Compositions and methods for antigen-specific therapy

Abstract

A platform for therapy provides a multi-target therapeutic molecule comprising an anti-CD3 antigen binding fragment of an anti-CD3 antibody linked with a linker to two or more auto-antigen(s). The auto-antigens include Dsg1, Dsg3, bullous pemphigoid antigen 180, bullous pemphigoid antigen 230, MuSK, anti-phospholipase A2 Receptor. A pharmaceutical composition comprises a disclosed multi-target therapeutic molecule is disclosed. Methods of treating diseases using the disclosed molecules are disclosed

Description

COMPOSITIONS AND METHODS FOR ANTIGEN-SPECIFIC THERAPYTECHNICAL FIELD

[0001] This disclosure relates to a platform for using a chimeric molecule with multiple immunecell binding moieties for therapy.BACKGROUND

[0002] The immune system is a powerful defense system for animals. Scientists are beginning to manipulate the immune system to make it attack antigens and cells in order to cure diseases. More is required to meet this goal.SUMMARY

[0003] In one aspect, this disclosure provides a chimeric molecule comprising a T-cell binding moiety and two or more auto-antigens of an autoimmune disease, each auto-antigen is linked to the T-cell binding moiety by linker. In some embodiments, the T-cell binding moiety is an anti- CD3 antibody, or an antigen-binding fragment thereof. In other embodiments, the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region.This disclosed chimeric molecule can be referred to herein also as “B-cell Selective Molecular Autoantigen-Receptor Targetors” (“B-SMAART”).

[0004] In another aspect, this disclosure provides a pharmaceutical composition comprising one or more disclosed chimeric molecules.

[0005] In another aspect, a method is provided of treating or preventing an autoimmune disease in a patient comprising administering a therapeutically or prophylactically effective amount of a disclosed pharmaceutical composition to said patient.

[0006] Numerous other aspects are provided in accordance with these and other aspects of the invention. Other features and aspects of the present invention will become more fully apparent from the following detailed description and the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A (patient PV327), FIG. IB (patient PV102), and FIG. 1C (patient PV114) show IgG reactivity in a longitudinal analysis for 3 patients. The y axis shows fold change inexpression levels of the various autoantibodies in the blood. The X-axis shows data point for same patient in different phases of disease: A= active disease; LTR=long term remission (>6m); R=remission.

[0008] FIG. 2A is a diagram of an embodiment of a disclosed chimeric molecule (a B- SMAART) and FIG. 2B is a diagram showing the mechanism of action of an embodiment of a disclosed B-SMAART.DETAILED DESCRIPTION

[0009] As used herein, the word “a” or “plurality” before a noun represents one or more of the particular noun.

[0010] For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow, unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term “about,” whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0011] “Effective amount,” “prophylactically effective amount,” or “therapeutically effective amount” refers to an amount of an agent or composition that provides a beneficial effect or favorable result to a subject, or alternatively, an amount of an agent or composition that exhibits the desired in vivo or in vitro activity. “Effective amount,” “prophylactically effective amount,” or “therapeutically effective amount” refers to an amount of an agent or composition that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, disorder or condition in a patient / subject, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations. In some embodiments, an effective amount or a therapeutically effective of a molecule results in the killing of deleterious autoreactive B cells.

[0012] As used herein, a “patient” and a “subject” are interchangeable terms and may refer to a human patient / subject, a dog, a cat, a non-human primate, etc.

[0013] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably and are known in the art and can mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.

[0014] The terms “antibody and antigen binding fragment(s) thereof’ are known in the art. These terms are used herein to include a fusion protein comprising an antibody or antigen binding fragment(s) thereof.

[0015] The term “antibody fragment,” “antigen-binding fragment of an antibody,” and the like are known in the art. The term “antibody fragment,” “antigen-binding fragment of an antibody,” and the like can, for example, refer to a fragment of an antibody that retains the ability to bind to a target antigen. Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), a Fd fragment, a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of an antigen-binding fragment of an antibody. See, e.g., Todorovska et al. (2001) J Immunol Methods 248(l):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1): 177- 189; Poljak (1994) Structure 2(12): 1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283. An antigen-binding fragment can also include the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. An antigen-binding fragment can comprise the CDRs of the light chain and heavy chain polypeptide of an antibody.

[0016] The term “antibody fragment,” “antigen-binding fragment of an antibody,” and the like also can include, e.g., single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al. (2000) Curr Pharm Biotech 1:253-263; Reichmann et al. (1999) J Immunol Meth 231:25-38; PCT application publication nos. WO 94 / 04678 and WO 94 / 25591; and U.S. patent no. 6,005.079. The term "antibody fragment" also includes single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

[0017] The term ‘an antigen-binding fragment” of an antibody can also include the entire Fc tail of an antibody with or without fucosylation.

[0018] The terms “self-antigen” and “auto- antigen” are used interchangeably herein.

[0019] All ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

[0020] All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” or “5 to 10” or “5-10” should be considered to include the end points 5 and 10.

[0021] The term “CD3” is known in the art and refers to cluster of differentiation 3 and is a protein complex and T cell co-receptor.

[0022] It is further to be understood that the feature or features of one embodiment may generally be applied to other embodiments, even though not specifically described or illustrated in such other embodiments, unless expressly prohibited by this disclosure or the nature of the relevant embodiments. Likewise, compositions and methods described herein can include any combination of features and / or steps described herein not inconsistent with the objectives of the present disclosure. Numerous modifications and / or adaptations of the compositions and methods described herein will be readily apparent to those skilled in the art without departing from the present subject matter.

[0023] 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 belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions controls.

