P0 (RPLPO, P05388), 60S acidic ribosomal protein Pl (RPLP1, P05386), 60S acidic ribosomal protein P2 (RPLP2, P05387), plasma protease Cl inhibitor (SERPING1, PO5155), complement Clq subcomponent subunit A (C1QA, P02745), complement Clq subcomponent subunit B (Cl QB, P02746), complement Clq subcomponent subunit C (C1QC, P02747), glycosylphosphatidylinositol-anchored high density lipoproteinbinding protein 1 (GPIHBP1, Q8IV16); a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13, Q76LX8), complement factor H (CFH, P08603), complement factor I (CFI, P05156), von Willebrand factor (VWF, P04275), coagulation factor VIII (F8, P00451), integrin alpha-lib (GPIIb) (ITGA2B, P08514), integrin beta-3 (GPIIIa) (ITGB3, P05106), platelet glycoprotein lb alpha chain (GPIb) (GP1BA, P07359), platelet glycoprotein lb beta chain (GP1BB, P13224), platelet glycoprotein IX (GP9, P14770), integrin alpha-2 (ITGA2, P17301), integrin beta-1 (ITGB1, P05556), platelet glycoprotein 4 (CD36, P16671), platelet glycoprotein V (GP5, P40197), P-selectin (SELP, QI 4242), platelet factor 4 (PF4, P02776); aquaporin-4 Ml (AQP4, P55087), aquaporin-4 M23 (AQP4, P55087), aquaporin-4 orthogonal arrays, myelin proteolipid protein (PLP1, P60201), myelin-oligodendrocyte glycoprotein (MOG, Q16653), myelin basic protein (MBP, P02686), myelin-associated oligodendrocyte basic protein (MOBP, QI 3875), myelin-associated glycoprotein (MAG, P20916), alpha-crystallin B chain (CRY AB, P02511), glial fibrillary acidic protein (GFAP, P14136), claudin-11 (Claudin-11, 075508), endoplasmic reticulum chaperone BiP (HSPA5, Pl 1021); acetylcholine receptor subunit alpha (CHRNA1, P02708), acetylcholine receptor subunit beta (CHRNB1, Pl 1230), acetylcholine receptor subunit gamma (CHRNG, P07510), acetylcholine receptor subunit delta (CHRND, Q07001), acetylcholine receptor subunit epsilon (CHRNE, Q04844), muscle skeletal receptor tyrosineprotein kinase (MUSK, 015146), low-density lipoprotein receptor-related protein 4 (LRP4, 075096), agrin (AGRN, 000468), acetylcholinesterase collagenic tail peptide (COLQ, Q9Y215), a-subunit of the voltage-gated potassium channel Kvl.4, titin (TTN, Q8WZ42), ryanodine receptor 1 (RYR1, P21817), ryanodine receptor 2 (RYR2, Q92736), ryanodine receptor 3 (RYR3, Q 15413), A-kinase anchor protein 12 (AKAP12, Q02952); glutamate receptor ionotropic, NMDA 1/ GluNl (GRIN1, Q05586), glutamate receptor ionotropic, NMDA 2A/ GluN2A (GRIN2A, Q12879), glutamate receptor ionotropic, NMDA 2B/ GluN2B (GRIN2B, Q13224), glutamate receptor ionotropic, NMDA 2C/ GluN2C (GRIN2C, Q14957), glutamate receptor ionotropic, NMDA 2D/ GluN2D (GRIN2D, 015399), cerebellar degeneration-related protein 2/ Yo antigen (CDR2, Q01850), ELAV-like protein 1 (ELAV-like protein 1, Q15717), ELAV-like protein 2 (ELAVL2, Q12926), ELAV-like protein 3 (ELAVL3, Q14576), ELAV-like protein 4 (ELAVL4, P26378), leucine-rich glioma-inactivated protein 1 (LGI1, 095970), contactin-associated proteinlike 2/ CASPR2 (CNTNAP2, Q9UHC6), contactin-2 (CNTN2, Q02246), dihydropyrimidinase- related protein 5/ CRMP5 (DPYSL5, Q9BPU6), glutamate receptor 1/ GluAl (GRIA1, P42261), glutamate receptor 2/ GluA2 (GRIA2, P42262), glutamate receptor 3/ GluA3 (GRIA3, P42263), glutamate receptor 4/ GluA4 (GRIA4, P48058), gamma-aminobutyric acid receptor subunit alpha- 1 (GABRA1, P14867), gamma-aminobutyric acid receptor subunit beta-3 (GABRB3, P28472), gamma-aminobutyric acid type B receptor subunit 1 (GABBR1, Q9UBS5), gamma-aminobutyric acid type B receptor subunit 2 (GABBR2, 075899), gamma-aminobutyric acid receptor subunit gamma-2 (GABRG2, P18507), metabotropic glutamate receptor 1 (GRM1, Q13255), metabotropic glutamate receptor 5 (GRM5, P41594), amphiphysin (AMPH, P49418), adenylate kinase isoenzyme 51 AK5 (AK5, AK5), dipeptidyl aminopeptidase-like protein 6 (DPP6, P42658), delta and Notch-like epidermal growth factor-related receptor (DNER, Q8NFT8), neurexin-3 (NRXN3, Q9Y4C0), RNA-binding protein Nova- 1/ Ri antigen (N0VA1, P51513), paraneoplastic antigen Mai (PNMA1, Q8ND90), paraneoplastic antigen Ma2 (PNMA2, Q9UL42), paraneoplastic antigen Ma3 (PNMA3, Q9UL41), modulator of apoptosis 1/ Ma4 (M0AP1, Q96BY2), microtubule-associated protein IB (MAP1B, P46821), dihydropyrimidinase-related protein 1 (CRMP1, Q14194); ganglioside GM1, ganglioside GMlb, ganglioside GDla, ganglioside GDlb, ganglioside GQlb, ganglioside GTla; protein-glutamine gammaglutamyltransferase 2 (TGM2, P21980), protein-glutamine gamma-glutamyltransferase E (TGM3, Q08188), protein-glutamine gamma-glutamyltransferase 6 (TGM6, 095932); thyroid peroxidase (TPO, P07202), thyrotropin receptor (TSHR, P16473), thyroglobulin (TG, P01266); islet cell autoantigen 1 (ICA1, Q05084); islet cell autoantigen 1-like protein (ICA1L, Q8NDH6); glutamate decarboxylase 1 (GAD1, Q99259), glutamate decarboxylase 2 (GAD-65) (GAD2, Q05329); receptor-type tyrosine-protein phosphatase-like N / IA2 (PTPRN, QI 6849); receptor-type tyrosine-protein phosphatase N2/ IAR (PTPRN2, Q92932), zinc transporter 8 (SLC30A8, Q8IWU4); steroid 21 -hydroxylase (CYP21A2, P08686), steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A1, P05093), 3 beta-hydroxysteroid dehydrogenase/Delta 5->4-isomerase type 2 (HSD3B2, P26439), adrenocorticotropic hormone receptor (MC2R, Q01718), NACHT, LRR and PYD domains-containing protein 5 (NLRP5, P59047), testis-specific gene 10 protein (TSGA10, Q9BZW7), hyaluronidase PH-20 (SPAM1, P38567), disintegrin and metalloproteinase domaincontaining protein 2 (ADAM2, Q99965), follicle-stimulating hormone receptor (FSHR, P23945), follitropin subunit beta (FSHB, P01225), glycoprotein hormones alpha chain (CGA, P01215), lutropin-choriogonadotropic hormone receptor (LHCGR, P22888), lutropin subunit beta (LHB, P01229); cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A1, P05108); cobalamin binding intrinsic factor (CBLIF, P27352), potassium-transporting ATPase alpha chain 1 (ATP4A, P20648), potassium-transporting ATPase subunit beta (ATP4B, P51164); desmoglein-1 (DSG1, Q02413), desmoglein-3 (DSG3, P32926), collagen alpha-l(XVII) chain/ 180 kDa bullous pemphigoid antigen (COL17A1, Q9UMD9), dystonin/ 230 kDa bullous pemphigoid antigen (DST, Q03001), collagen alpha-l(VII) chain (COL7A1, Q02388), envoplakin (EVPL, Q92817), epiplakin (EPPK1, P58107), periplakin (PPL, 060437), plectin (PLEC, Q15149), desmoplakin (DSP, P15924), desmocollin-1 (DSC1, Q08554), laminin gamma-1, laminin 5, laminin 6, ladinin- 1 (LAD1, 000515), integrin alpha-4 (ITGA4, P13612), integrin beta-6 (ITGB6, P18564), neuronal acetylcholine receptor subunit alpha-9 (CHRNA9, Q9UGM1); tumor necrosis factor (INF, P01375), lymphotoxin-alpha (LTA, P01374), granulocyte-macrophage colony-stimulating factor (CSF2, P04141), granulocyte colony-stimulating factor (CSF3, P09919), interleukin-1 alpha (ILIA, P01583), interleukin-1 beta (IL1B, P01584), interleukin-6 (IL6, P05231), interleukin- 17A (IL17A, Q16552), interleukin- 17F (IL17F, Q96PD4), interferon alpha-1/13 (IFNA1, P01562), interferon alpha-2 (IFNA2, P01563), interferon alpha-4 (IFNA4, P05014), interferon alpha-5 (IFNA5, P01569), interferon alpha-6 (IFNA6, PO5O13), interferon alpha-7 (IFNA7, P01567), interferon alpha-8 (IFNA8, P32881), interferon alpha-10 (IFNA10, P01566), interferon alpha-14 (IFNA14, P01570), interferon alpha-16 (IFNA16, P48551), interferon alpha-17 (IFNA17, P01571), interferon alpha-21 (IFNA21, P01568), interferon omega- 1 (IFNW1, P05000), interferon kappa (IFNK, Q9P0W0), interferon lambda- 1 (IFNL1, Q8IU54), interferon lambda-2 (IFNL2, Q8IZJ0), interferon lambda-3 (IFNL3, Q8IZI9), interferon lambda-4 (IFNL4, K9M1U5), interferon gamma (IFNG, P01579); cytochrome P450 2D6/ LKM-1 (CYP2D6, P10635), cytochrome P450 2C9 (CYP2C9, Pl 1712), cytochrome P4502A6 (CYP2A6, Pl 1509), UDP-glucuronosyltransferase 1A (UGT1A1, P22309), cytochrome P450 1A2 (CYP1A2, P05177), formimidoyltransferasecyclodeaminase (FTCD, 095954), dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial/ PDC-E2 (DLAT, Pl 0515), dihydrolipoyllysine- residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial/ 0GDC-E2 (DLST, P36957), lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial/ BCOADC-E2 (DBT, Pl 1182), pyruvate dehydrogenase complex protein X component, mitochondrial (PDX1, Pl 6451), dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT, P10515), nuclear autoantigen Sp-100 (SP100, P23497), coilin (COIL, P38432), nuclear pore membrane glycoprotein 210 (NUP210, Q8TEM1), lamin-B receptor (LBR, Q8MLV1), nuclear pore glycoprotein p62 (NUP62, P37198), o-phosphoseryl-tRNA(Sec) selenium transferase (SEPSECS, Q9HD40), or any parts thereof, any of their isoforms, any of their splicing variants, in either their unmodified or in their post-translationally modified (e.g., citrullinated, carbamylated, acetylated, glycosylated, phosphorylated, oxidated, y-carboxylated, among other), or proteolytically processed forms.

