EP4683943A1

EP4683943A1 - Compositions and methods for antigen-specific therapy

Info

Publication number: EP4683943A1
Application number: EP24719813.8A
Authority: EP
Inventors: Animesh SINHA; Elizabeth QUACKENBUSH
Original assignee: Bodhi Bio LLC
Current assignee: Bodhi Bio LLC
Priority date: 2023-03-23
Filing date: 2024-03-25
Publication date: 2026-01-28
Also published as: WO2024197296A1; AU2024240821A1

Abstract

A platform for therapy provides a chimeric molecule comprising a T-cell binding moiety linked with a linker to a HLA I or a HLA II molecule; the HLA I or HLA II molecule has an autoimmune disease-specific peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule. A pharmaceutical composition comprises a disclosed chimeric molecule. Methods of treating diseases using the disclosed molecules and compositions.

Description

COMPOSITIONS AND METHODS FOR ANTIGEN-SPECIFIC THERAPY

TECHNICAL FIELD

[0001] This disclosure relates to a platform for using a molecule to target antigen- specific T cells 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 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 one or more HLA I or HLA II molecules; each HLA I or HLA II molecule having an autoimmune disease- specific peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule; the HLA I or HLA II molecule 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 antigenbinding fragment to a T-cell receptor variable chain region.

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

[0005] In another aspect, this disclosure provides a method of treating or preventing an autoimmune disease in a patient in need thereof comprising administering to said patient a therapeutically or prophylactically effective amount of a disclosed pharmaceutical composition. [0006] Numerous other aspects arc 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 in expression 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.

DETAILED DESCRIPTION

[0008] As used herein, the word “a” or “plurality” before a noun represents one or more of the particular noun.

[0009] 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.

[0010] “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 amount is an amount of the disclosed pharmaceutical composition to kill disease-related T cells. [0011] 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.

[0012] The term “antibody fragment,” “antigen-binding fragment of an antibody,” and the like are known in the ail. 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, c.g., Todorovska ct 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. 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 Sei 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 Vn domains with modifications such that single domain antibodies are formed.

[0013] The term ‘an antigen-binding fragment” of an antibody can also include the entire Fc tail of an antibody.

[0014] The term “CD3” refers to cluster of differentiation 3 and is a protein complex and T cell co-receptor. CD3 is a protein complex involved in activating both the cytotoxic T cell (CD8⁺ T cells) and T helper cells (CD4⁺ T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the CD3-zeta (i^-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and the other CD3 molecules together constitute the TCR complex.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the ait 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, will control.

[0019] AUTOIMMUNE DISEASES

[0020] 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 2^nd or 3^rd leading 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. Treatment options for autoimmune diseases are limited and largely nonspecific or symptom oriented. No true cures are available and there is no consensus treatment guidelines. Targeted, individualized therapies are lacking.

[0021] 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. [0022] Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering skin disease, characterized by intraepithelial (suprabasalar) acantholysis, which is a loss of ccll-ccll adhesion. Quite a bit is known about PV. HLA genetic predisposition (HLA DRB 1*0402 and DQB 1*0503) is known; T cell (Th2 driven) and B cell subsets (producing IgG4 autoantibodies) are known. And the primary autoantibody targets (autoantigens) - Desmoglein (Dsg)-3 and Desmoglein-1 - are known.

[0023] 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.

[0024] 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.

[0025] Currently proposed antigen- specific therapy makes use of Chimeric Auto- Antibody Receptor T (CAAR-T) cells, an adaptation of the CAR-T cell strategy. 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 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.

[0026] 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 arc needed;

• potential for significant, life-threatening adverse effects;

• potential for long lived, permanence of therapy; • inability to tune down therapy;

• inability to readily adapt to multiple target therapy;

• inability to readily personalize to individual patients;

• inability to readily adapt to evolving autoimmune response in a given patient;

• exorbitant costs;

• potential risk of secondary cancer;

• commercial scalability challenges and

• complicated payer policies .

[0027] HUMAN LEUKOCYTE ANTIGENS (HLA)

[0028] Humans have two classes of HLA molecules: HLA class I antigens and HLA class II antigens.

[0029] HLA class I antigens are expressed on nucleated cells and platelets while the HLA class II antigens are expressed on antigen presenting cells (APC) such as B lymphocytes, dendritic cells, macrophages, monocytes, Langerhans cells, endothelial cells, activated T cells, and thymic epithelial cells.