[0024] AUTOIMMUNE DISEASES; AUTOIMMUNE ACTIVITIES

[0025] Autoimmune disease, prevalent in the population, is a major healthcare burden. An autoimmune disease is when a host’s immune system attacks one or more self-antigens (autoantigens). In the majority of autoimmune diseases the target auto-antigen(s) are not known. There are over 100 known human autoimmune diseases, affecting between 5-10% of the population. Autoimmune diseases are the 2ndor 3rdleading cause of morbidity and mortality and cost the US healthcare system over $100 billion annually. Lupus, pemphigoid, myasthenia gravis, multiple sclerosis, type 1 diabetes, and pemphigus vulgaris are just some examples of autoimmune diseases. In patients with severe COVID, a significant number have new autoantibodies upon recovery, including autoantibodies to interferon. Treatment options forautoimmune diseases are limited and largely non-specific or symptom oriented. No true cures are available and there are no consensus treatment guidelines. Approved targeted, individualized therapies are lacking.

[0026] Pemphigus is a group of IgG-mediated autoimmune diseases of stratified squamous epithelia, such as the skin and oral mucosa, in which acantholysis (the loss of cell adhesion) causes blisters and erosions. Pemphigus has three major subtypes: pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus.

[0027] Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering skin disease, characterized by intraepithelial (suprabasalar) acantholysis, which is a loss of cell-cell adhesion. Quite a bit is known about PV. An HLA genetic predisposition (HLA DRB 1*0402 and DQB 1*0503) is known; T cell (Tin driven) and B cell subsets (producing IgG4 autoantibodies) are known; and the primary autoantibody targets (autoantigens) - Desmoglein (Dsg)-3 and Desmoglein-1 - are known.

[0028] Dsg3 and Dsgl are keratinocyte-associated cell surface proteins relevant to cell-cell adhesion. Anti-Dsg3 and anti-Dsgl autoantibodies can be detected in human PV patients and can be followed by ELISA. The titers roughly correlate with disease activity and serve as disease biomarkers.

[0029] Current and proposed treatments of PV include general immunosuppression with, for example, steroids; immunoglobulin-focused therapy, such as intravenous IG or FcRn blockade; B-cell targeted therapies, such as anti-CD20 molecules, BTK inhibitors, or BAFF inhibitors; and antigen- specific therapies.

[0030] Currently proposed antigen-specific therapy makes use of exploratory Chimeric AutoAntibody Receptor T (CAAR-T) cells, an adaptation of the CAR-T cell strategy, as well as classic CAR-T cells recognizing CD19. CAAR-T cells are T-cells engineered to express autoantigen-based chimeric immunoreceptors. This platform directs T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR), without the requirement for endogenous T-cells (autologous or allogeneic). For PV, engineered human T cells expressing the PV autoantigen Dsg3 exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and specifically eliminate Dsg3-specific B cells in vivo in a PV mouse model.

[0031] Major hurdles, however, are associated with such cell-based therapies. These hurdles include:• complicated manufacturing process, involving harvesting of autologous T cells, engineering of cells, reinfusing cells and failing productions;• treatment time lag from start to finish;• complex patient referral pathway;• accredited CAAR-T cell specialty centers and trained staff are needed;• potential for significant, life-threatening adverse effects;• potential for long lived, permanence of therapy and related adverse events;• inability to tune down therapy;• inability to readily adapt to multiple target therapy;• inability to readily adapt to evolving autoimmune response in a given patient;• potential risk of secondary cancer;• exorbitant costs;• commercial scalability challenges; and• complicated payer policies.

[0032] HEMOPHILIA

[0033] Hemophilia A patients can be treated successfully with factor VIII. However, 5-30% of patients with hemophilia A (of all severities) develop inhibitory anti-factor VIII antibodies (inhibitors) following replacement therapy.

[0034] GENE THERAPY VECTORS

[0035] Gene therapy holds enormous promise. However, a patient’s immune system can be a hindrance to gene therapy. Viral capsids, viral- vector DNA (also referred herein as a DNA molecule carried by a viral vector), and even the transgene products themselves may be recognized as foreign by the immune system. Immunity against viral capsids, viral-vector DNA (also referred herein as a DNA molecule carried by a viral vector), and transgene products can limit the efficacy and restrict dosing of gene therapy.

[0036] MOLECULES, COMPOSITIONS, AND METHODS

[0037] In one aspect, this disclosure provides a chimeric molecule comprising a T-cell binding moiety and two or more auto-antigens of an autoimmune disease, each auto-antigen, or polypeptide or peptide fragment thereof, is linked to the T-cell binding moiety by linker. In some embodiments, the T-cell binding moiety is an anti-CD3 antibody, or an antigen-binding fragmentthereof. Tn another embodiment, the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region. In some embodiments, the T-cell receptor variable chain region is selected from V 2, V05.1, VP6, VP8, vpiO, and VP13.1. In certain embodiments, two or more auto-antigens are the same auto-antigens. In some embodiments, two or more auto-antigens are different.

[0038] Each autoantigen can be a full-sized protein, or a polypeptide fragment thereof (or variants / mutants of either a full- sized protein or smaller polypeptide) but still bind to its antibody or antigen-binding fragment thereof.

[0039] The chimeric molecule can be referred to herein also as “B-cell Selective Molecular Autoantigen-Receptor Targetors” (“B-SMAART”). The B-SMAART chimeric molecule is designed to bridge a cytolytic T-cell to an autoimmune B-cell.

[0040] In certain embodiments, the chimeric molecule further comprises an anti-CD19 antibody, or an antigen-binding fragment thereof, linked to the T-cell binding moiety or an auto-antigen, or polypeptide or peptide fragment thereof, by linker. In some embodiments, one or more of the antigen binding fragments is a single chain antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, or a single chain Fvfragment. In some embodiments, one or more of the antigen binding fragments is a fragment of an IgG molecule. In certain embodiments, an antibody or an antigen binding fragment with an Fc tail has reduced or no fucose moieties.

[0041] CD19 is known in the art. Briefly, CD19 is a protein found on the surface of B cells that is crucial for their development, activation, and proliferation.