Non-limiting examples of amino acid sequences encoding linkers or hinges, as used in BaiTEs or BaitEs, are given in Table 2.

TABLE 2. NON-LIMITING EXAMPLES OF LINKERS OR HINGES INCORPORATED INTO

BISPECIFIC (AUTO) ANTIGEN-IMMUNE EFFECTOR CELL ENGAGING ANTIBODIES

Non-limiting examples of amino acid sequences of TCR-CD3 complex proteins that are targeted by effector cell-binding domains of BaiTEs are given in Table 3. TABLE 3. NON-LIMITING EXAMPLES OF TCR-CD3 COMPLEX PROTEINS TARGETED BY THE EFFECTOR CELL-BINDING DOMAIN

Non-limiting examples of amino acid sequences encoding variable heavy (VH) chains and variable light (VH) chains, as used in effector cell-binding domains of BaiTEs or BaitEs, are given in Tables 4-5 TABLE 4. NON-LIMITING EXAMPLES OF VARIABLE LIGHT CHAIN PEPTIDES FROM

WHICH EFFECTOR CELL-BINDING DOMAIN IS DERIVED

TABLE 5. NON-LIMITING EXAMPLES OF VARIABLE HEAVY CHAIN PEPTIDES FROM

WHICH THE EFFECTOR CELL-BINDING DOMAIN IS DERIVED

Non-limiting examples of amino acid sequences of NK cell activating receptors that are targeted by effector cell-binding domains of BaitEs are given in Table 6. TABLE 6. NON-LIMITING EXAMPLES OF NK CELL ACTIVATING RECEPTORS TARGETED BY THE EFFECTOR CELL-BINDING DOMAIN

Non-limiting examples of amino acid sequences encoding fusion protein domains, as used in BaiTEs or BaitEs, are given in Table 7. TABLE 7. NON-LIMITING EXAMPLES OF FUSION PROTEIN DOMAINS (or any parts thereof)

Non-limiting examples of compositions of scFv BaiTEs and BaitEs are given in Tables 8-11. Table 8. Non-limiting compositions for VH-VL scFv BaiTEs with N-terminal antigenic domain Table 9. Non-limiting compositions for VL-VH scFv BaiTEs with N-terminal antigenic domain

Table 10. Non-limiting compositions for VH-VL scFv BaiTEs with C-terminal antigenic domain

Table 11. Non-limiting compositions for VL-VH scFv BaiTEs with C-terminal antigenic domain

Table 12. Exemplary sequences for scFv BaiTEs (provided using one non-limiting example for engager domain, linker, and signal peptide, respectively. In some embodiments, sequence may contain other or additional signal peptides, pro-peptides, or may lack pro-peptides.)

Ill

Example 2 - p2GPI-BaiTEs redirect human T cells to selectively kill anti-p2GPI B cells, but not other B cells

For the treatment of APS, BaiTEs that fuse an scFv specific for human CD3 (e.g., derived from 0KT3, UCHT1, UCHTlv9, L2K-07, hXR32, 26II6, or SP34) or CP (e.g., derived from JOVI. l) with different antigenic sequences of disease-relevant autoantigens (e.g., P2GPI, EPCR, or PT) were developed. It was demonstrated that BaiTEs can bind primary human T cells (FIGs. 11-12) in liquid phase. It was shown that BaiTEs bind to both human CD8+ and CD4+ T cells by flow cytometry and that the antigenic domain remains accessible for binding. B2GPI-BaiTEs bound to the surface of T cells were detected using a patient-derived monoclonal antibody specific for P2GPI (FIGs. 12).

In addition, it was demonstrated that BaiTEs bind B cells expressing autoreactive BCRs with expected specificity, but do not bind B cells expressing irrelevant BCRs (FIG. 13). For this, Ramos B cells were modified by CRISPR/Cas9 homology directed repair to express a patient- derived autoreactive anti-[32GPI DI BCR (Pl-117). Anti-02GPI Ramos B cells and unedited Ramos B cells (expressing irrelevant BCR) were incubated with P2GPI-BaiTEs incorporating different antigenic sequences of P2GPI, EPCR-BaiTE, or no BaiTE. Binding of BaiTEs to autoreactive Ramos B cells was confirmed using a DyLight 488-conjugated antibody by flow cytometry. Relevant BaiTEs successfully bound anti-p2GPI BCRs on the surface of autoreactive B cells (FIG. 13).

It was demonstrated that P2GPI-BaiTEs are potent drugs to redirect bystander T cells to engage and eliminate pathogenic anti-P2GPI DI B cells in APS, while not killing B cells expressing other BCRs (FIGs. 14-16). Human T cells were co-cultured with Ramos B cells expressing anti- P2GPI BCRs or unedited Ramos B cells (expressing irrelevant BCRs) in the presence or absence of purified p2GPI-BaiTE. In the presence of p2GPI-BaiTE, primary human T cells killed autoreactive anti-p2GPI Ramos B cells but not wild-type Ramos B cells. In the absence of BaiTE, neither autoreactive anti-p2GPI Ramos B cells nor wild-type Ramos B cells were killed by human T cells. B cells are stained with FITC anti-CD20 and autoreactive anti-P2GPI BCRs were detected using DyLight 649-conjugated StrepTactin XT (FIG. 14).