[0030] HLAs corresponding to MHC class I (A, B, and C) belong to HLA Class 1 group and present peptide antigens to killer T-lymphocytes (killer T-cells, which are also referred to as CD8-positive T-cells). HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR) belong to HLA Class II group and also present peptide antigens, but to T-helper cells (also called CD4-positive T cells).

[0031] Wild-type HLA class I molecule is a heterodimer and consists of two non-covalently linked polypeptide chains, a HLA-encoded a chain or heavy chain and a non-HLA encoded subunit, p2-microglobulin. The a chain has three regions, including a cytoplasmic region containing a peptide-binding groove (also referred to herein as a HLA peptide binding cleft) made from the al and a2 domains, a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane, and a highly conserved a3 immunoglobulin-like domain to which CD8 binds. A peptide-binding groove is formed between the al and a2 helices with a p-pleated sheet as its floor.

[0032] Wild type class II HLA molecule is also a heterodimer and is composed of two non- covalently associated polypeptide chains, an a chain and a P chain. A peptide binding groove (also referred to herein as a HLA peptide binding cleft) is formed between the al and pi domains with a P-plcatcd floor.

[0033] Wild type HLA I and wild type HLA II are dimer molecules. Either HLA I and HLA II tetramers with antigen-specific peptides have been made and have become widely applied molecular tools for in vitro use. For example, these tetramers detect antigen- specific T cells by binding to specific T cell receptors (TCR) expressed by the T cells and reagents can be generated to detect either antigen- specific CD4 or antigen- specific CD8 T cells.

[0034] As referred to herein, an HLA I molecule or an HLA II molecule HLA I and HLA II includes select domains thereof, monomer(s) comprising the select domains, a dimer and a tetramer and also includes fragments thereof, fusion proteins comprising these molecules, as long as the molecules are able to bind and present the peptide antigens to TCRs.

[0035] DISEASE-SPECIFIC PEPTIDES

[0036] Immunogenic recognition by T cells relics on the presentation of immunogenic peptides at the cell surface.

[0037] In the HLA I pathway, intracellular proteins are degraded into small peptides by the proteasome. These peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) protein complex. There, they can bind to human leukocyte antigen class I (HLA-I) molecules. After trafficking to the cell surface, the complexes may be recognized by CD8 T cells. HLA-I molecules bind short peptides, mainly 9-11 amino acids, and different HLA-I alleles have distinct binding specificities.

[0038] In the HLA II pathway, peptides coming from the degradation of phagocytosed extracellular proteins are presented on HLA-II molecules for recognition by CD4 T cells. The HLA II molecules bind longer peptides (12-20 amino acids) within an open-ended peptide- binding site. Unlike HLA-I, HLA-II molecules are mainly expressed on specific professional antigen-presenting cells (pAPCs), such as dendritic cells or B cells.

[0039] Known disease- specific peptides are located within the HLA peptide binding cleft of the HLA I or the HLA II molecule. A peptide bound to the HLA molecule portion of a disclosed chimeric molecule can be slightly modified and differs from ones known in the art to be presented by HLA for certain diseases, including, for example and without limitation, autoimmune diseases. [0040] Known disease- specific peptides located within the HLA peptide binding cleft of the HLA I or the HLA II molecule can be derived from auto-antigen proteins known in the art; that is, being a peptide from an auto-antigen protein, though such peptide may be modified as needed.

[0041] T-CELL RECEPTOR

[0042] T-cell receptor (“TCR”) is a protein complex found on the surface of T lymphocytes. TCR recognizes and binds disease-specific peptides located within the HLA peptide binding cleft of the HLA I or the HLA II molecule.

[0043] TCR is a heterodimer. In humans, in 95% of T cells the TCR consists of an alpha (a) chain and a beta (P) chain, whereas in 5% of T cells the TCR consists of gamma and delta (y/5) chains. Each chain has a variable and a constant domain. The variable domain has three hypervariable regions, known as complementarity determining regions (CDRs). The alpha chain is generated by VJ recombination and the beta chain is generated by VDJ recombination.