[0042] In certain embodiments, the chimeric molecule further comprises an anti-CD20 antibody, or an antigen-binding fragment thereof, linked to the T-cell binding moiety or an auto-antigen, or polypeptide or peptide fragment thereof, by linker. In some embodiments, one or more of the antigen binding fragments is a single chain antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, or a single chain Fvfragment. In some embodiments, one or more of the antigen binding fragments is a fragment of an IgG molecule. In certain embodiments, an antibody or an antigen binding fragment with an Fc tail has reduced or no fucose moieties.

[0043] CD20 is known in the art. Briefly, CD20 is B lymphocyte cell-surface molecule.

[0044] In some embodiments, the auto-antigen is Dsg3 or Dsgl, or portions of the auto-antigen Dsg3 or Dsgl. In some embodiments, the chimeric molecule comprises both Dsg3 and Dsgl, or portions of the auto-antigen Dsg3 and Dsgl. Dsg3 and Dsgl are known B cell target auto-antigens of patients with Pemphigus vulgaris (PV), an autoimmune blistering skin disease. In some embodiments, at least one of the autoantigens is an autoantigen selected from the group consisting of: ANXA9 (Annexin A9), ATP2cl (ATPase Secretory Pathway Ca2+ Transporting 1), C / N, C5a receptor 1, Cadherin 8 (CDH8), Cadherin 9 (CDH9), CCT7, CD32 (FCGR2A), CD33, CENP (A / B), CHRNA9, CHRND , CHRNE (Cholinergic Receptor Nicotinic Epsilon Subunit). COL21A1 (collagen type XXI alpha 1 chain), Collagen III (COL3A1), Collagens, Cytochrome B5 (CYB5B), DSC1 (Desmocollin-1), DSC2 (Desmocollin-2), DSC3 (Desmocollin-3), dsDNA+ssDNA, Dsgl (Desmoglein 1), Dsg3 (Desmoglein 3), Dsg4 (Desmoglein 4), E-Cadherin, FcIgE, FH (fumarase), GP1BA (Glycoprotein lb Platelet Subunit Alpha), HLA-DRA, HLA E, HSP60 Protein, Human Ml, Human M2, Human M3, Human M4, Human M5, Integrin alpha X, JUP. NDUFS1 (NADH-ubiquinone oxidoreductase), PDHA1 (pyruvate dehydrogenase El subunit alpha 1), PK3, PR3-MPO, Ro-SSA, RPP, SOD2 (superoxide dismutase 2), TG (thyroglobulin) ,TPO (Thyroid Peroxidase), and UlsnRNP. These auto-antigens are auto-antigens identified for PV, including Dsgl and Dsg3. In some embodiments, at least one of the auto-antigen is patient specific.

[0045] In some embodiments, two of the the auto-antigens are bullous pemphigoid antigen 180 and bullous pemphigoid antigen 230. Bullous pemphigoid antigen 230 are known auto-antigens of patients with bullous pemphigoid. In some embodiments, at least one of the auto-antigens is patient specific. In some embodiments, a disclosed chimeric molecule comprises muscle specific tyrosine kinase (MuSK). MuSK is a known auto-antigen of patients with myasthenia gravis (MG). In other embodiments, a disclosed chimeric molecule comprises an anti-phospholipase A2 Receptor (PLA2R). PLA2R is a known auto-antigen of patients with membranous nephropathy (MNEP), a disorder where the body’s immune system attacks the filtering membranes in the kidney. These membranes clean waste products from the blood.

[0046] In some embodiments, the chimeric molecule comprises Dsgl and / or Dsg3 and one or more of the auto-antigens from Table 2 other than Dsgl and Dsg3. In some embodiments, the chimeric molecule comprises the auto-antigen bullous pemphigoid antigen 180, bullous pemphigoid antigen 230, or both. In some embodiments, the chimeric molecule comprises Dsgl and Dsg3. In some embodiments, the components comprise Dsgl and / or Dsg3 and one or more of the auto-antigens from Table 2 other than Dsgl and Dsg3. In some embodiments, two of the the auto-antigens are bullous pemphigoid antigen 180 and bullous pemphigoid antigen 230. Insome embodiments, at least one of the auto-antigens is patient specific. In some embodiments, a disclosed chimeric molecule comprises muscle specific tyrosine kinase (MuSK). MuSK is a known auto-antigen of patients with myasthenia gravis (MG). In other embodiments, a disclosed chimeric molecule comprises an anti-phospholipase A2 Receptor (PLA2R). PLA2R is a known auto-antigen of patients with membranous nephropathy (MNEP), a disorder where the body’s immune system attacks the filtering membranes in the kidney. These membranes clean waste products from the blood.

[0047] In some embodiments, at least one of the auto-antigens is patient specific.

[0048] In another aspect, this disclosure provides a chimeric molecule comprising a T-cell binding moiety and an interferon molecule or a portion thereof linked to the T-cell binding moiety by linker, wherein said T-cell binding moiety is an anti-CD3 antibody or an antigenbinding fragment thereof or an antibody or antigen-binding fragment to a T-cell receptor variable chain region. In some embodiments, the T-cell binding moiety is an anti-CD3 antibody or an antigen-binding fragment thereof. In some embodiments, the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region. In some embodiments, the T-cell receptor variable chain region is selected from V02, V05.1, V06, Vp8, VpiO, and Vpi3.1. In certain embodiments, the chimeric molecule further comprises an antiCD 19 antibody or an antigen-binding fragment thereof, linked to the T-cell binding moiety or the interferon molecule or a portion thereof by linker. A pharmaceutical composition comprising one or more disclosed chimeric molecule comprising an interferon molecule or a portion thereof is provided. This disclosure also provides a method of treating a patient having autoantibodies to an interferon after a Covid-19 infection, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising one or more disclosed chimeric molecule comprising an interferon molecule or a portion thereof.

[0049] In another aspect, a pharmaceutical composition comprising one or more disclosed chimeric molecule is provided. In some embodiments, the pharmaceutical composition comprises two or more disclosed chimeric molecules, so as to treat or prevent a specific autoimmune disease.