Furthermore, it was demonstrated that p2GPI-BaiTE selectively depleted anti-P2GPI B cell clones of various B cell receptor surface antigen densities. Anti-p2GPI B cell clones expressing various densities of autoreactive B cell receptor (Pl -117) on their cell surface were generated and single cell clones established. In co-culture experiments, autoreactive anti-P2GPI Ramos B cells are efficiently killed at low BaiTE concentrations (e.g., 15 ng/mL), while wild-type cells are not depleted even at highestBaiTE concentrations (e.g., 1000 ng/mL). BaiTEs were effective at killing autoreactive B cells across a wide range of anti-P2GPI BCR cell surface densities, making them promising therapeutics even for low-antigen density targets like plasma cells (FIGs. 15-16). In addition, it was shown that p2GPI-BaiTE efficiently depletes anti-P2GPI B cell clones expressing different patient-derived monoclonal B cell receptors (Pl-117, Pl-190, and P2-6, respectively) (FIG. 17). In addition, it was shown that p2GPI-BaiTE efficiently depletes anti- 02GPI B cell clones expressing patient-derived monoclonal B cell receptors regardless of their isotype or subclass (e.g., IgM, IgA, IgGl, IgG3) (FIG. 19).

Finally, it was demonstrated that treatment with p2GPI-BaiTEs abrogates autoantibody production. Human T cells were co-cultured with Ramos B cells expressing one of three different anti-P2GPI B cell receptors (Pl-117, Pl-190, or P2-6) in the presence or absence of two different P2GPI-BaiTEs. At the end of the experiment, anti-P2GPI autoantibody levels were measured in cell culture supernatants. BaiTE3 effectively abrogated anti-p2GPI autoantibody production for all target B cell lines (FIG. 18).

Claims

WHAT IS CLAIMED IS:

1. A chimeric polypeptide comprising:

(a) an antigenic domain comprising an (auto)antigenic moiety, wherein the (auto)antigenic moiety is recognized by a B cell receptor (BCR); and

(b) an effector cell-binding domain, wherein the effector cell-binding domain specifically binds to and engages an immune effector cell.

2. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises a single-chain polypeptide or multi-chain polypeptide.

3. The chimeric polypeptide of claim 1 or 2, wherein the chimeric polypeptide further comprises a linker sequence, hinge, dimerization domain, bioconjugation domain, or any combination thereof.

4. The chimeric polypeptide of claim 1-3, wherein a first linker sequence, hinge, dimerization domain, bioconjugation domain, or any combination thereof is between the antigenic domain and the effector cell-binding domain.

5. The chimeric polypeptide of any one of claims 1-4, wherein the (auto)antigenic moiety is derived from a phospholipid-binding protein.

6. The chimeric polypeptide of claim 5, wherein the phospholipid-binding protein is beta-2 glycoprotein I ([32GPI), endothelial protein C receptor (EPCR), prothrombin (PT).

7. The chimeric polypeptide of claim 6, wherein the (auto)anti genic moiety comprises at least part of a domain of 02GPI, wherein 02GPI comprises domain I (DI), domain II (DII), domain III (Dill), domain IV (DIV), or domain V (DV); an amino acid epitope comprising a sequence of “RGGMR”; any of their orthologs; or any combination thereof.

8. The chimeric polypeptide of claim 6, wherein the (auto)antigenic moiety comprises at least part of an endothelial protein C receptor (EPCR); a full or part of an extracellular (EC) domain of EPCR; a modified extracellular (EC) domain of EPCR; or an EPCR in complex with a phospholipid.

9. The chimeric polypeptide of claim 6, wherein the (auto)antigenic moiety comprises at least part of prothrombin (PT) or posttranslationally modified prothrombin; or a PT in complex with a phospholipid.