[0044] MOLECULES, COMPOSITIONS, AND METHODS

[0045] This disclosure provides a chimeric molecule comprising a T-cell binding moiety and one or more HLA I or HLA II molecules; each HLA I or HLA II molecule having an autoimmune disease- specific peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule; the HLA I or HLA II molecule 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 antigenbinding fragment to a T-cell receptor variable chain region. In some embodiments, the T-cell receptor variable chain region is selected from V 2, V 5.LVP6, V 8, v iO, and V 13.1. In other embodiments, In some embodiments, the T-cell receptor variable chain region is one disclosed in Hsu et al., Sci. Transl. Med. 15, eadi0258 (2023) 29 November 2023 or one disclosed in Vantourout et al., Sci. Adv. 9, eadj6174 (2023) 6 December 2023. Other suitable target T-cell receptor variable chain region is disclosed and known in the art. See, e.g., US Patent Publication Nos. 20210380692, 20220064255, 20220064297, 20230035484, 20230048244, 20230031734, 20230048244, 20230127740, 20230142522, 20230174650, 20230227552, 20230374133, 20210277119.

[0046] The disclosed chimeric molecule is designed to bind to two or more targets. If the chimeric molecule can bind to three targets, it is also called a trimeric molecule. [0047] Antibodies to CD3 and antibodies to a T-cell receptor variable chain region are known and available in the art. Examples of antibodies to a T-ccll receptor variable chain region arc in, for example, Hsu et al., Sci. Transl. Med. 15, eadi0258 (2023) 29 November 2023 or one disclosed in Vantourout et al., Sci. Adv. 9, eadj6174 (2023) 6 December 2023, US Patent Publication Nos. 20210380692, 20220064255, 20220064297, 20230035484, 20230048244, 20230031734, 20230048244, 20230127740, 20230142522, 20230174650, 20230227552, 20230374133, 20210277119. Any anti-CD3 antibodies can be used and any antibodies to a T- cell receptor variable chain region can be used.

[0048] In some embodiments, the HLA I or the HLA II molecule is a domain or domains (select domains) of an HLA I or HLA II molecule. In some embodiments, the HLA II is a tetramer or a dimer. In some embodiments, the HLA I molecule or the HLA II molecule lacks transmembrane and intracellular domains. In other embodiments, the HLA I molecule or HLA II molecule is a multimer of the HLA I or HLA II molecules. See Chang, 328 Mol. Cells 2021: 44(5): 328-334. These multimers bind to their TCR with greater affinity than HLA I and HLA II in their natural form.

[0049] As referred herein, the “multimers” of HLA I and HLA II molecules include multimers as disclosed in Chang, 328 Mol. Cells 2021; 44(5): 328-334.

[0050] A disclosed chimeric molecule can comprise a domain or domains of an HLA I or HLA II molecule. The domain or domains of an HLA I or HLA II molecule comprise a peptide binding cleft and need not be a dimer or tetramer, but can be, for example, a monomer.

[0051] Given the known allelic variation in the major histocompatibility complex (MHC), a library of such molecules bound to the same or substantially the same peptide may be needed. [0052] In some embodiments, a DRB 1*0402 or a DQB 1*0503 peptide is bound to the HLA molecule. In other embodiments, a 15-20mer peptide derived from the outer 3 (membrane distal) domains of the desmoglein 3 protein molecule is bound to the HLA molecule.

[0053] In some embodiments, the autoimmune disease-specific peptide is derived from an autoantigen, that is, a part of an auto-antigen protein, though the peptide may be further modified as needed. In some embodiments, the auto-antigen is Dsg3 or Dsg of the autoimmune disease PV. In some embodiments, the autoantigens is an autoantigen selected from Table 2. These autoantigens in Table 2 are auto-antigens identified for PV. In some embodiments, one of the the auto-antigens is bullous pemphigoid antigen 180 or bullous pemphigoid antigen 230. In some embodiments, at least one of the autoantigens is patient specific, which means that a specific patient is determined to have a particular auto-antigen to which the patient has made autoantibody against. Methods of determining what auto-antigen a patient has is known in the ait. In some embodiments, the auto-antigen is muscle specific tyrosine kinase (MuSK). MuSK is a known auto-antigen of patients with myasthenia gravis (MG). In other embodiments, the autoantigen is 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.

[0054] If a non-human subject is meant to receive the disclosed chimeric molecule, then instead of human leukocyte antigen, the appropriate MHC molecule from that subject is used. The disclosure includes such chimeric molecules.

In some embodiments, the antigen binding fragment is a single chain antibody, a Fab fragment, a Fab’ fragment, a Flab’)? fragment, or a single chain F_v fragment. In some embodiments, the antigen binding fragment is a fragment of an IgG molecule. In some embodiments, the antibody or the antigen binding fragment with an Fc tail has reduced or no fucose moieties.