[0050] This disclosure also provides, in yet another aspect, a method of treating or preventing an autoimmune disease in a patient comprising administering a therapeutically or prophylactically effective amount of a disclosed pharmaceutical composition to said patient. In someembodiments, the autoimmune disease is Pemphigus vulgaris (PV) or its variants, and each chimeric molecule comprises one or more auto-antigens, or a polypeptide or peptide fragment of an auto-antigen, of Pemphigus vulgaris. In other embodiments, the autoimmune disease is bullous pemphigoid, wherein said chimeric molecule or molecules comprise auto-antigens specific to bullous pemphigoid. In certain embodiments, the autoimmune disease is a subtype of myasthenia gravis, wherein said chimeric molecule or molecules comprise auto-antigens specific to said subtype of myasthenia gravis. In yet other embodiments, the autoimmune disease is membranous nephropathy, wherein said chimeric molecule or molecules comprise auto-antigens specific to membranous nephropathy.

[0051] This disclosure also provides a method of treating or preventing a B-cell mediated disease in a patient in need thereof comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising a disclosed chimeric molecule comprising an anti-CD19 antibody or antigen binding fragment thereof, or an anti-CD20 antibody or antigen binding fragments thereof.

[0052] In some embodiments, the disclosed chimeric molecule comprises a component with an antibody backbone or is a fusion protein comprising an antibody or an antigen binding fragment thereof.

[0053] In some embodiments, one or more of the antigen binding fragments is a single chain antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, or a single chain Fvfragment. In certain embodiments, one or more of the antigen binding fragments is a fragment of an IgG molecule. In some embodiments, one or more of the antigen-binding fragment has reduced or no fucose moieties. The alteration involves having fucose moieties removed from the antibody moieties of the disclosed multi-specific molecule or have the antibody made such that low or no fucose is added to it. Low or no fucose on the antibody moieties of the disclosed multi- specific molecule can be accomplished by methods known in the art. including, without limitation, producing the disclosed multi- specific molecule in specific cell lines that lack or are deficient in enzyme(s) responsible for fucosylation, or in the pathway for generating fucose, or enzymatically removing fucose moieties by treating the disclosed multi- specific molecule with glycosidase and glycosynthase enzymes.

[0054] The autoantigen can be a full- sized protein or smaller polypeptides (or variants / mutants of either a full-sized protein or smaller polypeptide) but still bind to its antibody or antigen-binding fragment thereof. Tn some embodiments, the size of the auto-antigen is optimized for binding to its cognate B-cell receptor but does not get bound by a patient’s circulating autoantibodies to the auto-antigen. The auto-antigen component of the chimeric molecule can be a recombinant polypeptide. The known auto-antigens are known in the art and can be obtained by, for example, recombinant DNA technology or other methods known in the art. The auto-antigens may also be purchased or gifted.

[0055] The antibodies to CD3 and TCR are known in the art or can be obtained or generated by routine laboratory techniques. CD3 and TCR proteins are known and can be obtained or generated by routine laboratory techniques. The anti-TCR antibody and an antigen binding fragment thereof are any protein molecules that bind a specific TCR. The anti-TCR antibody, or an antigen binding fragment thereof, binds to T-cells expressing a specific disease-associated TCR on the T-cell surface. Generating fragments of a protein, fusion protein, antigen binding fragments of an antibody by, for example, recombinant DNA technology, is routine in the art.

[0056] Anti-CD19 antibodies, or antigen-binding fragments thereof, are known in the art. Any suitable anti- CD19 antibodies, or antigen- binding fragments thereof, can be used.

[0057] Anti-CD20 antibodies, or antigen-binding fragments thereof, are known in the art. Any suitable anti- CD20 antibodies, or antigen- binding fragments thereof, can be used.

[0058] Any suitable linker can be used. The linker is one that generally makes a covalent bond or covalent bonds with the protein(s) or DNA. The linker is one that links two protein molecules or fragments together or a protein molecule and a DNA molecule or fragments thereof.

[0059] In some embodiments, the linker is a peptide. A peptide linker can be composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The linker can be poly-glycine or poly-glycine with one or more Ser and / or Thr. In some embodiments, the linker is GGGS or GGGSGGGS. A peptide linker can be generated as part of the multi-target therapeutic molecule by recombinant DNA technology.

[0060] In some embodiments, the linker is an amino acid, such as for example, a glycine.

[0061] hr other embodiments, the linker is a chemical non-peptide moiety. Proteins are typically cross-linked in a chemical reaction involving a cross-linker and side chains of amino acids. The reactivity of amino groups, thiols and carboxylic acids, render them as prime targets for crosslinking. The cross-linker can be a molecule with two reactive groups on either end, separated by a spacer. These reactive groups can target either primary amino groups (found in the side chainof lysine and at the protein N-terminus) or thiols (cysteine side chain). A small molecule, 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), can be used to activate carboxylic acids (aspartate, glutamate, protein C-terminus) to cross-link with amines (lysine, protein N-terminus). This directly cross-links atoms of the protein(s) with each other in a “zero-length” cross-link. Other cross-linkers have been synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and l-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. Other examples of chemical linkers include, without limitation, Bis(sulfosuccinimidyl) suberate (BS3), polyethylene glycol (PEG), which can be used as a single or branched chained moiety in a pegylation reaction, block sulfhydryls, such as N-Ethylmaleimide (NEM) and S-methyl methanesulfonothioate (MMTS), N-Succinimidyl-S- acetylthioacetate (SATA), and 2-Iminothiolane-HCl (Trant’ s reagent). In some embodiments, the cross linker is an acid-sensitive m-aconityl group, such as, for example, cis- aconitic anhydride. Diener, E., Diner, U., Sinha, A., Xie, S., and Vergidis, R. 1986, Science 231(4734): 148-150; Diener, U„ Diener, E„ Sinha. A., Xie, S„ and Vergidis, R. 1986. Selective suppression of murine lymphocyte function by daunomycin conjugated via an acid sensitive spacer to target specific carriers. In: Mediators of Immune Regulation and Immunotherapy (S.K. Singhal and T.L. Delovitch, Eds.), Elsevier Science Publications, Amsterdam, p. 177-181. These chemical non-peptide linkers are attached to proteins chemically by reactions known in the art. See, e.g., Id.