10. The chimeric polypeptide of claim 4, wherein the (auto)antigenic moiety further comprises a second linker sequence or hinge within the antigenic domain.

11. The chimeric polypeptide of any one of claims 1-10, wherein the (auto)antigenic moiety is derived from proteinase 3 (PR3), myeloperoxidase (MPO), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG), coagulation factor VIII (FVIII), muscle skeletal receptor tyrosine-protein kinase (MuSK), phospholipase A2 receptor (PLA2R), disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), E3 ubiquitin-protein ligase TRIM21 (TRIM21), signal recognition particles (SRP), 3- hydroxy-3 -methyl glutaryl-coenzyme A reductase (HMGCR), protein-arginine deiminase type-4 (PAD4), desmoglein 1 (DSG1), or desmoglein 3 (DSG3).

12. The chimeric polypeptide of any one of claims 1-10, wherein the (auto)antigenic moiety is derived from any one of the autoantigenic peptides from Table 1.

13. The chimeric polypeptide of any one of claims 1-12, wherein the (auto)antigenic moiety comprises a mutation or modification in a nucleic acid sequence or an amino acid sequence of the (auto)antigenic moiety.

14. The chimeric polypeptide of claim 13, wherein the mutation or modification (i) inhibits or abrogates specific or unspecific binding of the (auto)antigenic moiety to tissues, cells, membranes, receptors, or ligands other than their cognate B cell receptors, (ii) inhibits or abrogates enzymatic activity, or (iii) alters other undesired biological function of the antigen.

15. The chimeric polypeptide of any one of claims 1-14, wherein the (auto)antigenic moiety is a post-translationally modified peptide.

16. The chimeric polypeptide of claim 15, wherein the posttranslational modification is citrullination/deimination, carb amyl ati on, acetylation, glycosylation, deamination, phosphorylation, oxidation, or y-carboxylation.

17. The chimeric polypeptide of any one of claims 1-14, wherein the (auto)antigenic moiety comprises a lipid or phospholipid.

18. The chimeric polypeptide of any one of claims 1-14, wherein the (auto)antigenic moiety comprises a single-stranded or double-stranded nucleic acid or an oligonucleotide.

19. The chimeric polypeptide of any one of claims 1-18, wherein the (auto)antigenic moiety comprises one or more (auto)antigen(s).

20. The chimeric polypeptide of any one of claims 1-19, wherein the effector cell-binding domain is derived from an antibody, antibody fragment, or immunoligand.

21. The chimeric polypeptide of claim 20, wherein the effector cell-binding domain comprises an immunoglobulin variable light chain, variable heavy chain, or both.

22. The chimeric polypeptide of any one of claims 20-21, wherein the effector cell-binding domain comprises a single-chain antibody.

23. The chimeric polypeptide of claim 22, wherein the effector cell-binding domain comprises a single-chain variable fragment (scFv).

24. The chimeric polypeptide of claim 23, wherein the scFv comprises a variable light chain and a variable heavy chain.

25. The chimeric polypeptide of any of the claims 20-24, wherein the effector cell-binding domain binds to a protein or epitope of the T cell receptor (TCR)-CD3 complex.

26. The chimeric polypeptide of any of the claim 20-25, wherein the effector cell-binding domain binds to CD3s, CD3y, CD35, Ca (TRAC), any C|3 alleles (TRBC1, TRBC2), Va alleles (TRAV1-TRAV41), any V0 alleles (TRBV1-TRBV30), any Cy alleles (TRGC1, TRGC2), C6 (TRDC), any Vy alleles (TRGV1-9), any V5 alleles (TRDV1-3), or TCR Vy9V52 of the T cell receptor (TCR)-CD3 complex.

27. The chimeric polypeptide of any of the claims 20-26, wherein the effector cell-binding domain binds to a surface activating receptor or co-receptor on an immune effector cell.

28. The chimeric polypeptide of claim 21, wherein the effector cell-binding domain further comprises a third linker sequence.

29. The chimeric polypeptide of claim 28, wherein the third linker sequence is between the variable light chain and the variable heavy chain.

30. The chimeric polypeptide of any one of claims 1-29, wherein the effector cell-binding domain comprises one or more effector cell binding domain(s).