[0055] In another aspect, this disclosure provides a pharmaceutical composition comprising one or more disclosed chimeric molecules. In some embodiments, the pharmaceutical composition comprises two or more disclosed chimeric molecules.

[0056] This disclosure provides a method of treating or preventing a patient with a disease, such as, for example and without limitation, an auto-immune disease, in which a disease-specific peptide that is presented by a HLA I or a HLA II molecule is known. The method comprises administering a therapeutically or prophylactically effective amount of a disclosed pharmaceutical composition to said patient. In such case, the disclosed chimeric molecule can be referred to herein also as “T-cell Selective Molecular Autoantigen-Receptor Targetors” (“T- SMAART”).

[0057] In some embodiments, the disease is the autoimmune disease Pemphigus vulgaris (PV) or its variants. In such embodiments, the disclosed chimeric molecule or molecules comprise HLA- 1 or HLA-2 molecules with a PV-specific peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule. [0058] In some embodiments, the disease is the autoimmune disease bullous pemphigoid. Tn these embodiments, the disclosed chimeric molecule or molecules comprise HLA-1 or HLA-2 molecules with bullous-pemphigold-disease-specific-peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule.

[0059] In some embodiments, the autoimmune disease is a subtype of myasthenia gravis. In these embodiments, the components of the disclosed chimeric molecule or molecules comprise HLA- 1 or HLA-2 molecules with peptide specific for said subtype of myasthenia gravis bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule.

[0060] In some embodiments, the disease is the autoimmune disease membranous nephropathy. In these embodiments, the components of the disclosed chimeric molecule or molecules comprise HLA-1 or HLA-2 molecules with membranous-nephropathy-disease-specific -peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule.

[0061] In some embodiments, the disclosed T-SMAART has an antibody backbone.

[0062] The antibodies to CD3 are known in the art or can be obtained or generated by routine laboratory techniques. CD3 protein is known and can be obtained or generated by routine laboratory techniques.

[0063] HLA I and HLA II dimers and tetramers are known in the art and can be obtained or generated by routine laboratory techniques. When referred to herein, including in the claims, HLA I and HLA II dimers and tetramers include fragments thereof, fusion proteins comprising these molecules, as long as the molecules are able to bind and present a peptide antigen. A molecule comprising select domain(s), which molecule can be a monomer, of HLA I or HLA II can be constructed by routine laboratory techniques; this molecule is also able to bind and present a peptide antigen.

[0064] Some disease-specific peptides that bind to the HLA peptide binding cleft are known. These peptides can be obtained or made by routine methods.

[0065] 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 F_v fragment. 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 antibody or the antigen binding fragments with an Fc tail 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 moictics 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 multispecific molecule with glycosidase and glycosynthase enzymes.

[0066] 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.

[0067] 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. A peptide linker can be generated as part of the multi-target therapeutic molecule by recombinant DNA technology.

[0068] In 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 chain of 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 1 -hydroxy-7 -azabenzotriazole 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 (Traut’s reagent). In some embodiments, the cross linker is an acid-sensitive cz -aconityl group, such as, for example, czT-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.

[0069] The size of the linker and the size of each component can be optimized as needed. [0070] Methods of making the disclosed chimeric 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 case of a peptide linker, the disclosed chimeric molecule, or at least part of it, can be made as a large fusion protein.

[0001] The disclosure provides a highly specific, potentially less toxic strategy to create a “targeted bullet” for the treatment of autoimmune diseases. The disclosed chimeric 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.

[0002] 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.

[0003] Methods of making the disclosed chimeric 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 chimeric molecule, or at least part of it, can be made as a large fusion protein.

[0004] 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-proprictary mouse model, such as 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, along with quantitation of PV-targeting T cells. 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.

[0005] The disclosed chimeric molecules can be tested in human subjects. 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.

[0071] FORMULATING AND ADMINISTERING COMPOSITIONS

[0072] 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. [0073] The composition for use in a disclosed method may be in any suitable form and may be formulated for any suitable means of delivery.

[0074] In some embodiments, the disclosed composition is provided in a form suitable for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or any other 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.

[0075] 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 arc 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 comprise one or more needles and/or one or more syringes. The kit may further comprise instructions for use.

[0076] 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”).

[0077] 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.

[0078] In certain embodiments, the composition further comprises suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other pharmaceutically acceptable excipients.

[0079] 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).