[0062] The auto-antigen can be an antigenic determinant of the auto-antigen, one that maintains the ability to bind to its B cell receptor. The auto-antigen, or its antigenic determinant, can be part of a fusion protein. The auto-antigen can be full-length or can be fragments and can be variants or mutants of either the full-length auto-antigen or fragments of the auto-antigen, so long as the auto-antigen is still recognized by the auto-antibody directed to it. The autoantigen^) can be chimeric / fusion proteins. In some embodiments, the auto-antigen is fused to an Fc domain of an immunoglobulin. In some embodiments, the auto-antigen comprises N-terminal auto-antigen and C-terminal human IgG Fc domain.

[0063] In some embodiments, the auto-antigen(s) on the disclosed chimeric molecule binds to B- cells expressing on their surface B-cell receptors to the auto-antigens, such as to Dsg3 and to Dsgl. In some embodiments, The auto-antigen on the disclosed chimeric molecule serves as lure to attract auto-reactive B cells expressing auto-antigen specific B cell receptors on their surface.

[0064] The auto-antigen can be a full-sized protein, or smaller polypeptides (or variants / mutants of either a full-sized protein or smaller polypeptide) but still bind to its antibody or antigenbinding fragment thereof. In some embodiments, the size, structure and position of the autoantigen are optimized for binding to its cognate B-cell receptor but not by a patient’s circulating auto-antibodies to the auto-antigen. The known auto-antigens are known in the art and can be obtained by, for example, recombinant DNA technology or other methods known in the art. The auto-antigens may be purchased or gifted.

[0065] In some embodiments, the anti-CD3 antibody, or an antigen (CD3) binding fragment thereof, is any protein molecule that binds CD3, which is found on all T-cells. The anti-CD3 antibody, or an antigen (CD3) binding fragment thereof, binds to T-cells expressing CD3 on their surface. Generating fragments of a protein by. for example, recombinant DNA technology, is routine in the art.

[0066] In some embodiments, the auto-antigen is Dsg3. In other embodiments, the auto-antigen is Dsgl. The auto-antigen can be an antigenic determinant of Dsg3 or Dsgl that maintains the ability to bind to its B-cell receptor. In further embodiments, the multi-target therapeutic molecules comprise anti-CD3 antibody, or an antigen-binding fragment thereof, with a linker to Dsg3 and to Dsgl. In further embodiments, the multi-target therapeutic molecules comprise an anti-CD3 antigen binding fragment of an anti-CD3 antibody (in certain embodiments, the antigen binding fragment is an FAB domain to CD3), with a linker to Dsg3 and / or to Dsgl and to antiCD 19 antibody, or an antigen-binding fragment thereof.

[0067] In some embodiments, one component is the auto-antigens Dsg3 or Dsgl of the autoimmune disease PV. In some embodiments, at least one of the auto-antigens is an autoantigen selected from Table 2. These auto-antigens in Table 2 are auto-antigens identified for PV other than Dsgl and Dsg3. In some embodiments, the components comprise Dsgl and / or Dsg3 and one or more of the autoantigens from Table 2. In some embodiments, one of the autoantigens is bullous pemphigoid antigen 180 or bullous pemphigoid antigen 230. In some embodiments, the components comprise Dsgl and Dsg3. In some embodiments, the components comprise Dsgl and / or Dsg3 and one or more of the autoantigens from Table 2. In some embodiments, two of the the auto-antigens are bullous pemphigoid antigen 180 and bullous pemphigoid antigen 230. In some embodiments, at least one of the autoantigens is patient specific. In some embodiments, a disclosed chimeric molecule comprises muscle specifictyrosine kinase (MuSK). MuSK is a known, dominant auto-antigen of patients with a sub-type of myasthenia gravis (MG). In other embodiments, a disclosed chimeric molecule comprises an anti-phospholipase A2 Receptor (PLA2R). PLA2R is a known auto-antigen of patients with membranous nephropathy (MNEP), a disorder where the body’s immune system attacks the filtering membranes in the kidney. These membranes clean waste products from the blood.

[0068] The auto-antigen(s) can be chimeric / fusion proteins. In some embodiments, the autoantigen has a poly-histidine tag (such as a his-6 tag), located at any suitable location, such as the N-terminus or the C-terminus of the auto-antigen. In other embodiments, other tag is fused to the auto-antigen, including, without limitation, the HQ tag, HN tag, HAT tag, maltose binding protein tag, strep tag, GST tag. The auto-antigen(s) on the disclosed multi-target therapeutic molecule binds to B-cells expressing on their surface B-cell receptors to the auto-antigens, such as to Dsg3 and to Dsgl. The auto-antigen on the disclosed multi-target therapeutic molecule serves as lure to attract auto-reactive B cells expressing auto-antigen specific B cell receptors on their surface. The anti-CD3 antibody attracts killer T cells and brings them in close proximity to the autoreactive B cells to facilitate their elimination. The anti-CD19 moiety could further serve to bring T cells and B cells in close proximity. Generating fragments of a protein or fusion protein by, for example, recombinant DNA technology, is routine in the art.

[0069] In another aspect, this disclosure provides a pharmaceutical composition comprising a disclosed multi-target therapeutic molecule.

[0070] The size of the linker, the size of each component and the distance between components can be optimized as needed.

[0071] Similar to bispecific antibodies in some embodiments, this approach facilitates formation of a “synapse” between T cells and antigen-specific B cells, to facilitate T cell mediated, antigenspecific elimination of autoreactive B -lymphocytes.