31. The chimeric polypeptide of any one of claims 1-30, further comprising a fusion protein domain.

32. The chimeric polypeptide of claim 31, wherein the fusion protein domain comprises a protein molecule that extends half-life of the chimeric polypeptide in vivo.

33. The chimeric polypeptide of claim 32, wherein the protein molecule 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.

34. The chimeric polypeptide of claim 31, wherein the fusion protein domain comprises a protein molecule that comprises a payload or toxin.

35. The chimeric polypeptide of any one of claims 31-34, wherein the fusion protein domain further comprises a fourth linker sequence.

36. The chimeric polypeptide of any one of claims 31-35, wherein the fusion protein domain is expressed N-terminal to both the antigenic domain(s) and the effector cell-binding domain(s).

37. The chimeric polypeptide of any one of claims 31-35, wherein the fusion protein domain is expressed C-terminal to both the antigenic domain(s) and the effector cell-binding domain(s).

38. The chimeric polypeptide of any one of claims 31-35, wherein the fusion protein domain is expressed in-between the antigenic domain(s) and the effector cell-binding domain(s).

39. The chimeric polypeptide of any one of claims 1-38, wherein the antigenic domain(s) are expressed N-terminal to the effector cell-binding domain(s).

40. The chimeric polypeptide of any one of claims 1-38, wherein the antigenic domain(s) are expressed C-terminal to the effector cell-binding domain(s).

41. The chimeric polypeptide of any one of claims 1-40, wherein the immune effector cell is a human immune cell.

42. The chimeric polypeptide of claim 41, wherein the immune effector cell is a T cell.

43. The chimeric polypeptide of claim 41, wherein the immune effector cell is an NK cell.

44. A nucleic acid molecule encoding the chimeric polypeptide of any one of claims 1-43.

45. A recombinant vector comprising the nucleic acid molecule of claim 44.

46. A cell comprising the nucleic acid molecule of claim 44 or the recombinant vector of claim 45.

47. A pharmaceutical composition comprising: any of the chimeric polypeptides of claims 1-43, the nucleic acid molecules of claim 44, the recombinant vector of claim 45, or the cell of claim 46; and a pharmaceutically acceptable carrier.

48. A method of treating a B cell-mediated disorder in a subject, the method comprising: administering to the subject the recombinant vector of claim 45, the cell of claim 46, or the pharmaceutical composition of claim 47.

49. The method of claim 48, wherein the B cell-mediated disorder comprises an allergy.

50. The method of claim 48, wherein the B cell-mediated disorder is an autoimmune disease.

51. The method of claim 50, wherein the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease.

52. The method of claim 50, wherein the autoimmune disease is antiphospholipid syndrome (APS) or catastrophic antiphospholipid syndrome.

53. The method of claim 51, wherein the systemic autoimmune disease is rheumatoid arthritis, a spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, lupus nephritis, Sjogren’s disease, scleroderma/ systemic sclerosis, an idiopathic inflammatory myopathy, myositis, dermatomyositis, anti synthetase syndrome, an immune-mediated necrotizing myopathy, IgG4-related disease, vasculitis, granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, anti-glomerular basement membrane disease, Henoch-Schonlein purpura, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, or giant cell arteritis. The method of claim 51, wherein the organ-specific autoimmune disease is acquired haemophilia, autoimmune encephalitis, anti-N-methyl-d-aspartate (NMDA) receptor encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune and paraneoplastic encephalitis, Addison’s disease, celiac disease, Graves’ disease, Hashimoto thyroiditis, immune thrombocytopenia purpura, multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody-related diseases, neuromyelitis optica (NMO) spectrum disorders, myasthenia gravis, Lambert-Eaton myasthenia syndrome, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, other autoimmune blistering diseases, autoimmune membranous nephropathy, primary membranous nephropathy, primary biliary cirrhosis, thrombotic thrombocytopenic purpura, or type I diabetes mellitus The method of any claims of 48-54, wherein the subject is an individual at risk of an autoimmune disease. The method of claim of 55, wherein the subject has preclinical autoimmunity or a disease-associated autoantibody.