[0080] In some embodiments, the disclosed composition can be administered intravenously, intraperitoneally or intramuscularly, but other suitable routes of administration are also possible. In some embodiments, the disclosed composition is administered subcutaneously or intravenously.

[0081] Water may be used as a carrier and diluent in the composition. The use of other pharmaceutically acceptable solvents and diluents in addition to or instead of water is also acceptable. [0082] 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 carrier 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.

[0083] 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.

[0084] The composition for use in a disclosed method may be administered systemically. Suitable routes of administration include, for example, parenteral administration, such as intravenous, intraperitoneal administration. However, depending on a dosage form, the disclosed composition may be administered by other routes.

[0085] The disclosed composition can be co-administered with another appropriate agent or therapy.

[0086] EXAMPLES

[0087] 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.

[0006] EXAMPLE 1 - EXAMPLE 1 Assessment of Auto- Ab Specificity in PV Patients - development of a multiplexed platform to comprehensively identify autoantigens in an autoimmune disease

[0007] Multiplexed protein microarrays were used to probe PV patient or negative control sera. [0008] 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.

[0009] Table 1

[0010] 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.

[0011] See Sajda, T el al. Proc Natl Acad Sci. 2016 Feb 16; 113(7): 1859-64.

[0012] IgG Reactivity was compared for PV patients vs. controls. Thirty five antigens were identified with significantly increased IgG autoreactivity in the PV group. These auto-antigens are shown in Table 2.

[0013] Table 2 AUTO ANTIGENS

[0014] 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 appeal’s 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.

[0088] 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

CLAIMS What is claimed is:

1. A chimeric molecule comprising a T-cell binding moiety and one or more HLA I or HLA II molecules; each HLA I or HLA II molecule having an autoimmune disease- specific peptide bound to a HLA peptide binding cleft of the HLA I or the HLA II molecule; the HLA I or HLA II molecule 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 antigenbinding fragment to a T-cell receptor variable chain region.

2. The chimeric molecule of claim 1, wherein the HLA I molecule or the HLA II molecule lacks transmembrane and intracellular domains.

3. The chimeric molecule of claim 1, wherein the HLA I molecule or HLA II molecule is a multimer.

4. 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.

5. 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.

6. The chimeric molecule of claim 5, wherein the T-cell receptor variable chain region is selected from V02, VP5.1,VP6, Vp8, VpiO, and VP 13.1.

7. The chimeric molecule of any of the preceding claims, wherein the antigen binding fragment is a single chain antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, or a single chain F_v fragment.

8. The chimeric molecule of any of the preceding claims, wherein the antigen binding fragment is a fragment of an IgG molecule.

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

10. The chimeric molecule of any of the preceding claims, wherein the autoimmune diseasespecific peptide is derived from an auto-antigen of the auto-immune disease.

11. The chimeric molecule of claim 10, wherein the autoimmune disease-specific peptide is derived from Dsg3 or Dsgl.

12. The chimeric molecule of claim 10, wherein the autoimmune disease-specific peptide is derived from an auto-antigen selected from the group consisting of:

13. The chimeric molecule of claim 10, wherein the autoimmune disease- specific peptide is derived from bullous pemphigoid antigen 180 or bullous pemphigoid antigen 230.

14. The chimeric molecule of any of claims 1-13, wherein the autoimmune disease-specific peptide is patient specific.

15. A pharmaceutical composition comprising one or more chimeric molecule of any of claims 1-14.

16. The pharmaceutical composition of claim 15, wherein said composition comprises two or more chimeric molecules.

17. A method of treating or preventing an autoimmune disease in a patient in need thereof comprising administering to said patient a therapeutically or prophylactically effective amount of a pharmaceutical composition of any of claims 15-16.

18. The method of claim 17, wherein the autoimmune disease is Pemphigus vulgaris (PV) or its variants and the autoimmune disease- specific peptide is derived from an auto-antigen of PV.

19. The method of claim 17, wherein the autoimmune disease is bullous pemphigoid and the autoimmune disease- specific peptide is derived from an auto-antigen of bullous pemphigoid.

20. The method of claim 17, wherein the autoimmune disease is a subtype of myasthenia gravis and the autoimmune disease-specific peptide is derived from an auto-antigen of the subtype of myasthenia gravis.

21. The method of claim 17, wherein the autoimmune disease is membranous nephropathy and the autoimmune disease- specific peptide is derived from an auto-antigen of membranous nephropathy.