[0072] The disclosure provides a highly specific, potentially less toxic strategy to create a '‘targeted bullet” for the treatment of autoimmune diseases. The disclosed multi-target therapeutic molecule can be:• antigen- specific (multiple auto-antigens could be linked);• can be modified for any autoimmune disease where the antigen targets are known; and• can be individualized for a given patient.

[0073] The disclosed molecule, method and system do not require harvesting of autologous patient lymphocytes; do not require genetic engineering of patient T cells; and do not require reinfusion of autologous T cells.

[0074] Methods of making the disclosed multi-target therapeutic molecules are known in the art. For example, recombinant DNA technology can be used to make the separate protein molecules; chemical reactions to link proteins to each other with a linker or a protein and a DNA together with a linker are known and are used to link these moieties to each other. In the case of a peptide linker, the multi-target therapeutic molecule, or at least part of it, can be made as a large fusion protein.

[0075] The approach disclosed herein allows auto-antigen components of the chimeric molecule to serve as a lure to attract autoreactive B cells expressing the auto-antigen specific B-cell receptor (BCR) on the surface of these B-cells. In some embodiments, some of the other component(s) of the chimeric molecule brings killer T cells in close proximity to the autoreactive cells so as to facilitate their elimination. The disclosed chimeric molecule creates an immune synapse between a CD3+T cell and the correct BCR-bearing B-cell, leading to T-cell mediated killing of autoreactive B -lymphocytes only.

[0076] The disclosed chimeric molecules can be constructed by methods known in the art. The chimeric molecule constructs can be screened via in vitro cell-killing assays and INF-gamma secretion. Assay for the disclosed chimeric molecules can use cell targets that are nonproprietary autoantibody- secreting hybridomas; human donor T cells from peripheral blood samples; and the analysis of killing activity can be done in the presence of human PV serum antibodies, for embodiments in which the chimeric molecule is directed to PV. Animal studies using a non-proprietary mouse model, such as a PV mouse model, can be done on the disclosed chimeric molecules. Quantitation of serum anti-DSG-3 Ig in PV mice post-infusion of a disclosed chimeric molecule can be performed. Also, immunofluorescence of animal mucosa samples to detect IgG deposition after infusion of a disclosed chimeric molecule can be performed. Histologic mucosal blister formation (i.e., acantholysis) from the animals can be done after infusion of a disclosed chimeric molecule. Serial quantification of hybridoma burden by bioluminescence imaging can be performed in animals administered the disclosed chimera. Characterization of off-target activity can be performed.51Cr release assay can be performed to measure cytotoxicity of a disclosed chimeric molecule against human HaCat keratinocytes.Microscopic analysis of human skin xenografts as cellular targets can be performed after infusion of a disclosed chimeric molecule.

[0077] The disclosed chimeric molecules can be tested in human patients. Safety of the disclosed chimeric molecules can be monitored in these subjects. The efficacy of the chimeric molecules in these subjects can be monitored and assessed, such as change in autoantibody titer; PV Disease Area Index (PDAI) for a PV-specific disclosed chimeric molecule’ indices of remission: serologic and clinical; PK data; and PK / Immunogenicity / Pharmacodynamic assessments.

[0078] FORMULATING AND ADMINISTERING COMPOSITIONS

[0079] The disclosed composition may be administered to a subject in need thereof by any suitable mode of administration, any suitable frequency, and at any suitable, effective dosage.

[0080] The composition for use in a disclosed method may be in any suitable form and may be formulated for any suitable means of delivery.

[0081] In some embodiments, the disclosed composition is provided in a form suitable for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or any other appropriate route of injection. In some embodiments, compositions for injection are provided in sterile and / or non-pyrogenic form and may contain preservatives and / or other suitable excipients, such as sucrose, sodium phosphate dibasic heptahydrate or other suitable buffer, a pH-adjusting agent such as hydrochloric acid or sodium hydroxide, and polysorbate 80 or other suitable detergent.

[0082] When provided in solution form, in some embodiments, the composition for use in a disclosed method is provided in a glass or plastic bottle, vial or ampoule, any of which may be suitable for either single or multiple use. The bottle, vial or ampoule containing the disclosed composition may be provided in kit form together with one or more needles of suitable gauge and / or one or more syringes, all of which preferably are sterile. Thus, in certain embodiments, a kit is provided comprising a liquid solution as described above, which is packaged in a suitable glass or plastic bottle, vial or ampoule and may further comprising one or more needles and / or one or more syringes. The kit may further comprise instruction for use.

[0083] The disclosed composition can be produced by methods employed in accordance with general practice in the pharmaceutical industry, such as, for example, the methods illustrated in Remington: The Science and Practice of Pharmacy (Pharmaceutical Press; 21st revised ed. (2011) (hereinafter “Remington”).

[0084] In some embodiments, the disclosed composition comprises at least one pharmaceutically acceptable vehicle or excipient. These include, for example, diluents, carriers, excipients, fillers, disintegrants, solubilizing agents, dispersing agents, preservatives, wetting agents, preservatives, stabilizers, buffering agents (e.g. phosphate, citrate, acetate, tartrate), suspending agents, emulsifiers, and penetration enhancing agents such as DMSO, as appropriate. The composition can also comprise suitable auxiliary substances, for example, solubilizing agents, dispersing agents, suspending agents and emulsifiers.

[0085] In certain embodiments, the composition further comprises suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other pharmaceutically acceptable excipients.

[0086] A complete description of pharmaceutically acceptable excipients can be found, for example, in Remington's Pharmaceutical Sciences (Mack Pub., Co., N.J. 1991) or other standard pharmaceutical science texts, such as the Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017).

[0087] In some embodiments, the disclosed composition can be administered intravenously, intraperitoneally or intramuscularly, but other suitable routes of administration are also possible.

[0088] Water may be used as a earner and diluent in the composition. The use of other pharmaceutically acceptable solvents and diluents in addition to or instead of water is also acceptable.

[0089] Large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids, can also be used as earner compounds for the composition. Pharmaceutically acceptable earners in therapeutic compositions may additionally contain liquids, such as water, saline, glycerol or ethanol. Moreover, the said compositions may further comprise excipients, such as wetting agents or emulsifiers, buffering substances, and the like. Such excipients include, among others, diluents and carriers conventional in the art, and / or substances that promote penetration of the active compound into the cell, for example, DMSO, as well as preservatives and stabilizers.

[0090] The composition for use in a disclosed method may be presented in various dosage forms depending on the object of application; in particular, it may be formulated as a solution for injections.

[0091] The composition for use in a disclosed method may be administered systemically. Suitable routes of administration include, for example, parenteral administration, such as intravenous or intraperitoneal administration. However, depending on a dosage form, the disclosed composition may be administered by other routes.

[0092] The disclosed composition can be co-administered with another appropriate agent or therapy.

[0093] EXAMPLES

[0094] For this invention to be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

[0095] EXAMPLE 1 Assessment of Auto-Ab Specificity in PV Patients - development of a multiplexed platform to comprehensively identify autoantigens in an autoimmune disease

[0096] Multiplexed protein microarrays were used to probe PV patient or negative control sera.

[0097] Array 1.0: 15 auto-antigens were tested on 80 patients / controls; 5 disease associated targets were identified.Sajda T., Hazelton J., Patel M., Seiffert- Sinha K. Steinman L., Robinson W.H., Haab B.B., and Sinha A.A. 2016. Multiplexed autoantigen microarrays identify HLA as a key driver of anti- desmoglein and -non-desmoglein reactivities in Pemphigus. PNAS 113(7): 1859-64.Sinha, A.A. and Sajda, T. 2018. The evolving story of autoantibodies in Pemphigus vulgaris: development of the “super compensation hypothesis”. Front. Med. 5:218. doi:10.3389 / fmed.2018.00218. Array 2.0: 50 auto-antigens were tested on 675 patients / controls; 35 disease-associated targets were identified.

[0098] Table 1

[0099] Reactivities were stratified by clinical subtypes, with static parameters such as age, sex, HLA expression and disease onset, and with dynamic parameters such as disease activity, morphology, and disease duration.

[0100] See Sajda, T et al. Proc Natl Acad Sei. 2016 Feb 16;113(7): 1859-64.

[0101] IgG Reactivity was compared for PV patients vs. controls. Thirty five antigens were identified with significantly increased IgG autoreactivity in the PV group. These autoantigens are shown in Table 2.

[0102] TABLE 2 Auto-antigens identified for PV

[0103] The auto-antigens from Table 2 are: ANXA9 (Annexin A9), ATP2cl (ATPase Secretory Pathway Ca2+ Transporting 1), C / N, C5a receptor 1, Cadherin 8 (CDH8), Cadherin 9 (CDH9), CCT7, CD32 (FCGR2A), CD33, CENP (A / B), CHRNA9, CHRND , CHRNE (Cholinergic Receptor Nicotinic Epsilon Subunit), COL21A1 (collagen type XXI alpha 1 chain), Collagen III (COL3A1), Collagens, Cytochrome B5 (CYB5B), DSC1 (Desmocollin-1), DSC2 (Desmocollin-2), DSC3 (Desmocollin-3), dsDNA+ssDNA, Dsgl (Desmoglein 1), Dsg3 (Desmoglein 3), Dsg4 (Desmoglein 4), E-Cadherin, FcIgE, FH (fumarase), GP1BA (Glycoprotein lb Platelet Subunit Alpha), HLA-DRA, HLA E, HSP60 Protein, Human Ml, Human M2, Human M3, Human M4, Human M5, Integrin alpha X, JUP, NDUFS1 (NADH-ubiquinone oxidoreductase), PDHA1 (pyruvate dehydrogenase El subunit alpha 1 ), PK3, PR3- MPO, Ro-SSA, RPP, SOD2 (superoxide dismutase 2), TG (thyroglobulin), TPO (Thyroid Peroxidase), and UlsnRNP.

[0104] Multiple non Dsg3 and Dsgl auto-antibodies were found to be correlated with disease activity. The pattern is similar in each patient, with an average of 9 auto-antigens. It appears that the set of antigens driving disease activity differs in each patient. Individual patients have unique auto-antigenic profiles. See FIGS. 1A, IB, and 1C.

[0105] EXAMPLE 2 - Exemplary Embodiments

[0106] FIG. 2A illustrates an embodiment of the disclosed chimeric molecule; FIG. 2B illustrates its mode of action.

[0107] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, while only certain features of the invention have been illustrated and described, many modifications and changes will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A chimeric molecule comprising a T-cell binding moiety and two or more auto-antigens of an autoimmune disease, each auto-antigen is linked to the T-cell binding moiety by linker, wherein said T-cell binding moiety is an anti-CD3 antibody, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment to a T-cell receptor variable chain region, wherein any two auto-antigens are the same auto-antigens or different.

2. The chimeric molecule of claim 1, wherein one of the auto-antigens is Dsg3 or Dsgl.

3. The chimeric molecule of claim 1, wherein two of the auto-antigens are Dsg3 and Dsgl.

4. The chimeric molecule of claim 1, wherein two of the auto-antigens are Dsg3 and Dsg3 or Dsgl and Dsgl.

5. The chimeric molecule of claim 1, wherein at least one of the autoantigens is an auto-antigen selected from the group consisting of: ANXA9 (Annexin A9), ATP2cl (ATPase Secretory Pathway Ca2+ Transporting 1), C / N, C5a receptor 1 , Cadherin 8 (CDH8), Cadherin 9 (CDH9), CCT7, CD32 (FCGR2A), CD33, CENP (A / B), CHRNA9, CHRND , CHRNE (Cholinergic Receptor Nicotinic Epsilon Subunit), COL21A1 (collagen type XXI alpha 1 chain), Collagen III (COL3A1), Collagens, Cytochrome B5 (CYB5B), DSC1 (Desmocollin-1), DSC2 (Desmocollin- 2), DSC3 (Desmocollin-3), dsDNA+ssDNA, Dsgl (Desmoglein 1), Dsg3 (Desmoglein 3), Dsg4 (Desmoglein 4), E-Cadherin, FcIgE, FH (fumarase), GP1BA (Glycoprotein lb Platelet Subunit Alpha), HLA-DRA, HLA E, HSP60 Protein, Human Ml, Human M2, Human M3, Human M4, Human M5, Integrin alpha X, JUP, NDUFS1 (NADH-ubiquinone oxidoreductase), PDHA1 (pyruvate dehydrogenase El subunit alpha 1), PK3, PR3-MP0, Ro-SSA, RPP, SOD2 (superoxide dismutase 2), TG (thyroglobulin) ,TPO (Thyroid Peroxidase), and UlsnRNP.

6. The chimeric molecule of claim 1 , wherein two of the auto-antigens are bullous pemphigoid antigen 180 and bullous pemphigoid antigen 230.

7. The chimeric molecule of any of the preceding claims, wherein at least one of the autoantigens is patient specific.

8. The chimeric molecule of any of the preceding claims, wherein the T-cell binding moiety is an anti-CD3 antibody or an antigen-binding fragment thereof.

9. The chimeric molecule of any of the preceding claims, wherein the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region.

10. The chimeric molecule of claim 9, wherein the T-cell receptor variable chain region is selected from vp2, vp5.1, VP6, VP8, vpiO, and Vpi3.1.

11. The chimeric molecule of any of the preceding claims, further comprising an anti-CD19 antibody, or an antigen-binding fragment thereof, or an anti-CD20 antibody, or an antigenbinding fragment thereof, linked to the T-cell binding moiety or an auto-antigen, or polypeptide or peptide fragment thereof, by linker.

12. The chimeric molecule of any of the preceding claims, wherein one or more of the antigen binding fragments is a single chain antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, or a single chain Fvfragment.

11. The chimeric molecule of any of the preceding claims, wherein one or more of the antigen binding fragments is a fragment of an IgG molecule.

12. The chimeric molecule of any of the preceding claims, wherein the antibody or antigen binding fragment with an Fc tail has reduced or no fucose moieties.

13. A pharmaceutical composition comprising one or more chimeric molecules of any of claims 1-12.

14. The pharmaceutical composition of claim 13, wherein the composition comprises two or more chimeric molecules.

15. A method of treating or preventing an autoimmune disease in a patient comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition of claim 12 or claim 13 to said patient.

16. The method of claim 15, wherein the autoimmune disease is Pemphigus vulgaris (PV) or its variants and each chimeric molecule comprises one or more auto-antigens, or a polypeptide or peptide fragment of an auto-antigen, of Pemphigus vulgaris.

17. The method of claim 15, wherein the autoimmune disease is bullous pemphigoid and each chimeric molecule comprises one or more auto-antigens, or a polypeptide or peptide fragment of an auto-antigen, of bullous pemphigoid.

18. The method of claim 15, wherein the autoimmune disease is a subtype of myasthenia gravis and each chimeric molecule comprises one or more auto-antigens, or a polypeptide or peptide fragment of an auto-antigen, of the subtype of myasthenia gravis.

19. The method of claim 15, wherein the autoimmune disease is membranous nephropathy and each chimeric molecule comprises one or more auto-antigens, or a polypeptide or peptide fragment of an auto-antigen, of membranous nephropathy.

20. A chimeric molecule comprising a T-cell binding moiety and an interferon molecule or a portion thereof linked to the T-cell binding moiety by linker, wherein said T-cell binding moiety is an anti-CD3 antibody or an antigen-binding fragment thereof or an antibody or antigenbinding fragment to a T-cell receptor variable chain region.21 . The chimeric molecule of claim 20, wherein the T-cell binding moiety is an anti-CD3 antibody or an antigen-binding fragment thereof.

22. The chimeric molecule of claim 20, wherein the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region.

23. The chimeric molecule of claim 22, wherein the T-cell receptor variable chain region is selected from V02, V05.1, V06, V08, V01O, and V013.1.

24. The chimeric molecule of any of claims 20-23, further comprising an anti-CD19 antibody or an antigen-binding fragment thereof, linked to the T-cell binding moiety or the interferon molecule or a portion thereof by linker.

25. A pharmaceutical composition comprising one or more chimeric molecules of any of claims 20-24.

26. The pharmaceutical composition of claim 25, comprising two or more chimeric molecules.

27. A method of treating a patient having autoantibodies to an interferon after a Covid-19 infection, comprising administering a therapeutically effective amount of a pharmaceutical composition of claim 25 or claim 26.

28. A method of treating or preventing a B-cell mediated disease in a patient in need thereof comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising the chimeric molecule of claim 13 or claim 14.

29. A chimeric molecule comprising a T-cell binding moiety and a viral vector administered to a subject for gene therapy or a DNA molecule of a viral vector administered to a subject for gene therapy linked to the T-cell binding moiety by linker, wherein said T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region.

30. The chimeric molecule of claim 29, wherein the T-cell binding moiety is an antibody or antigen-binding fragment to a T-cell receptor variable chain region.

31. The chimeric molecule of claim 30, wherein the T-cell receptor variable chain region is selected from V 2, V05.1, V06, V08, V01O, and V013.1.

32. The chimeric molecule of any of claims 29-31, further comprising an anti-CD19 antibody or an antigen-binding fragment thereof, linked to the T-cell binding moiety or the interferon molecule or a portion thereof by linker.

33. A pharmaceutical composition comprising one or more chimeric molecules of any of claims 29-32.

34. The pharmaceutical composition of claim 33, comprising two or more chimeric molecules.

35. A method of to improve gene therapy in a patient in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition of claim 33 or 34.

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